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predominant male sex hormones drive the development andmaintenance of male characteristics by binding to androgen receptor AR As androgensare systemically distributed throughout the whole anism they affect many tissues andcell types in addition to those in male sexual ans It is now clear that the immunesystem is a target of androgen action In the lungs many immune cells express ARs andare responsive to androgens In this review we describe the effects of androgens and ARson lung myeloid immune cells”monocytes and macrophages”as they relate to healthand disease In particular we highlight the effect of androgens on lung diseases such asasthma chronic obstructive pulmonary disease and lung fibrosis We also discuss thetherapeutic use of androgens and how circulating androgens correlate with lung diseaseIn addition to human studies we also discuss how mouse models have helped to uncoverthe effect of androgens on monocytes and macrophages in lung disease Although therole of estrogen and other female hormones has been broadly analyzed in the literaturewe focus on the new perspectives of androgens as modulators of the immune systemthat target myeloid cells during lung ‚ammationEdited byFlavia BazzoniUniversity of Verona School ofMedicine and Surgery ItalyReviewed byPaola ParronchiUniversity of Florence ItalyTim WillingerKarolinska Institutet SwedenSandra O GollnickUniversity at Buffalo United StatesCorrespondenceNicola HellernhellerjhmieduKeywords androgen androgen receptor monocyte macrophage asthma lung sex difference sex hormoneSpecialty sectionThis was submitted toCytokines and Soluble Mediators inImmunitya section of the journalFrontiers in ImmunologyReceived March Accepted June Published August CitationBecerraDiaz M Song M and Heller N Androgen and AndrogenReceptors as Regulators of Monocyteand Macrophage Biology in theHealthy and Diseased LungFront Immunol 103389fimmu202001698INTRODUCTIONThe immune system is essential for maintaining homeostasis within tissues and ans andprotecting them against threats such as harmful pathogens or cancerous transformation Itcomprises both innate and adaptive components The innate immune system is made up of theinnate lymphoid innate lymphoid cells [ILCs] natural killer cells [NKs] and lymphoid tissueinducers [LTi] and innate myeloid subsets The innate immune system consists of a networkof immune cells and molecules that provide rapid firstline defense against pathogens In contrastthe adaptive immune response made up of B and T lymphocytes takes days or even weeks tobecome established Innate immune cells express pattern recognition receptors that recognizeunique and conserved pathogenassociated molecular patterns such as lipopolysaccharide LPSviral ssRNA and fungal glucan B and T cells have evolved to recognize a finer repertoireof self and nonselfantigens that facilitate pathogenspecific actions immunologic memorygeneration and host immune homeostasis regulation To accomplish this the adaptiveimmune response involves a tightly regulated interplay between T and B lymphocytes andFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyantigenpresenting cells of the myeloid lineage such as dendriticcells DCs monocytes and macrophages Myeloid cells arisefrom the bone marrow The type and magnitude of the immuneresponse is ‚uenced by biological sex and age and thereforediï¬ers between males and females Sex diï¬erences in the functionof the immune system arise from both genetic chromosomalsex diï¬erences and diï¬erences mediated by the action of maleand female sex hormones Because the concentration of sexhormones changes over the lifespan and throughout the courseof the menstrual cycle in women the function of the immunesystem also changes during diï¬erent stages of life Innate myeloidimmune cells like other cell types express sex hormone receptorsand are responsive to sex hormones Sex hormones are synthesized from cholesterol through adefined enzymatic cascade predominately in the gonads and theadrenal glands Sex hormones are also produced in othertissues including the brain placenta mammary glands liver andadipose tissue “ In addition to driving sexual developmentof egg and sperm production sex hormones are responsiblefor the development of male and female secondary sexualcharacteristics like breast development and growth of facial hairthat occur during puberty Androgens include testosteronedihydrotestosterone DHT androstenedione androstenedioland dehydroepiandrosterone DHEA with DHT being the mostpotent The concentration of androgens in circulation isabout sevenfold higher in adult men than in adult women Estradiol and progesterone are the predominantfemale sex hormones synthesized by the ovaries andadrenal glands Both male and female sex hormones are boundto the plasma proteins albumin and sex hormone bindingglobulin SHBG and only a small percentage exists as freehormone “ Thus the bioavailability of sex hormones isregulated by their biosynthesis and also the amount of albuminand SHBGImportantly sex hormones mediate not only anatomicdiï¬erences between women and men but also direct sexdiï¬erences in immune responses leading to diï¬erent risks forimmunologic diseases Overall women have a greaterrisk for autoimmune diseases such as systemic sclerosis andsystemic lupus erythematosus whereas men are morelikely to die of infectious and parasitic diseases Moreovermen have a greater risk of nonreproductive cancers “Both gender and sex are important mediators of these andother health and disease diï¬erences observed between men andwomen While gender refers to the array of socially constructedroles attitudes personality traits and behaviors sex representsa biological characteristic of an individual includingthe hormonal milieu and chromosome complement Ingeneral estrogens are considered to have pro‚ammatoryproperties and androgens are thought to have anti‚ammatoryproperties In the United States and worldwide relevant evidence highlights important epidemiologic sexdiï¬erences in incidence susceptibility and severity of a numberof diseases that aï¬ect the respiratory tract In this reviewwe will focus on how male sex hormones the androgensmodulate the response of myeloid cells in the lung and howthis modulation impacts the outcome of diï¬erent diseases ofthe lungSEX DIFFERENCES IN HUMAN LUNG ANDLUNG DISEASESsex mediates diï¬erencesBiologicalin the incidence andpathophysiology of lung diseases These diï¬erences arise fromsex diï¬erences in the structure and function of the lung itselfand also in the immune cells that populate the lung and arerecruited to it during ‚ammation Before birth the female lunghas several structural advantages over the male lung Surfactantis produced earlier and although the female lung is smaller ithas more alveoli per unit area Neonatal females have higherexpiratory flow rates than do male neonates when corrected forsize Thus male sex is a major risk factor for the developmentof respiratory distress syndrome bronchopulmonary dysplasia inneonates “ and asthma in childhood In addition to the contribution of structural diï¬erences ofthe lung between the sexes sex diï¬erences in lung function andlung diseases are also dependent on the action of sex hormonesWe have summarized some broad concepts that define howtestosterone and estrogen aï¬ectlung macrophage functionand how this may contribute to the outcome of particularlung diseases in Figure As testosterone rises after pubertythe immunosuppressive eï¬ects of this hormone on protectiveimmune responses to infectious diseases in males can worsenpulmonary disease This would be exemplified by tuberculosisor ‚uenza Some of these eï¬ects are a result of androgeneï¬ects on critical ‚ammatory macrophage functions althoughthe eï¬ects on the adaptive immune system also have a significantcontribution to the overall outcome Thus testosterone appearsto play a key immunoregulatory role in lung macrophagesTestosterone™s immunoregulatory properties also appear to bedependent on the amount of cellular expression of AR andon the concentration of the hormone Low concentrations oftestosterone have been noted in patients with asthma COPD andtuberculosis Low testosterone may also be linked to insufficientcontrol of tissuedamaging ‚ammatory responses seen inCOPD and pulmonary fibrosis Estrogen tends to promotewound healing responses in macrophages Dysregulation ofwound healing responses and overactive tissue remodelingmacrophages in the lung could be broadly used to describe theTh2 response in allergic asthma which is worse in womenCancer could also be considered an aberrant wound healingresponse driven by M2like tumor associated macrophages Wehave highlighted here how sex hormones contribute to changesin lung macrophage function that contribute to lung diseaseHowever it should be pointed out that not every sex diï¬erencein lung disease is due to direct eï¬ects on macrophages but on thebroader coordinated immune response as a wholeAsthmaBefore puberty the structural diï¬erences in the lung as wellas gender diï¬erences likely account for the higher incidence ofFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyFIGURE Sex differences in lung diseases discussed in this Review and how they may be connected to the effects of androgens and estrogens on ‚ammatorymacrophages in the lungasthma in boys than in girls With the onset of puberty male andfemale sex hormones and their eï¬ects on the structural cells ofthe lung and on the immune system contribute to the incidenceof asthma The incidence and severity of asthma aregreater in adult women than in adult men and greaterin female than in male mice Female sex hormones suchas estrogen appear to worsen asthma although a straightforwardcorrelation between amount of female sex hormone and asthmasymptoms has not been concluded Androgens have multipleimmunoregulatory and bronchodilatory functions and maycontribute to or be biomarkers for better lung function inmen Accordingly serum testosterone is low in men withmoderate to severe asthma “ In one study each ngdLincrease in serum testosterone correlated with a CI P decrease in the likelihood of having asthma On the other hand high concentrations of testosterone andcyclic AMP in sputum of asthmatic women during the lutealphase of the menstrual cycle were thought to play a role inpremenstrual exacerbations The idea that sex hormonesmay be a causal factor in asthma was significantly strengthenedby a recent study of adults that quantified serum sexhormones and asthma outcomes That study showed thatlow testosterone in both women and men was associated with anincreased incidence of asthma The other interesting finding wasthat higher testosterone was protective against asthma in obesewomen Obesity is a risk factor for asthma “ Thereforehow high body mass index BMI and circulating sex hormonestogether aï¬ect asthma requires further investigationAnother androgen dehydroepiandrosterone DHEA alsoknown as androstenolone is an endogenous steroid hormoneand one of the most abundant circulating steroids in humansIt is a precursor for the synthesis of both testosterone andestrogen DHEA is sulfated at the C3 position into DHEAS by the action ofthe sulfotransferase enzymes SULT2A1and SULT1E1 in the adrenal glands The amount of DHEAS in the circulation is ˆ¼“ times those of DHEADHEA became of interest to the asthma field because womenwith severe asthma had very low concentrations of DHEAS and DHEAS concentration correlated with lung function Interestingly DHEAS is suppressed by oral or inhaledglucocorticoids the mainstay therapy for asthma HumanDHEA peaks at around age and then follows an agedependentdecline until they reach prepubertal concentrations Reducedsecretion of DHEA with age has been related to a numberof ageassociated conditions Replacement of DHEA has beenconsidered as a possible therapeutic that could activate protectiveresponses in an aging immune system DHEA is known todownregulate Th2‚ammatory cytokines while upregulatingIL2 synthesis in concanavalin Astimulated peripheralblood mononuclear cells from adult males with atopic dermatitis Thus it was hypothesized that it would be a usefultreatment for atopic diseases including asthma and the results ofthe clinical trials for DHEA in asthma patients show promiseThe results are discussed in a later section titled œEï¬ects ofandrogen exposure on monocytes macrophages in humans withlung diseaseCOPDSex diï¬erences also have been reported in chronic obstructivepulmonary disease COPD a heterogeneous chronic andprogressive respiratory disorder that includes chronic bronchitisand emphysema Chronic exposure of the airways to insultssuch as cigarette smoke leads to epithelial cell injury destructionof pulmonary capillary vasculature acceleration of epithelial cellsenescence and airway remodeling The loss of lung complianceultimately leads to COPD COPD was previously thoughtto aï¬ect mostly elderly men primarily because of the higherprevalence of smoking in men However as smoking ratesincreased in women the number of COPD cases in womenexceeded that of men These diï¬erences are not only basedon gender as women develop more severe COPD with earlyonset disease years and have greater susceptibility toCOPD with lower tobacco exposure Moreover increasingage in female smokers leads to a faster annual decline inFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyforced expiratory volume in the first second when compared tothat of male smokers even when they smoke fewer cigarettes Similarly pulmonary fibrosis is another lung disease thatmanifests sex diï¬erences with men being more aï¬ectedthan women It is characterized by destruction of thepulmonary parenchyma and deposition of extracellular matrixwith alterations in phenotype of both fibroblasts and alveolarepithelial cells InfluenzaThe lungs are also the target of respiratory viruses such as‚uenza A œï¬‚u respiratory syncytial virus and coronavirusessuch as severe acute respiratory syndrome and the MiddleEast respiratory syndrome The viruses infectthe airwayepithelial cells and cause damage to the epithelial barrierby themselves or as a result ofthe immune response tothe viralinfection Sex diï¬erences have been noted in theimmune response to ‚uenza A virus and to the ‚uenzavaccine In general women have a more robust protectiveimmune response to ‚uenza virus and vaccine than do menAlthough this elevated response is helpful in clearing viruswomen of reproductive age also experience higher mortalityand hospitalizations “ possibly from collateral tissuedamage to the lungs The vigorous immune response in womenalso means that women experience more adverse events aftervaccination Indeed a systems biology approach identifiedthat high testosterone was correlated with a blunted responseto the flu vaccine in men As testosterone wanes in elderlymen mortality increases Since the male immune responseto the virus is also less robustthe incidence of seasonalflu is generally higher in men than in women in developedcountries according to the World Health anization It is not yet known how fluctuations in sex hormones acrossthe menstrual cycle and lifespan aï¬ect the immune system™sresponse to the ‚uenza virus in humans Mouse studieshave revealed that estrogen is protective at high but notlow concentrations On the other hand testosteronereplacement in gonadectomized or aged male mice enhancedsurvival rates Despite these findings in mouse modelsstudies examining the eï¬ect of sex hormones on cellular andmolecular mechanisms in human immune cells during ‚uenzainfection are lackingTuberculosisLike ‚uenza infection tuberculosis TB a lung disease causedby Mycobacterium tuberculosis exhibits notable sex diï¬erencesin the number of cases worldwide with men being almosttwice as frequently aï¬ected than women Both sexand gender diï¬erences impact the incidence of TB AlthoughTB aï¬ects less women than men in adulthood womenin their economically active years “ years old have ahigher TB incidence compared to women in other age groups This indicates that factors associated with gender such asexposure to the bacteria are important in this disease Howeverbecause male predominance does not occur in children thissuggests that biological factors such as male sex hormones alsoplay a significant role This is supported by a study ofmedically castrated men who experienced a significantly smallerproportion of death from TB compared to in intactmen Understanding how androgens lead to the greatersusceptibility of men to TB is critical as TB is still one ofthe leading fatal infectious diseases worldwide and may alsomay favor the development of other diseases such as lungcancer Lung CancerLung cancer is a very complex disease that depends on anumber of variants such as sex gender race and socioeconomicstatus The development of lung cancer is also related toenvironmental factors such as pollution due to industrializationand urbanization An additional genderassociated riskfactor significantly linked to developing lung cancer is cigarettesmoking Historically more men develop lung cancer andsuï¬er lung cancerassociated deaths compared to women However the incidence of lung cancer has changed notably inboth women and men In men lung cancer incidence startedto increase in the 1920s and started to decrease in the early1990s while in women the mortality rates and incidence beganto rise in the 1960s Changes in smoking habits in the lastseveral decades with a rise in the number of women who smokecorrelate with an increase in the incidence of lung cancer in thisdemographic group Smoking is definitely a key factor inthe development of lung cancer however recent studies showa higher incidence of lung cancer in young women comparedto young men even when the prevalence of cigarettesmoking among young women has approached but not exceededthat among men This suggests that the higher incidenceof lung cancer in women is not explained simply by genderdiï¬erences in smoking habits a deeper analysis of diï¬erencesmediated by sex such as greater sensitivity to tobacco smoke inwomen is warranted Furthermore men and women develop diï¬erent specifictypes of lung cancer Malignant mesothelioma is more commonin men while women develop more adenocarcinoma particularly nonsmall cell lung cancer NSCLC Womenhave a superior survival rate for lung cancer compared tomen Tumorassociated macrophages are critical in tumorprogression yet how androgens ‚uence macrophage behaviorin lung cancer and in responses to treatment must be addressedmore deeply to develop better therapies and increase survivalrates in menTHE MYELOID IMMUNE SYSTEM IN LUNGHEALTH AND DISEASEAlveolar MacrophagesThe lungs are a primary interface with the external environmentThe delicate structures needed for gas exchange make themsusceptible to damage from invading pathogens and toxicmolecules Some insults to the lung can lead to the developmentof chronic conditions such as allergic asthma As a protectivemechanism alveolar macrophages clearspace ofinfectious toxic or allergenic ps to maintain homeostasisin the alveoli Thus alveolar macrophages have a dualthe airFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyfunction as ‚ammatory cells phagocytosing and killinginhaled bacteria or viruses and also as controllers ofthe‚ammatory immune response minimizing alveolar damageResident alveolar macrophages are seeded embryonically fromyolk sac and fetalliver monocytes “ In asthma andother lung diseases recruited alveolar macrophages derived fromblood monocytes can turn into pathogenic cells worseningthe condition Mouse alveolar macrophages arecharacterized by high surface expression of Siglec F and produceTGF TGF both supports AM development and theirmaintenance of immune homeostasis by induction of Tregs andsuppression of B and T cell proliferation Another importantfunction of AM is the clearance of surfactant AM from male andfemale mice respond diï¬erently to surfactant protein A SPA SPA acts as an opsonin and is important in clearanceof pathogens Sex diï¬erences in AM responses to surfactantcould aï¬ect bacterial clearance and regulate the production ofpro‚ammatory mediators The molecular mechanisms thatmediate these diï¬erences and how sex hormones change thisimportant AM function is an open questionIn the human lung there appears to be more diversity inthe subtypes of lung macrophages compared to mice The maindeterminant of the frequency of subtypes of macrophages inhumans appears to be their anatomicallocation within thelung AM are the predominantimmune cells in the lungairways bronchi and bronchioalveolar space Flow cytometricpanels have employed HLADR CD163 CD169 and CD206to diï¬erentiate between AM IM and monocytes Human AMwere identified as large highly autofluorescent CD14 CD16cells that also express CD206 CD169 and MARCO There appear to be two populations of AM distinguished byeither high or low expression of CD163 More recent approachesto characterize the macrophage populationsin the lunginvolve singlecell transcriptomic analysis Althoughmacrophages show a large variation in the transcriptionalphenotype expression of MARCO CCL18 APOC1 APOEPPARG and MRC1 was found in macrophages from healthydonors while CHI3L1 MARCKS IL1RN PLA2G7MMP9 and SPP1 were highly expressed in macrophages frompulmonary fibrosis patients Thus a second contributor todiversity is likely the activation state of the cells There are nodata that describe sex diï¬erences in human AM responses andthe eï¬ect of sex hormones on these cells From our mouse andhuman MDM studies we would predict that androgens augmentthe immune homeostatic functions of these cells in the malelung Further work is still needed to standardize characterizationof the diï¬erent subpopulations of human lung macrophagepopulations and their role in maintaining healthy lung functionand in diseaseIMsInterstitial MacrophagesInterstitial macrophagesanother macrophagepopulation found in the lung They are a minor populationof monocytederived macrophages which comprise“ of lung macrophages and are localized in the lungparenchyma IMs contribute to maintaining homeostasisthrough the spontaneous release of IL10 a cytokine thataredampens ‚ammation IMs can prevent the developmentof aberranttype allergic responses triggered by inhaledallergens and have been related to reduction of asthma Diï¬erent subpopulations of IMs have been foundin the lung however their characterization has not arrived at aconsensus due to difficulties in their identification and isolationIn the mouse lung diï¬erent subpopulations of IMs have beendescribed based on the expression of surface markers One reportdescribed three diï¬erent subpopulations of IMs based on thediï¬erential expression of pro‚ammatory cytokines chemokineligands MHCII CD11c CD206 and Lyve1 other groupidentified two subpopulations based on similar markers butincluding CX3CR1 Moreover IMs subpopulations canbe also described based on the diï¬erent anatomic locationsthese cells populate inside the mouse lung parenchyma Further work is needed to better characterize and define thediï¬erent IM populations as the diï¬erent subtypes may havediï¬erent functions during the ‚ammatory process Smallerin size than their AM counterparts human IMs express moreof the monocytic marker CD14 than AM perhaps suggestingtheir monocytic origin and have lower expression of CD169than human AM The responses of IM to androgen will dependon their expression of AR which has not been measured Thiswill be a challenge due to difficulties in clearly identifying thispopulation and its subpopulations from the monocytic AMand other myeloid populations in the lungMonocytesMonocytes are produced in the bone marrow along with anumber of other myeloid cells Myeloid cells originate fromcommon pluripotent hematopoietic stem cells and representthe major subset of white cells in circulation These cellscomprise basophils neutrophils eosinophils DCs monocytesand macrophages among others Monocytes are releasedinto circulationthen blood monocytes are recruited into‚amed tissue and can mature into macrophages or dendriticcells There are two main subsets of mouse monocytesœclassical or Ly6Chigh monocytes that originate directly fromLy6C precursors and œnonclassical or Ly6Clow monocytesthat derive from Ly6Chigh monocytes The origin ofLy6C low monocytes was demonstrated by Sunderkotteret al by tracking the maturation of DiIlabeled Ly6Chighmonocytes into DiIlabeled Ly6Clow monocytes Thisprocess depends on the transcription factor Nr4a1 whichregulates the development and survival of Ly6Clow monocytes These two monocyte subsets mirror the human CD14classical and CD16 nonclassical monocyte populationsrespectively Ly6Chigh monocytes highly express thechemokine receptor CCchemokine receptor CCR2 whereasLy6Clow monocytes highly express CX3CR1 ImportantlyCCR2 expression is required for Ly6C monocyte egress fromthe bone marrow into the circulation and entry into non‚amed and ‚amed tissues “ from the blood As monocytes migrate into tissue they mature into macrophagesdeveloping unique tissuedependent morphology and functions They lose expression of Ly6C and gain expression ofMHC class II becoming more efficient antigenpresenting cellsFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biology Some authors have proposed the concept of œtissuemonocytes which are monocytes that can enter nonlymphoidans without obligatory diï¬erentiation into macrophagesTherefore monocytes are much more than simply precursorsfor macrophagesIn human lungs monocytes which can be both beneficialand pathogenic in a variety of pulmonary diseases arepresent at steady state Multiplecolor cytometric analysison cells obtained from diï¬erent anatomical locations of the lungof healthy subjects nonsmokers with normal lung function andabsence of disease or infection revealed that while intermediatemonocytes CD14CD16 are more frequent in the airwaysclassical monocytes CD14CD16ˆ’ are more frequent in blood Moreover the diï¬erent monocyte subsets produced TNFα to diï¬erent degrees upon stimulation with TLR ligands and Thus the anatomic location where samples are obtainedshould be considered and reported when working with humanbronchoscopies as this may alter the type and abundance ofmonocytes and macrophages found Accurate identification ofmonocytes in the lung compartments in humans has been achallenge because monocytic œcontamination from the bloodvessels Overcoming this challenge Desch et alperformed a flow cytometric phenotyping study and identifiedtwo additional lung monocyte populations by analyzing lungsobtained from donors who died of nonpulmonary causes CD14 CD206ˆ’ CD1cˆ’ CD1aˆ’ intravascular monocyteswere similar to CD14 blood monocytes and CD14 CD206CD1cˆ’ CD1aˆ’ monocytes were described as tissue œmonocytesThese studies highlightthe beginningof understanding the complexity of lung monocyte subtypesand their functions depending on the ‚ammatory state ofthe lungthat we are just atOther myeloid populationslike DCs occupy the lungparenchyma at steady state and their relative numbers changeduring ‚ammation We refer readers to previous excellentreviews in this journal that cover the importance of DCs inimmune responses in the lung and how they are aï¬ectedby sex diï¬erences Therefore we will not discuss DCs here “Macrophage ActivationPolarization is a very important eï¬ector characteristic observedin monocytes and macrophages Polarization refers to the changein phenotype and function of monocytes and macrophagesas they are exposed to diï¬erent‚ammatory milieus orfactors in the tissue microenvironment To understand theeï¬ects of the diï¬ering ‚ammatory or tissue environments onmonocytemacrophage phenotype and function researchershave used cytokines and other factors in vitro to mimic diï¬erent‚ammatoryand tissue microenvironments Monocytesand macrophages stimulated with interferonγ LPS TNFαinterleukin IL12colonystimulating factor promote a pro‚ammatory macrophagephenotype denoted as M1 polarization The activation state wasalso known as œclassical activation M1polarized macrophagesmediate immunity to intracellular infections such as viruses andand granulocytemacrophagebacteria and they are generally considered tumoricidal “ M1 macrophages accomplish these functions by inducingproduction of nitric oxide reactive nitrogen intermediatesreactive oxygen species and hydrogen peroxide “ Incontrast activation of macrophages with IL4 or IL13 as inextracellular parasitic infections and allergic reactionsleadsto M2 polarization or œalternative activation of macrophages M2 macrophages produce ‚ammatory mediatorsand chemokines such as chitinaselike proteins IL13 CCL17 CCL18 CCL22 and CCL24 which activateTh2 cells and promote eosinophil ltration into the lungs In allergic asthma a Th2‚ammatory response to inhaledallergens drives lung macrophages toward an M2 phenotypeIncreased number and percent of M2 macrophages havebeen correlated with asthma severity and a decline in lungfunction in humans and mouse models “ SimilarlyM2 macrophages are the predominant subset seen in pulmonaryfibrosis and are responsible for fibrogenesis During COPDthe number of macrophages in airwayslung parenchymabronchoalveolar lavage fluid and sputum increases This increase may occur as a result of enhanced monocyterecruitment from circulation in response to chemokines suchas CCL2 and CXCchemokine ligand1 which are increased inthe sputum and bronchoalveolar lavage fluid of patients withCOPD Unlike in allergic asthma and pulmonary fibrosismacrophages in COPD are polarized toward an M1 profile In addition to aï¬ecting men and women diï¬erently anothercommonality of COPD is that macrophages both in the alveolarspace and in lung tissue present an altered activation phenotypeDiï¬erent concentrations of cytokines TNFα IL1 IL6 IL IL12 and chemokines CCL2 CCL5 CCL7 CCL13 CCL22IL8 CXCL9 and CXCL10 are found comparing smokers tohealthy subjects “ Thus the external provoking stimulusuniquely shapes macrophage phenotype and functionWhile the M1M2 designations are useful for in vitro studieswith stimulation with defined cytokines the in vivo phenotypeof macrophages exists on a spectrum somewhere in betweenthese two welldefined opposing phenotypes or does not fitthe paradigm at all For example M1 and M2 markers canexist simultaneously within the same cell in some cases “ The key factors dictating the macrophage phenotypeor activation state are the stage ofthe immune responseand the soluble factors and interactions in a particular tissuemicroenvironment For example the lung environment is richin GMCSF TGF and PPARγ and is critical for developmentof mature AMs after birth in both mice “and humans “ Furthermoreinteractions betweenCD200 on type II alveolar epithelial cells and CD200R on thesurface of the AM deliver regulatory signals to the AM toprevent pro‚ammatory signaling and macrophage activation Thus macrophage nomenclature has evolved as ourunderstanding of the phenotypes and functions of diï¬erenttypes of tissue resident macrophages recruited monocytes andmonocytederived macrophages advances Indepth studies ofthe eï¬ects of androgens and other sex hormones on tissuemacrophage plasticity and phenotype have yet to be carried outFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyMECHANISMS OF ANDROGEN SEXSTEROID ACTIONEFFECTS OF ANDROGEN EXPOSURE ONMONOCYTES MACROPHAGES IN VITROBecause androgens are lipophilic steroid hormones they caneasily diï¬use across cell membranes withoutthe need forreceptormediated import Androgens in circulation arefound mostly bound to sex hormonebinding globulin andalbumin Free unbound steroid sex hormones can signalthrough two diï¬erent mechanisms the classical ARlocate

Thyroid_Cancer

"Follicular dendritic cell sarcoma FDCS is a rare mesenchymal tumor that mostly occurs in systemiclymph nodes FDCS in the uterine cervix has not yet been reportedCase presentation A 49yearold woman was referred to our department with a cervical tumor which washistologically suspected to be undifferentiated carcinoma She underwent hysterectomy salpingooophorectomyand pelvic lymphadenectomy after neoadjuvant chemotherapy with paclitaxel and carboplatin The resectedspecimen contained high numbers of spindle cells and was immunohistochemically confirmed to be FDCS Thetumor was completely resected and recurrence was not detected at a 16month followupConclusion FDCS is an extremely rare malignant tumor in the uterine cervix and an accurate diagnosis andcomplete resection are essential for a good prognosisKeywords Follicular dendritic cell sarcoma Cervical cancerBackgroundFollicular dendritic cell sarcoma FDCS is a rare mesenchymal tumor that was initially reported in []Three types of tumors are derived from dendritic cellsFDCS derived from follicular dendritic cells that presentantigens to B lymphocytes in lymph follicles [] interdigitating dendritic cell sarcoma derived from the Tcellzones of lymphoid ans such as the paracortex anddeep cortex of the lymph nodes and fibroblastic reticular cell sarcoma derived from a reticular network oflymphoid ans [] An accurate diagnosis is challenging without an appropriate series ofimmunohistochemistry therefore some tumors may be diagnosed asundifferentiated carcinoma The accuracy of a diagnosisinfluences the prognosis of patients because of the lowresponse rate to established chemotherapy and radiotherapy [“] Correspondence myoshihara1209mednagoyauacjp2Department of Obstetrics and Gynecology Nagoya University GraduateSchool of Medicine Tsurumacho Showaku Nagoya Aichi JapanFull list of author information is available at the end of the Although previous studies described FDCS in the cervical lymph nodes liver stomach and tonsils FDCS inthe uterine cervix has not yet been reported We hereinpresent a 49yearold woman diagnosed with FDCS inthe uterine cervix which was successfully treated bycomplete surgery without postoperative adjuvant therapybased on a precise pathological diagnosis This is thefirst case of FDCS arising from the uterine cervix andwe described the time course of this patient from herinitial admission to diagnosis and treatment We alsoemphasized the importance of considering FDCS as adifferential diagnosis for cervical tumors because an accurate diagnosis and complete resection are essential fora good prognosisCase presentationA 49yearold woman gravida para with abnormalgenital bleeding was referred to our department with acervical mass She had a previous history of uterine myomectomy and cervical polypectomy which were both benign diseases Pelvic magneticimagingrevealed a — mm solid tumor developing from theresonance The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cNakamura BMC Women's Health Page of findingsrevealed aIntraoperativeand carboplatin AUC5 mgbody Massive genitalbleeding occurred months after the admission therefore the patient underwent extended total hysterectomybilateral salpingooophorectomy and pelvic lymphadenectomysmallamount of bloody ascites and the resected specimen hada necrotic mass at the posterior side of the cervix Fig Tumor cells exhibited the same histopathological characteristics as those in the previous biopsy Fig 3a Additionalimmunohistochemistry revealed cells that werepositive for both CD68 and FDC Fig 3b which wasconsistent with the diagnosis of FDCS The tumor wascompletely resected and lymphovascular invasion wasnot detected There was also no evidence of lymph nodemetastasis She did not receive adjuvant therapy and herCA19“ level decreased to within almost normal limitsTable Tumor recurrence was not detected at the month followup after admission The consent of the patient for publication was recorded according to the Ethics Committee of Nagoya University and the principlesof the Declaration of HelsinkiDiscussionThis is the first case of FDCS in the uterine cervix Afterreported surgery and chemotherapy we reached a finaldiagnosis Our experience demonstrates the difficultiesassociated with accurately diagnosing FDCS due to alack of familiarity with the pathologies of rare tumorsincluding sarcoma Although FDCS in the gynecologicalsystem is markedly rarer than other carcinomas it needsto be considered as a differential diagnosis because of itspotentiallyappropriatetreatmentprognosiswithoutfatalFDCS has been reported in approximately casesworldwide with an age range of to years and amalefemale ratio of The primary lesion of FDCS isFig Surgical specimen of the tumor The tumor with a necroticlesion arose from the posterior side of the cervix arrowheadsFig Magnetic resonance imaging of the patient at the initialpresentation A solid tumor developing from the posterior of theuterine cervix was confirmed arrowheadsposterior of the uterine cervix Fig and — and — mm spaceoccupying lesions were also detectedaround the rectus which were suspected to be lymphnode metastases Chestabdominal computed tomography CT showed no enlarged lymph nodes or distantmetastasis except for the tumors already detected Positron emission tomographyCT revealed the accumulation offluorodeoxyglucose maximum standardizeduptake value at the cervical tumor only Serumtumor marker levels were as follows carcinoembryonicantigen ngml cancer antigen125 UmLcancer antigen199 Uml squamous cell carcinomaantigen ngml soluble interleukin2 receptor Uml The other results of the blood examination including renal function liver enzymes and electrolyteswere within normal limitsBiopsy of the cervical tumor was performed and ahistopathological examination indicated that the tumorhad atypically spindle ovoid and polygonal cells with aneosinophilic hyalinerich cytoplasm with whorled andcordlike forms Nuclei were oval to round and had mildatypia Inflammatory cells were scattered to various degrees around tumor cells The results of immunohistochemical staining were as follows cytokeratin AE1AE3positive epithelial membrane antigen EMA negativeCAM52 negative cytokeratinMNF116 positive S100negative vimentin positive actin negative desminnegative cluster of differentiation CD negative CD8negative CD10 negative CD5 negative CD20 negativeCD79a negative PgR PgR inhibin negative thyroid transcription factor1 negative EpsteinBarr virusencoded RNA in situ hybridization negativeSince the tumor was suspected to be undifferentiatedcarcinoma of the uterine cervix the patient receivedchemotherapy with four cycles of paclitaxel mgm2 0cNakamura BMC Women's Health Page of Fig The histopathology of the cervical tumor The tumor had atypically spindle ovoid and polygonal cells with an eosinophilic hyalinerichcytoplasm with whorled and cordlike forms Nuclei were oval to round and had mild atypia a Tumor cells were positive for FDC bmostly in the lymph nodes in the neck axilla and mediastinum The remainder of FDCS originate from extralymphatic lesions such as the liver mesenterium stomach smallintestine pharynx tonsils retroperitoneumand ovary [“] however FDCS in the uterine cervixhas not yet been reported in the English literatureDue to the wide range of primary lesions there is noknown specific initial symptom of FDCS some patientsmay exhibit symptoms associated with an increase intumor volume such as lymph node swelling [] Sincethere are no specific findings in blood examinations orimaging studies difficulties are associated with makingan accurate diagnosis prior to the initiation of treatmentThe diagnosis of FDCS is pathologically confirmed usingbiopsy or resected specimens however due to the rarityof this tumor it is important to conduct appropriate immunohistochemistry in consideration of FDCS from thefindings of hematoxylin and eosin staining The tumorcells of FDCS are spindle to epithelioid in shape with thecoexistence of multinucleated cells Single nuclear inflammatory cells infiltrate and form bundles flowers andswirls structures Some giant cells and ReedSternbergcells are also detected in these tumors To reach a definitive diagnosis of FDCS FDC markers such as CD21CD35 KiM4p and CNA42 are used to distinguish itfrom other tumors that may have a mesenchymal structure such as undifferentiated carcinoma meningiomaand paraganglioma [ ] Alternatively FDCS may befound within lesions of Castleman™s disease particularlyits hyalinevascular type [] It has been reported thatFDCS occurred after the excision of a lesion of hyalinevascular type Castleman™s disease and occupied most ofthe lesions It is also important to distinguish FDCSfrom inflammatory pseudotumorlike FDCS associatedwith EpsteinBarr virus This tumor is often found in theabdominal ans particularly in the liver and spleenand has the characteristics of positive FDCS markersand the detection of EBV by in situ hybridization It progresses more slowly than FDCS and longterm survivalhas been reported even after recurrence []The treatment for cervical cancer principally involvessurgeryradiation and chemotherapy New surgicaltechniques such as laparoscopic radical hysterectomyand sentinel lymph node biopsy have also become available [ ] On the other hand a basic therapeuticstrategy for FDCS is prioritized to guarantee completesurgical tumor resection because of the low responserate to established chemotherapy and radiotherapy [“] Saygin examined patients with FDCS andreported 2year survival rates of and forearlylocally advanced and distant metastasis diseaserespectively Patients who underwent complete tumorresection had a better prognosis than those with unresectable localized tumors Furthermore no prognosticdifference was observed between the surgery group andpostoperative radiotherapy groups A large tumor sizelarger than cm and lymphoplasmacytic cell invasionhave been identified as poor prognostic factors [] InTable Level of tumor markers in each followup periodTumor marker Normal rangeCA125 UmL AdmissionCA199 UmL CEA UmL SCC ngmL At surgery m after admissionAt final followup m after admissionsIL2R UmL Abbreviation CA cancer antigen CEA carcinoembryonic antigen SCC squamous cell carcinoma antigen sIL2R soluble interleukin2 receptor 0cNakamura BMC Women's Health Page of the present case as the tumor remained localized andwas completely resected the patient could follow favorable clinical course without any tumor recurrenceConclusionFDC sarcoma is a rare tumor that may develop in theuterine cervix FDCS needs to be considered when confirming a mesenchymal cervical tumor and appropriateimmunohistochemistry needs to be performed for bothan accurate diagnosis and selection of a therapeuticstrategyAbbreviationsFDC Follicular dendritic cell sarcoma FDCS Follicular dendritic cellCT Computed tomography CD Cluster of differentiationAcknowledgmentsThe authors thank Dr Sakata and Dr Hirata for supporting the managementof the case with patience and knowledgeAuthors™ contributionsTN and MY developed the study concept interpreted the data and wrotethe manuscript ST and HK participated in designing the study and plannedthe investigations FK drafted the study design and supervised all of thework All authors have approved the final manuscriptShia J Chen W Tang LH Carlson DL Qin J Guillem JG Extranodalfollicular dendritic cell sarcoma clinical pathologic and histogeneticcharacteristics of an underrecognized disease entity Virchows Arch “Ruco LP Gearing AJ Pigott R Pomponi D Burgio VL Cafolla A et alExpression of ICAM1 VCAM1 and ELAM1 in angiofollicular lymph nodehyperplasia Castleman's disease evidence for dysplasia of folliculardendritic reticulum cells Histopathology “ Maeda K Matsuda M Suzuki H Saitoh HA Immunohistochemicalrecognition of human follicular dendritic cells FDCs in routinely processedparaffin sections J Histochem Cytochem “Arber D Kamel O van de Rijn M Davis RE Medeiros LJ Jaffe ES et alFrequent presence of the EpsteinBarr virus in inflammatory PseudotumorHum Pathol “ Casarin J Bogani G Papadia A Ditto A Pinelli C Garzon S et alPreoperative Conization and risk of recurrence in patients undergoinglaparoscopic radical hysterectomy for early stage cervical cancer amulticenter study J Minim Invasive Gynecol “ Rossetti D Vitale SG Tropea A Biondi A Lagan  AS New procedures for theidentification of sentinel lymph node shaping the horizon of futureManagement in Early Stage Uterine Cervical Cancer Updat Surg “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsFundingNo funding was obtained for this studyAvailability of data and materialsNot applicableEthics approval and consent to participateNot requiredConsent for publicationWritten consent to publish this information was obtained from studyparticipantsCompeting interestsAll authors declare that there are no competing interestsAuthor details1Department of Obstetrics and Gynecology Anjo Kosei Hospital Anjo AichiJapan 2Department of Obstetrics and Gynecology Nagoya UniversityGraduate School of Medicine Tsurumacho Showaku Nagoya AichiJapanReceived April Accepted August References Monda L Warnke R Rosai J A primary lymph node malignancy withfeatures suggestive of dendritic reticulum cell differentiation A report of cases Am J Pathol “Tew JG Kosco MH Burton GF Szakal AK Follicular dendritic cells asaccessory cells Immunol Rev “Saygin C Uzunaslan D Ozguroglu M Senocak M Tuzuner N Dendritic cellsarcoma a pooled analysis including cases with presentation of ourcase series Crit Rev Oncol Hematol “Pileri SA Grogan TM Harris NL Banks P Campo E Chan JK Tumoursof histiocytes and accessory dendritic cells an immunohistochemicalapproach to classification from the international lymphoma study groupbased on cases Histopathology “Chan JK Fletcher CD Nayler SJ Cooper K Follicular dendritic cell sarcomaClinicopathologic analysis of cases suggesting a malignant potentialhigher than currently recognized Cancer “ 0c"

Thyroid_Cancer

"Pluripotent stem cellsDirected differentiationIn vitro disease modellingLungAirwayMechanical cuesContentsChronic lung diseases remain major healthcare burdens for which the only curative treatment is lung transplantation In vitro human models are promising platforms for identifying and testing novel compounds to potentiallydecrease this burden Directed differentiation of pluripotent stem cells is an important strategy to generate lungcells to create such models Current lung directed differentiation protocols are limited as they do not recapitulate the diversity of respiratory epithelium generate consistent or sufficient cell numbers for drug discoveryplatforms and establish the histologic tissuelevel anization critical for modeling lung function In this review we describe how lung development has formed the basis for directed differentiation protocols and discussthe utility of available protocols for lung epithelial cell generation and drug development We further highlighttissue engineering strategies for manipulating biophysical signals during directed differentiation such that futureprotocols can recapitulate both chemical and physical cues present during lung development Elsevier BV All rights reservedOverview of key developmental stages Lung anogenesis molecularly defining lung fate in the embryo Branching morphogenesis and other mechanical cues generated during lung development Introduction Human embryology as a blueprint for lung directed differentiation Directed differentiation of lung epithelia inspired by embryology Mouse embryonic stem cell derived lung epithelia Modeling airway and lung diseases for drug discovery Opportunities to exploit mechanical cues for improving directed differentiation protocols in the future Micropatterning in 2D Stem cell behaviour on substrate topographies Micropatterning in 3D anoid systemsSubstrate textureHuman pluripotent stem cellderived lung epithelia Creation of human proximal lung epithelia Comparisons of proximal airway directed differentiation protocols Creation of human distal lung epitheliaComparisons of distal lung directed differentiation protocols Limitations of current directed differentiation protocols Ž Correspondence to G Karoubi Latner Thoracic Surgery Research Laboratories Toronto General Hospital College St Toronto ON M5G 1L7 CanadaŽŽ Correspondence to A P McGuigan Institute for Biomaterials and Biomedical Engineering University of Toronto College Street Toronto ON M5S 3G9 CanadaEmail addresses golnazkaroubiuhnresearchca G Karoubi alisonmcguiganutorontoca AP McGuigan101016jaddr2020080050169409X Elsevier BV All rights reservedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxAcknowledgementsReferencesFuture outlook IntroductionEndstage lung disease is the third leading cause of morbidity andmortality worldwide [] and produces a significant burden onhealthcare systems due to extensive resource expenditures for diseasemanagement and as lung transplantation is the only curative treatmentoption Such diseases include acute respiratory distress syndromechronic obstructive pulmonary disease cystic fibrosis and pulmonaryfibrosis Chronic pulmonary diseases result in million global deathsper year [] Patients who receive transplants face continued complications associated with chronic immunosuppression and graft rejectionwith the transplant survival rates at and years being and respectively [] Furthermore since lungs function as an important barrier between the internal and the external environments they are a critical site for bacterial and viral infections and disease transmissionparticularly relevant given the current COVID19 pandemic There istherefore a critical need to better elucidate the mechanisms of infectiondisease progression host response and cellular repair in the lung to enable the development of novel targeted therapeutics for lung diseaseTissueengineered models have emerged as a technology to addressthis challenge and shown some success in drug identification and toxicology studies For example commercially available airway epithelialmodels such as EpiAirwayTM MatTek Life Sciences serve as convenientplatforms with airliquid interface culture capabilities for assessing theeffect of chemical and physical stimuli [“] Other examples includethe Alveolus LungChip and Airway LungChip systems Emulate Incoriginally developed in the Ingber laboratory which mimic theepithelialendothelial interface of the airway and provide a more dynamic platform for testing new anti‚ammatory compounds inasthma [] and new small molecule targets to decrease cancerassociated pulmonary edema [] More complex models have alsobeen reported which involve selfassembly of heterogeneous progenitor cells into 3D structures termed anoids [] These anoidmodels can recapitulate aspects of human lung development in termsof tissue structures and protein expression and therefore present apromising opportunity for drug screening []A challenge in developing such human in vitro lung models to screenfor drugs however is the requirement for large batches of similarhuman cells as a starting population for tissue manufacturing to ensureminimal heterogeneity between test wells [] Achieving this is especially challenging when using primary human lung cells which exhibitconsiderable heterogeneity across donors and have a limited ability togrow and differentiate reliably [] Furthermore primary cells areoften extracted from diseased donors which is not ideal for conductingcontrolled studies due to the wide range of therapeutic and environmental factors these cells have already been exposed to Directed differentiation of pluripotent populations has the potential to create vastnumbers of cells from either healthy or diseased patients It allows introduction of specific diseaseassociated mutations via CRISPRCas9gene editing to recapitulate and understand pathologies in a controlledmanner As such directed differentiation enables the generation of anattractive cell source for drug screening platforms and personalized disease models that may provide insight into tissue regeneration mechanisms [“]Directed differentiation protocols to manufacture specific cell populations from pluripotent stem cells PSCs have been developed to meetthe need for a homogeneous human cell source Older lung directed differentiation protocols from the late 2000s have been proven inefficientdue to the nonstandardized methods through which they derive lungendoderm from embryoid bodies [“] A series of more standardizedstepwise protocols have since emerged in the last decade that provideavenues for developing airway and lung epithelia albeit with variableefficiencies [“] The first ‚uential directed differentiation protocol to produce lung epithelia used human PSCs in [] whichwas further supported by two prominent studies conducted usingmouse PSCs in [] These protocols have continued to be enhanced through adaptations related to the selection of growth factorsand small molecules the chronology of morphogen delivery as wellas innovations in enabling platforms such as cell sorting 3D cultureand singlecell analyses to efficiently derive normal and diseased lungepithelia from human PSCs [“] Despite such advancements limitations pertaining to heterogeneity in the resultingpopulations still exist which are likely attributed to variability across directed differentiation trials PSC cell lines or the persistence of contaminating cell populations belonging to other lineages While protocolshave progressed to some degree in differentiating proximal airwayand distal alveolar epithelia they remain limited Overall many unanswered questions remain with regards to the identity maturity andfunctionality of resulting cell types as well as their utility for tissue engineering and drug testing approaches Therefore these protocols mustbe optimized further to reliably produce large numbers of spatially relevant and functional lung and airway epithelial cells that appropriatelyrespond to both chemical and mechanical stimuli in the context of disease modeling and drug discoveryIn this review we discuss the directed differentiation protocols thatattempt to recapitulate lung development and disease and highlightpossible opportunities to enhance these protocols in the future Wefirst describe development of native lung tissue and the patterningevents that occur that differentiation models attempt to mimic andhighlight how human lung embryology has served as the blueprint tocreate the common pathway of lung directed differentiation protocolsWe then discuss the evolution of directed differentiation protocols tofind opportunities for creating specific populations of airway and lungepithelia through targeted manipulation of key signaling pathways in2D and 3D models We further describe how these models have beenused to recapitulate different airway and lung diseases Finally we discuss how tissue engineering and biophysical cues using biomaterialscan be utilized during lung directed differentiation to mimic patterningcues present in development to augment current differentiationprotocols Human embryology as a blueprintdifferentiationforlung directed Overview of key developmental stagesDirected differentiation protocols have been designed to mimicin vivo human lung development [] Indeed in vitro models of lungdevelopment have provided unique insight into human lung development [] As human lung development has been described at greatlength in earlier reviews [] we provide a brief overview as followsschematically represented in Fig During early embryogenesis at days post fertilization a process called gastrulation begins with the appearance of a structure called primitive streak through which cells migrate to form the primary embryonic germ layers definitive endodermmesoderm and ectoderm [“] Definitive endoderm expandsthereby forming the primitive gut tube comprised of three endodermalregions foregut midgut and hindgut [] This is when lungPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxFig Schematic of human lung development from an epithelial perspectivedevelopment begins at approximately four weeks into embryonic lifewith the outgrowth of foregut endoderm [] and continues througheight years of postnatal life [] There are five stages to lungdevelopment Embryonic weeks “ The future lung buds emerge from the ventral side of the primitive foregut endoderm into the surroundingmesenchyme and develop into embryonic lung buds with early trachea and bronchi [“] Pseudoglandular weeks “ Branching of the airway continuesleading to formation of conducting and terminal bronchioles whilethe proximal airway epithelium begins to develop [] Canalicular weeks “ Development of the respiratory or gasexchanging airways is initiated primitive alveoli form and the future distal epithelium begins to thin as distal epithelial markers areexpressed [] Saccular weeks “ Emergence of sacshaped distal airwayswhich develop crests with muscle and elastin to create indentationsThese distal airways extend to form alveoli by weeks [] The developing epithelium and vasculature within the future alveolus continue to merge closer together to facilitate future gas exchange andfurther differentiation of alveolar epithelial cells AEC I and II takesplace Alveolar periods week “ years True alveoli are seen in week and the majority of alveolarization takes place through sacculeseptation a process by which the sacshaped distal airways changetheir internal architecture and create thin walls intraluminallySeptation leads to an increase in surface area of the gas exchangingportion of the developing lung and prepares the fetus to breath airduring this stage [] Lung anogenesis molecularly defining lung fate in the embryoDuring the embryonic period early lung is genetically defined by theexpression of transcription factor NK2 Homeobox NKX21 and Srybox SOX2 [“] During human lung development it has beenfound that the lung buds and branches given off during thepseudoglandular period are mostly SOX2SOX9 [] BothSOX2 and SOX9 are individual markers of the early proximal or distal lineage respectively [“] Over the course of the canalicular and saccular periods of development weeks “ these double positivepopulations downregulate one SOX protein and maintain expressionof the other as these cells mature towards proximal or distal lineages[] The proximal airway closer to the mouth is comprised of apseudostratified columnar epithelium that is responsible for theconducting airway function debris and pathogen removal ciliatedcells mucus production goblet cells prevention of airway ‚ammation club cells and humidification of air as it passes through to the distal lung compartment [“] The squamous distal epitheliumcomposed of alveolar epithelial cells AEC I and II facilitates the respiratory function of the lung as air in the epithelial compartment is broughtinto close apposition to blood from the pulmonary vasculature it alsosecretes surfactants which play an immunologic role and decrease thesurface tension present at the airliquid interface thereby preventing alveolar collapse [] In humans a number of cell types are found in theproximal airway each identified with specific markers Table This includes basal cells tumor protein p63P63 keratinKRT5 nerve growthfactor receptorNGFR integrin α6ITGA6 integrin β4ITGB4 ciliatedcells Forkhead BoxJ1FOXJ1 acetylated tubulinAcTUB goblet cellsmucin 5ACMUC5AC mucin 5BMUC5B club cells club cell secretoryproteinCCSP or SCGB1A1 and pulmonary neuroendocrine cellsPNECs synaptophysinSYP chromogranin ACHGA On the otherhand homeodomainonly protein HOPX identifies the distal lungalong with AEC I cells T1α podoplaninPDPN aquaporin 5AQP5while AEC II cells are recognized via surfactant protein B SPC prosurfactant protein C proSPC or SPC and HT2280 []One mechanism by which lung epithelia begin to mature is based onchemokine secretions from the surrounding mesenchyme and the developing heart field which are well reviewed here [] Key players including fibroblast growth factors FGFs [“] WNTs [“]and bone morphogenetic proteins BMPs [“] are known to inducethe differentiation of early lung progenitors in a controlled manner Forexample in mouse it has been found that FGF10 plays a role in bud outgrowth [] and drives lung progenitors towards a distal fate []through canonical WNT signaling [] Proximal epithelia developbecause they are located further away from distally located FGF reservoirs in the mesenchyme in a mechanism that appears dependent onconcentration gradients [] BMP4 plays a key role in lung bud formation from foregut endoderm and establishment of both dorsoventralback to front and proximodistal top to bottom patterning in the nascent lung [] BMP4 is also present at high levels in distal bud tips andepithelia including AEC II cells [] however its inhibition promotesa proximal fate and along with BMP2 inhibition ciliated cell development [] Branching morphogenesis and other mechanical cues generated duringlung developmentWhile the cell fate of early proximal and distal lineages is directedthrough chemical signals the lung epithelium itself undergoes markedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxTable Epithelial populations in native human airways and lungsRegionProximal AirwayDistal LungCell TypeAssociated Markers for Cell CharacterizationCiliated CellGoblet CellClub CellBasal CellAlveolar epithelial cell type I AEC IAlveolar epithelial cell type II AEC IIFOXJ1 AcTUBMUC5AC MUC5BCCSP SCGB1A1 SCGB3A2P63 KRT5 NGFR ITGA6 ITGB4HOPX PDPN AQP5SPB SPC HT2280Cell Proportions in Native Lung“ []“ []“ []“ [] [] []changes in architecture a process known as branching morphogenesis[] From the simple tube of the anterior foregut endoderm to thecomplex tubular structure of the adult a highly stereotyped mechanismof branching morphogenesis facilitates the outgrowth division placement and structure of lung airway [] Branching morphogenesis ofthe lung is driven by three simple and iteratively used processes domain branching planar and orthogonal bifurcation [] The first formof branching is domain branching along a primary branch buds formin a linear and sequential fashion from proximal to distal The nextform of branching is planar bifurcation in which the tip of the formingtube bifurcates to create two new tips which subsequently elongateand bifurcate again creating four tips The last process of branching isknown as orthogonal bifurcation In this process the initial planar bifurcation is followed by a rotation around the planar axis which createstwo new tips through bifurcation A critical gene in this processSprouty has been found to attenuate Erk12 signaling thereby alteringthe orientation of cell division and future tube elongation [] Othercritical genes and regulatory networks associated with FGF signalingalso contribute to controlling the periodicity of the branched network[] Although elements such as domain specification bifurcation rotation and branch generation remain largely undetermined [] newtechnologies involving highresolution live imaging tension sensingand forcemapping are opening paths to further explore and explainthe branching morphogenesis phenomenon []The early structure of the lung gives rise to a striking architecturalseparation of future SOX2 proximal lineages and SOX9 distal lineages at least in mice [] The diameter of tube generated duringbranching morphogenesis in the pseudoglandular and canalicularstages has a small degree of variance within each stage as measuredfrom electron micrograph sources of fetal human tissue [] This suggests that the branching program is rigorous in its control of lung structure and that tubes themselves may have instructive potential on thedeveloping epithelia Once the basic an structure has formed thelung continues to be exposed to mechanical cues as it continues to mature In several cases these cues have been shown to be essential forcorrect an function In utero the fetal lung is a secretory an thatonly converts to an absorptive one to prepare for breathing afterbirth through a change in the activity of chloride and sodium channelslate in development Fetal lung secretions result in a static fluid pressureof around cmH2O in the developing terminal sacs of the fetus whichpropels branching morphogenesis outwards into the developing thoracic cavity [] Lack of amniotic fluid in the developing lung alters the expression of distal epithelial markers and consequentlyresults in the creation of smaller than normal lungs pulmonary hypoplasia [] highlighting the importance of this mechanical pressureduring lung development In addition cyclic strain is generated fromfetal breathing movements FBM in utero that prime the airway foruse after birth FBM are detectable from the tenth week of pregnancyand begin as infrequent and erratic activity with long quiescent periodsAs development continues these quiescent periods decrease andsustained periods of fetal breathing occur These breathing movementsvary with the fetal sleep cycle and can be chemically tuned [] andalter the volume of terminal sacs by around [] againhighlighting the importance of mechanical signals ‚uencing lung development Finally a novel FGF10FGFR2dependenttensionalmechanism has been shown by which distal epithelial cells in the lungaccumulate motor proteins at the apex of the cell thereby becoming resistant to compression from increasing fluid pressure within the tubelumen Cells under this tension are more likely to become AEC II cellswhile those under compression become AEC I cells [] Interestinglywhile the above examples highlight the importance of specific mechanical signals in the growth development and differentiation of the lungPSC directed differentiation protocols of the lung are primarily based onmimicking the sequential chemical changes that occur during lungdevelopment Directed differentiation of lung epithelia inspired by embryologyEarly attempts to create lung epithelia from PSCs began in mouseand did not attempt to mimic the stepwise changes in chemical signaling that occur during development Rather groups focused on applyinglunglike physical cues such as airliquid interface [] These protocols while successful in generating NKX21 positive populationsalso produced contaminating cells expressing pluripotency markersOCT4 NANOG SSEA4 TRA160 TRA181 These early attempts solidified that further optimization particularly related to the chemical cuesapplied was needed to reliably create lung progenitors from pluripotentsources without remnant pluripotent contaminating cells More successful directed differentiation protocols were rationalized from the detailed understanding of the chemical changes during lung embryologyIn this section we describe in detail the different differentiation protocols currently available that evolved from this approach Mouse embryonic stem cell derived lung epitheliaAlthough mouse models do not fully recapitulate human lung development they have served as guides for earlier iterations of PSC directeddifferentiation protocols and have identified critical chemical cues forlung anogenesis Broadly speaking these protocols begin by drivingstem cells towards a definitive endoderm fate SOX17 and FOXA2mimicking the preembryonic period of human lung developmentweeks “ through high doses of the nodal activating molecule ActivinA [“] Foregut endoderm is then induced via transforminggrowth factor beta TGFβ inhibition either alone [] or with BMP inhibition [] for a short period a process called anteriorization as duringthe embryonic period of human lung development weeks “ Thisforegut endoderm FOXA2SOX2 is subsequently induced to generate NKX21 cells putative lung progenitors by stimulating theretinoic acid RA BMP WNT and FGF signaling pathways []These lung progenitors are further matured as demonstrated by increased NKX21 expression through application of corticosteroids[] In brief each protocol begins with PSCs guided through definitiveendoderm followed by anteriorization to foregut endoderm and subsequent ventralization to generate NKX21 cells [] These protocolsformed the basis and backbone for the creation of human lung epitheliafrom PSCsGiven the structural and cellular complexity of the lung it is reasonable that the earliest protocols focused on mouse However there aresubtle differences that highlight how human models are different interms of structure patterning and differentiation For example thePlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxentire human conducting airway is comprised of a pseudostratified epithelium even at diameters less than 05mm [] In contrastconducting airways in mice only exhibit pseudostratified epitheliumwith accompanying submucosal glands and cartilage in the most proximal portion of the airway and transition directly into alveolar sacs []This difference in histology affects the residing cell populations as evidenced by the lack of basal cells in the lower portion of the proximal airways of mice [] Similarly mouse models suggest thatSOX2SOX9 progenitors are quite rare and their cell fate is ambiguous [] However evidence from directed differentiation of humanlung epithelia [] which has been confirmed in vivo []reveals that SOX2SOX9 progenitors are common in the developinglung buds and that branch tips of the pseudoglandular staged lunggive rise to both proximal and distal epithelia [] Moreover specificprotein markers have been found to differ in both timing and locationof expression between human and mouse models proSPC in mouseis expressed early and throughout the developing mouse epithelium[] while in human proSPC is rarely detected early in development and is only robustly found later in distal epithelia [] These examples highlight that while there are similarities development andpatterning of mouse and human lungs is different and these differencesrequire human models to be fully appreciated Human pluripotent stem cellderived lung epitheliaHuman PSC protocols have generally followed the same differentiation chronology as that of mouse directed differentiation wherein definitive endoderm anterior foregut endoderm and NKX21 lungprogenitors are produced sequentiallyDifferent groups have adhered to their own methods of generatingdefinitive endoderm which primarily involves exposing PSCs to highconcentrations of Activin A Slight variations such as introducing WNTagonism through WNT3a or CHIR99021 prior to [] or alongside[] Activin A or additional exposure to BMP4 and FGF2[] during this stage exist across protocols for differentially inducing primitive streak and its anteriorization towards producing definitive endoderm In addition the use of embryoid bodies which arelimited by user experience and technique has resulted in a widerange of production efficiencies for achieving this stage from CKITCXCR4EPCAM cells [] to CKIT CXCR4 cells []Recent advances in commercial products have led to development ofstandardized 2D culturebased media STEMdiff Definitive EndodermKit STEMCELL Technologies which allow reliable derivation of of definitive endoderm []Similarly generation of both anterior foregut endoderm andtoventralized lung progenitor populations has been subjectmuch investigation and modification Earlier work suggested thatSOX2FOXA2 ± in their case anterior foregut endoderm canonly be induced by subjecting definitive endoderm to TGFβ and BMP inhibition [] Subsequent studies however attempted to anteriorize definitive endoderm to foregut endoderm through TGFβ inhibition alone SOX2 a combination of endogenous WNT TGFβ and BMP inhibition not quantified [] and via Sonic Hedgehog SHH and FGF2signaling FOXA2 EPCAM [] A comparison of the lattertwo strategies demonstrated that SHH and FGF2 are insufficient inproducing reliable NKX21 lung progenitors [] possibly becauseFGF2 is involved in promoting thyroid lineages [] In general TGFβand BMP inhibition [] is the basis for currently applied endodermanteriorization strategies [“]Factors involved in early versions of ventralization in directed differentiation protocols included WNT3a FGF7 FGF10 BMP4 epidermalgrowth factor EGF and RA have now been reduced based on elimination studies [] As such CHIR99021 CHIR WNT agonist BMP4and RA are necessary and sufficient for producing lung progenitorsfrom anterior foregut endoderm derived from both mouse and humanPSCs [] Despite finding that FGF7 and FGF10 are nonessential for inducing NKX21 expression they continue to be used forventralization in some protocols [] Although each protocol differsin terms of the duration of each phase NKX21 lung progenitors aregenerally achieved by days with the exception of a study by deCarvalho in which they maintained their cultures for an additional days in FGF7 FGF10 and CHIR99021 to attain NKX21FOXA2 lung progenitors In all cases these lung progenitors are theneither sorted or directly guided towards proximal or distal progeny in2D or 3D culture systems Ideally products of directed differentiationprotocols should mimic the cell proportions present in human airwaysand lungs Table however current protocols have not progressedthat far While these protocols continue to be refined the percentageof select cell populations generated from these protocols have beensummarized in Table Creation of human proximal lung epitheliaProtocols to create proximal lung epithelia have focused on the production of the four major cells types present ciliated goblet club andbasal cells see Table for a summary of markers for each cell type Motivation for creating proximal epithelia in the field has primarily been todevelop patientspecific cystic fibrosis CF models [] andor toproduce epithelia with multiciliated cell populations for protocol validation [] A shift towards human PSCderived CF models hasbeen critical as mouse models do not accurately represent CF diseaseprogression and phenotypes seen in humans [“] As such thefirst evidence of human PSC proximalization using CF patientderivedPSCs was shown by Mou who exposed anterior foregut endodermto BMP4 GSK3iXV WNT agonist FGF2 and RAsupplemented B27 togenerate NKX21 cells by Day Although contaminatingneuroectodermal and distal lung NKX21SOX9 cells were presentday populations included proximal NKX21SOX2 progenitorsSubcutaneous implantation of this population in immunodeficientmice for days resulted in emergence of NKX21P63 cells howeverno mature epithelial markers for ciliated goblet and club cells werefoundWong employed a longer 2D differentiation approach to produce mature proximal airway epithelia in vitro Through a processthey called œproximal specification they generated day lung progenitors via low levels of BMP4 mimicking signaling gradients in theairway FGF7 and FGF10 which began expressing proximal genes Further culture with FGF7 FGF10 and FGF18 resulted in upregulated geneexpression of KRT5 P63 FOXJ1 SOX17 cystic fibrosis transmembraneconductance regulator CFTR and SCGB1A1 to a lesser extent alongwith low levels of distal SOX9 and SPC by day Protein expressionamounted to NKX21 panKRT P63 FOXJ1 cells These cells were subsequently matured in airliquid interface ALI culture for weeks week of submerged culture withFGF18 followed by weeks of ALI culture to generate CFTRpanKRT FOXJ1 with coexpressing CFTR and CFTRLHS28 cells The resulting epithelium ranged from being squamous to cuboidal with sparse pseudostratified regions implying thatthis protocol lacked specific maturation cues Contaminating thyroidthyroglobulin and PAX9 liver HNF4 and AFP and pancreaticPDX1 lineages were detected through quantitative PCR while percentages of goblet club and basal cell populations barring gene expression analysis were not evaluatedA similar 2D culture approach was employed by Firth to generate proximal lung progenitors which were subsequently matured intomulticiliated epithelia They optimized lower concentrations of BMP4required during the ventralization phase day “

Thyroid_Cancer

The role of serum inflammatory cytokines andberberine in the insulin signaling pathwayamong women with polycystic ovarysyndromeHongying Kuang12˜¯ Yuwei Duan34˜¯ Dan LiID5a Yanwen Xu34b Wenxia Ai2 Wei Li1Ying Wang1 Sha Liu2 Mushan Li2 Xiaoqiu Liu2 Manqi Shao2 The First Affiliated Hospital of Heilongjiang University of Chinese Medicine Harbin China HeilongjiangUniversity of Chinese Medicine Harbin China The First Affiliated Hospital of Sun Yatsen UniversityGuangzhou China Guangdong Provincial Key Laboratory of Reproductive Medicine Guangzhou China Department of Acupuncture the Third Affiliated Hospital Beijing University Of Chinese Medicine BeijingChina˜¯ These authors contributed equally to this worka Current address The First Affiliated Hospital of Sun Yatsen University Guangzhou Chinab Current address Reproductive Medicine Center The First Affiliated Hospital Sun Yatsen UniversityGuangzhou Guangdong China 3509437qqcom DL Xuyanwenlivecn YXAbstractObjectiveTo study the role of selected serum inflammatory cytokines and berberine in the insulin signaling pathway among women with polycystic ovary syndrome PCOSMethodsSelected serum inflammatory cytokines were analyzed in the p cells which were interfered by berberine from infertile women who were to be treated with In Vitro FertilizationIVF Intracytoplasmic Sperm InjectionEmbryo Transfer icsiet Among them patientshad PCOS infertility and were nonPCOS patients whose infertility resulted from fallopian tube and male factors The elisa method was used to detect the changes in the expression levels of inflammatory factors in the cells The correlations between the seruminflammatory cytokine expression levels and the corresponding clinical hormones were analyzed The changes in the expression mRNA and protein levels of the serum inflammatorycytokines were studied by realtime quantitative PCR and protein printing Fluorescencemicroscope and flow cytometry were used to detect the glucose uptake capacity of ovariangranulosa cells in PCOS patients under the action of insulin after berberineResultsIn the PCOS group IL17a P IL1Ra P00001 and IL6 P were significantly higher than those in the nonPCOS group In the nonPCOS group AMH level wasnegatively correlated with inflammatory cytokines IL17a r 0819P IL1a r a1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Kuang H Duan Y Li D Xu Y Ai W Li W The role of serum inflammatorycytokines and berberine in the insulin signalingpathway among women with polycystic ovarysyndrome e0235404 101371journalpone0235404Editor Meijia Zhang China Agricultural UniversityCHINAReceived November Accepted June Published August Copyright Kuang This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscript and its SupportingInformation filesFunding This work was supported by theOutstanding talents funding project of HeilongjiangUniversity of Chinese Medicine China [grantnumber 2018JC05] the Foundation for youngacademic leaders of Heilongjiang University ofChinese Medicine China [grant number2018RCD08] and the Foundation for HeilongjiangProvince Postdoctoral Research Startup ChinaPLOS ONE 101371journalpone0235404 August PLOS ONE 0c[grant number LBHQ18119] the Project ofUndergraduate Colleges and Universities YouthInnovation Talents by Education Department ofHeilongjiang Province China [grant numberUNPYST2018227] the Natural ScienceFoundation of Heilongjiang Province China [grantnumber H2016083]Competing interests The authors have declaredthat no competing interests existThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway0716P IL1b r 0678P IL2 r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantly correlated with the levels of the examined inflammatory cytokines Berberine significantlyreduced the expression level of mTOR mRNA P and increased the expressionlevel of IRS1 mRNA P in the PCOS granule cellsConclusionIn this study we find that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 cause women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors alter their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroyingthe normal ovulation and fertilization system of women leading to polycystic ovary syndrome characterized by menstrual thinning and abnormal ovulation Berberine can improvethe sensitivity of insulin by regulating the signal pathway of insulin receptor substrate1IRS1 and mammalian target of rapamycin mTOR in PCOS patients and achieve a therapeutic effect of treating PCOS IntroductionPolycystic ovary syndrome PCOS is the most common endocrinopathy affecting reproductive aged women It affects reproduction infertility irregular menstruation hirsutism etcmetabolism insulin resistance impaired glucose tolerance etc and psychological characteristics anxiety depression and deterioration in quality of life [] Berberine BBR as a quaternary ammonium salt extracted from plants such as Coptis chinensis and Phellodendronchinensis is currently used in the treatment of diabetes hyperlipidemia and PCOS [] Recentstudies have found that berberine has good hypoglycemic and hypolipidemic effects and is aneffective insulin sensitizer Berberine reduces the synthesis of steroid hormones and theexpression of ovarian aromatase through the action on the hypothalamuspituitaryovarianaxis HPOA improves the insulin resistance status of PCOS patients reduces body weightinduces ovulation and regulates menstruation thereby increasing pregnancy rate and livebirth rate [“] Clinical observations have demonstrated that even with longterm use of berberine its side effects are transient and mild suggesting that BBR is safe to use in PCOSpatients and a very promising plantbased compound for treating PCOS patients []Patients with PCOS have been found to be under a chronic lowgrade inflammation statusincluding high levels of leukocytes and disorder of the proinflammatory cytokines [] Interleukin IL6 is a multipotent cytokine that mediates inflammatory response by controllingcell differentiation migration proliferation and apoptosis thus playing a role in the development of insulin resistance [] IL17a is the œsignature cytokine secreted by the Th17 CD4ve T cell subset Activation of Th17type responses is important not only for host immunecontrol of extracellular bacterial and fungal infections but also associated with chronic inflammation and autoimmunity [] The IL1RA protein is a naturally occurring antagonist of proinflammatory cytokines These proinflammatory cytokines are involved in the underlyingmechanism of various chronic inflammatory conditions [] Therefore we hypothesize thatinflammatory factors are one of the important factors influencing the formation of PCOS andberberine may be an important drug that regulates PCOS inflammatory factorsPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayAntiMu¨llerian hormone AMH is an indicator of a patient™s ability to respond quicklyand efficiently to ovarian reserve In women AMH is produced by the granulosa cells of theovarian follicles and its secretion begins at puberty and lasts until menopause [] PCOS ischaracterized by hyperandrogenism and follicular blockade These two characteristics may bedue to an imbalance between AMH and follicle stimulating hormone [] Circulating insulinlevels in patients with PCOS increase thereby inducing follicular developmental disorderswhich in turn lead to ovarian polycystic ovary formation and higher than normal AMH HighAMH is one of the indicators of stubborn anovulation [] Research has shown that serumAMH level is relatively stable throughout and between menstrual cycles [] More and morestudies have used AMH as a biomarker for PCOS []In the present study we examined the effect and mechanism of serum inflammatory cytokines including IL6 IL17a IL1RA etc on insulin sensitivity of PCOS In addition weassessed whether berberine can improve insulin sensitivity of PCOS by antagonizing the proinflammatory effect of serum inflammatory cytokines Materials and methods SampleSerum samples and granulosa cells were collected from infertile women who were to betreated with In Vitro Fertilization IVF Intracytoplasmic Sperm InjectionEmbryo Transfericsiet Among them patients had PCOS infertility and were nonPCOS patientswhose infertility resulted from fallopian tube and male factors The nonPCOS women wererequired to have regular menstrual cycles The essays were analyzed by Guangdong ProvincialKey Laboratory of Reproductive Medicine at the first affiliated hospital of Sun YatSen University Data collection time “ ParticipantsInclusion criteria age Diagnosis of PCOS according to Rotterdam StandardRotterdam Diagnostic Criteria for PCOS Ovarian Ovulation Disorder Manifests Oligomenorrhea or Amenorrhea Clinically or biochemically determined androgen level increasesmore than 2nmolL or clinically manifested hirsute acne excluding Kaohsiung caused byother diseases Ovarian morphology showed polycystic changes Only of the above items can be met IVFET treatmentExclusion criteria Having orally taken drugs in the past three months that affect the results such as contraceptives or other hormone drugs insulin sensitizers and lipidlowering drugs Suffering from other androgen excess related diseases including congenital adrenal hyperplasia with hydroxylase deficiency androgen secreting tumor excessive use of androgenproducing drugs Cushing syndrome severe insulin resistance thyroid dysfunction andhyperprolactinemia Having a history of anic diseases of heart lung liver kidney and other important ansor patients with mental diseases and other reasons that may interfere the present studyoutcomesPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway Ethics statement The study was approved by the ICE for Clinical Research andAnimal Trials of the First Affiliated Hospital of Sun Yatsen University reference [] No and was conducted in accordance with the ethical standards of the Declaration ofHelsinki All participants have signed written informed consent and there were no minors ReagentBerberine was provided by Yuanye BioTechnology Co Ltd Shanghai China2NBDG N13195 Invitrogen DMEMF12 “ Gibco and Fetal bovine serumSV3008702 Hyclone were purchased from Thermo Fisher Scientific Waltham USA Thefollowing is a list of used reagents and their manufacturers™ informationRealtime quantitative PCR kits ROCHE CAT Reverse transcriptase PROMEGA CAT M1701RIPA pyrolysis fluid yantian Bi CAT P0013BBcl2 abcam CAT ab196495BAD abcam CAT ab90435BAX abcam CAT ab182733ACTINβ abcam CAT sc70319 Methods Granulosa cell acquisition and grouping The follicular fluid was centrifuged at rpm for minutes at room temperature The supernatant was discarded and the precipitate after centrifugation was resuspended by phosphate buffer saline PBS The cell suspension was slowly centrifuged to percoll level at room temperature with 1800rpm for 10minAfter centrifugation the white floc was sucked out and washed with PBS three times at1200rpm for 5min at room temperature We discarded the supernatant and added typeIV collagenase and blew it evenly Digestion was performed at ˚C for 1520min We addedthe same amount of culture solution to stop digestion and blew it evenly The filtrate was filtered by micron cell filter and centrifuged at room temperature with 1000rpm for 4minThe supernatant was discarded the cells were suspended again with PBS 3ml erythrocytelysate was added to mix well and the mixture was allowed to stand at ˚C for 10min It wasthen centrifuged at 1000rpm for 4min at room temperature Some white precipitate whichwas verified as granulocytes were observed after centrifugation After the seed plate of PCOSwomen granulosa cells was seeded the fluid was changed once hours After hours solvent or berberine was added for continuous intervention for hours which led to the PCOScontrol group and PCOS berberine intervention group Detection of serum inflammatory cytokines The Merck luminex testing platformwas used to detect serum cytokines according to the manufacturer™s instructions Realtime quantitative PCR At the end of berberine treatment Trizol method wasused to extract total RNA in the cells which was then used to reverse transcription of mRNATotal RNA was isolated from cells using Trizol regent Takara BioInc Tokyo Japan and I mgmRNA was reverse transcribed to cDNA using a reverse transcription Takara Bio Inc TokyoJapan and subjected to quantitative PCR which was performed with the BioRad CFX96Touch TM RealTime PCR Detection System BioRad CA using iQ TM SYBR Green Supermix BioRad CA and threshold cycle numbers were obtained using BioRad CFX ManagerSoftware Real time PCR was carried out using the following conditions min at ˚C min at ˚C and cycles of s at ˚C min at ˚C using 1μl of cDNA reverse transcribed as mentioned above Quantifast SYBR green PCR kit Qiagen Cat No and500nM of forward and reverse primers The following primers were usedPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayβactin GCCGTTCCGAAAGTTGCCTT GAGCGCGGCGATATCATCAAMPK ACAGCCGAGAAGCAGAAACA TTGCCAACCTTCACTTTGCCmTOR CTTAGAGGACAGCGGGGAAG TCCAAGCATCTTGCCCTGAGSTAT3 CTGAAACGGGCTTCAGGTCA TCCAGGGAGAAAGGGAGTCASOCS3 TCTGTCGGAAGACCGTCAAC CCTTAAAGCGGGGCATCGTAIRS1 TCTCTTCCCACGGCGATCTA TGACACTGCGGAAGGAACTCWe used the 2ΔΔCT method to calculate the relative expression level of target genes Western blot experiment After the end of each cell culture the cells were lysedusing RIPA lysate in an ice bath and the protein lysate was collected After the total proteinconcentration was determined by BCA method polyacrylamide gel electrophoresis was carried out according to the standard of μg of total protein per group After the electrophoresisthe protein in the gel was transferred to the polyvinylidene fluoride PVDF membraneblocked with skim milk powder and then incubated with the corresponding dilutedprimary antibody at ˚C overnight and finally labeled with the corresponding horseradish peroxidase After incubation for one hour at room temperature the ECL luminescentsolution was added and detected by autoradiography using a Biored gel imaging systemImmunoreactive protein bands were visualized with enhanced chemiluminescence ECL on aChemiDoc MP Imaging System Blots were scanned and quantified with the Image analysissoftware BioRad Image Lab All specific protein band densities were normalized to βactin amounts Detection of glucose uptake capacity in granulosa cells Granulosa cells of bothnonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granulosacells were randomly divided into two groups one group was cultured normally and the othergroup was added with berberine with the final concentration of 100μM After 24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of100nM were replaced at ˚C After 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and the difference in fluorescence density between different groups was compared by using flowjov10 Statistical methodsData were represented by mean±standard deviation SD and SPSS statistical softwarewas used for analysis Analysis of variance ANOVA was used to test betweengroup differences and the pvalues were corrected by Bonferroni p value less than was considered statistically significant Results Analysis of clinical baseline indicators of PCOS group and nonPCOSgroupAs shown in Table compared with the nonPCOS group the PCOS group had significantlyhigher BMIP LHP00001 LHFSHP00001 FPGP AMHP00001 the number of follicles obtainedP00001 ICSI mature eggsP and thenumber of normal fertilization P FSH levels were significantly lower in the PCOSgroup than those in the nonPCOS group P Detection of serum inflammatory cytokinesAs shown in Table IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group IL10 P IL13PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Comparison of clinical indicators between PCOS and nonPCOS groupsnonPCOS n PCOS n PvalueAge years oldMenarche ageSBP mmHgDBPmmHgPulse sminBMI kgm2FSH mIUmlLH mIUmlLHFSHE2 pgmlPRL ngmlT nmolLFPG mmollAMHngmleggs obtainedICSI mature eggsNormal fertilized eggsMeanSDMedianMeanSDMedian101371journalpone0235404t001Table Comparison of serum inflammatory cytokines between the PCOS and nonPCOS groupsIL10 pgmlIL13 pgmlIL17a pgmlIL1Ra pgmlIL1α pgmlIL1β pgmlIL2 pgmlIL6 pgmlIL8 pgmlTNFα pgml101371journalpone0235404t002nonPCOSn ±±±±±±±±±±±PCOSn ±±±±±±±±±PvalueP IL1α P IL1β P IL8 P and TNFα P inthe two groups was not statistically significantly different Correlation analysis between serum inflammatory cytokines anddiagnostic clinical indicatorsWe examined the correlation between serum inflammatory cytokines and clinical serum hormones and other levels in PCOS and nonPCOS patients In the nonPCOS group AMH levelwas negatively correlated with inflammatory cytokines IL17ar 0819P IL1ar 0716P IL1br 0678P IL2r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantlyPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaycorrelated with the levels of these inflammatory cytokines Among the diagnostic clinical indicators except AMH level there was no significant correlation between other clinical indicatorsand the expression level of inflammatory cytokines in the nonPCOS group and the PCOSgroup Tables “Table Correlation analysis of BMI FSH and other inflammatory cytokines in the nonPCOS groupnonPCOSBMIIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarP101371journalpone0235404t003FSHPrLHLHFSHrPrTable Correlation analysis of E2 PRL and other inflammatory cytokines in the nonPCOS groupnonPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarPrPrPr101371journalpone0235404t004Table Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the nonPCOS groupnonPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t005PPPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Correlation analysis of BMI FSH and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαBMIFSHLHLHFSHrPrPrPr101371journalpone0235404t006Table Correlation analysis of E2 PRL and other inflammatory cytokines in the PCOS groupPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrPrPrPr101371journalpone0235404t007PPTable Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t008 Detection of protein imprinting in granulosa cellsIt can be seen from Fig 1A that there was no significant difference in the expression levels ofAMPK P PAMPK P and ACC P protein in PCOS groupPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Granule cell protein expression level B Gray expression analysis of protein expression A Representativewestern blot images and B Summary of expression changes among relative genes Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than wasconsidered statistically significant101371journalpone0235404g001PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Fluorescence density map of sugar uptake capacity of granular cells B Fluorescence density analysis Granulosa cells ofboth nonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granule cells were randomly divided into twogroups one group was cultured normally and the other group was added with berberine with final concentration of 100μM After24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of 100nM was replaced at ˚CAfter 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and thedifference in fluorescence density between different groups was compared by using flowjov10 Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than was considered statisticallysignificant101371journalpone0235404g002compared with the nonPCOS group while the expression levels of mTORP00001 PmTORP00001 and STAT3P00001 were significantly increased At the same time theprotein expression level of IRS1P was significantly decreased Detection of granulated cell sugar uptake capacityAs shown in Fig 2A and 2B compared with granulosa cells obtained from the nonPCOSgroup the fluorescence intensity of granulosa cells in PCOS group was significantly reducedP00001 and the fluorescence density in the cells increased significantly after berberineP00001 Detection of expression of key factors of glucose metabolism ingranulosa cellsAs shown in Fig 1B the increased expression level of AMPK mRNA after the addition of berberine was significantly different from that in the PCOS group P Compared with thenonPCOS group the level of mTOR in PCOS granule cells was significantly increasedP and the expression level of IRS1 mRNA was significantly reduced P Berberine significantly reduced the expression level of mTOR mRNA in PCOS granule cellsP and increased the expression level of IRS1 mRNA in PCOS granule cellsP Discussion The correlation between selected serum inflammatory cytokines andPCOSThis study finds that IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group Studies have shown thatPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayPCOS associated with lowgrade chronic inflammation interleukin17A IL17A is one of themajor members of proinflammatory cytokines and is mainly involved in the development ofinflammatory and autoimmune diseases Within the immune process the genetic factors ofIL17A play a major role in the susceptibility of PCOS [] The level of IL1Ra is significantlyincreased in patients with PCOS which can lead to decreased insulin resistance and blood glucose metabolism causing obesity and metabolic syndrome in PCOS patients [] Tarkun [] studied the serum levels of IL6 in patients with PCOS higher than the normal control group and IL6 was significantly associated with insulin resistance and fasting blood glucose The increase of IL6 level is an important inflammatory factor inducing the occurrenceof PCOS AMH levels and inflammatory cytokines in PCOS patientsThis study finds that in the nonPCOS group AMH level was negatively correlated withinflammatory cytokines such asIL17a r 0819P 0004IL1a r 0716P 0002IL1b r 0678P 0031IL2 r 0765P 001IL8 r 0705P However in thePCOS group AMH levels were not significantly correlated with the levels of various inflammatory cytokineStudies have found that AMH plays a key role in the anovulatory mechanism of PCOS []AMH can promote the growth of preantral follicles to the small sinus stage in vitro whileincreasing the production of steroid hormones and paracrine factors as well as oocyte maturation AMH is a key follicular paracrineautocrine factor that has a positive effect on preantralfollicle survival and growth [] The alienation of AMH function affects the regulation of follicles and promotes the increase of small follicles in the body Due to the abnormal expressionof the number and function of follicles the regulation of the ovary on the matrix is weakenedand the follicular irregular growth is induced without follicular atresia The expression of normal levels of inflammatory cytokines may promote normal cell apoptosis Elevated inflammatory cytokines IL17aIL1aIL1bIL2IL8 may disrupt the ovarian follicle atresiaweakening the ability of apoptosis and inhibiting the maturation of oocytes In turn the AMHfunction is out of control and the correlation between AMH and inflammatory factor levels isinducedIt can be concluded that AMH levels under normal conditions can effectively regulate thelevel of inflammatory factors and promote the body™s own metabolism and reproductive function The expression of high AMH levels in PCOS patients causes a loss of correlation withinflammatory factors The abnormal level of AMH increases the level of inflammatory factorsresulting in a continuous low concentration of systemic inflammatory state in the humanbody leading to metabolic diseases such as insulin resistance and glycolipid metabolism andreproduction disfunction The effects of Berberine on granulosa cells™ insulin resistanceOur study finds that berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients Berberine can increase insulin sensitivity in patients reduce blood sugar improveinsulin resistance and achieve the therapeutic effect of treating PCOS Berberine is the mainactive ingredient of Chinese herbal medicine Coptis Cork and Mink and has been used totreat diarrhea metabolic disorders and infertility []Berberine regulates glucose metabolism through a variety of mechanisms and signalingpathways such as increased insulin sensitivity activation of the adenosine monophosphateactivated protein kinase AMPK pathway regulation of the intestinal microbiota andPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayinhibition of liver sugar Exogenous stimulates peripheral cell glycolysis promotes intestinalglucagon like protein1 GLP1 secretion upregulates liver lowdensity lipoprotein receptormRNA expression and increases glucose transporter [“]PCOS is a multifactorial endocrine disease that affects reproduction and metabolism Studies in dheainduced PCOS mouse models have found that mTOR and pmTOR serine2448are highly expressed in the ovary In normal mice in another study mTOR protein levels inthe corpus luteum of PCOS patients were the same as in healthy women However comparedwith healthy patients receiving insulin stimulation the expression of mTOR protein in the corpus luteum of PCOS patients is less so another link between PCOS and mTOR is metabolicdisorders during PCOS [] The combination of activation of the mTOR pathway and oxidative damage to DNA that causes replication stress is a particularly effective factor in promoting aging and aging [] Berberine inhibits the level of DNA damage signals [“] It canreduce the expression and activation of γH2AX including tumor A and TK6 cells as wellas normal WI38 cells or mitogenic human lymphocytes [] It can also reduce intracellularreactive oxygen species and mitochondrial transmembrane potential which is a sign of mitochondrial activation [“] However these drugs also significantly reduce phosphorylationlevels of Ser235 of ribosomal S6 protein RpS6 Ser2448 of mTOR and Ser65 of 4EBP1These data indicate that the reduction of mTOR S6K signal which in turn reduces the translation rate and is accompanied by a reduction in oxidative phosphorylation which leads to areduction in ROS and a reduction in oxidative DNA damage [] The mechanism by whichBRB exerts these effects may be by targeting mitochondriaThe protective effect of berberine on islet function may involve two pathways On the onehand berberine can improve insulin sensitivity in patients with PCOS with IR as the core []On the other hand berberine can promote insulin secretion from islet cells of PCOS patientsand protect islet cells through antioxidant activity [] Kong [] found that themolecular mechanism of berberine on insulin resistance by upregulating the expression ofinsulin receptors confirmed that berberine can reduce fasting blood glucose and fasting seruminsulin At the same time they also found that berberine activated its promoter through protein kinase CPKCdependent increasing insulin receptor mRNA and protein expressionIn fat and muscle cells berberine may stimulate cells by upregulating glucose transporter type GLUT1 expression and inhibiting retinol binding protein4 RBP4 At the same timeberberine also has a certain effect on the phosphorylation of IRS1 which can finally alleviateinsulin resistance []In other words berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients The mechanism by which berberine regulates the metabolism of glycolipids is not tostimulate the patient to increase insulin secretion but to increase the glucose consumption ofthe patient™s cells and improve glucose tolerance At the same time by increasing insulin sensitivity in patients further lowering blood sugar and improving insulin resistance and achievingthe effect of treating PCOS ConclusionIn this study we found that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 caused women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors altered their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroying thenormal ovulation and fertilization system of women and leading to polycystic ovary syndromecharacterized by menstrual thinning and abnormal ovulation Berberine can improve thePLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaysensitivity of insulin by regulating the signal pathway of mTOR mRNA and IRS1 mRNA inPCOS patients and achieve the therapeutic effect of treating PCOSSupporting informationS1 Raw ImagesPDFAuthor ContributionsConceptualization Yuwei Duan Wei LiData curation Wei LiFormal analysis Wei LiFunding acquisition Hongying KuangSoftware Manqi ShaoWriting “ original draft Hongying Kuang Yuwei Duan Wenxia AiWriting “ review editing Dan Li Yanwen Xu Wenxia Ai Wei Li Ying Wang Sha LiuMushan Li Xiaoqiu Liu Manqi ShaoReferencesTeede H Deeks A Moran L Polycystic Ovary Syndrome A complex Condition with PsychologicalReproductive and Metabolic Manifestations That Impacts on Health Across the Lifespan BMC Med ZHANG F MA T CUI P Diversity of the Gut Microbiota in DihydrotestosteroneInduced PCOSRats and the Pharmacologic Effects of Diane35 Probiotics and Berberine [J]Frontiers in microbiology IMENSHAHIDI M HOSSEINZADEH H Berberine and barberry Berberis vulgaris A clinical review [J] “LI M F ZHOU X M The Effect of Berberine on Polycystic Ovary Syndrome Patients with Insulin Resistance PCOSIR A MetaAnalysis and Systematic Review [J] LI W LI D KUANG H Berberine increases glucose uptake and intracellular ROS levels by promoting Sirtuin ubiquitination [J] Biomedicine pharmacotherapy Biomedecine pharmacotherapie RONDANELLI M INFANTINO V Polycystic ovary syndrome management a review of the possibleamazing role of berberine [J] “ Benson S Obesity Depression and Chronic Lowgrade Inflammation in Women with PolycysticOvary Syndrome Brain Behav Immun “ 101016jbbi200707003PMID Rehman K Akash MSH Liaqat A Role of Interleukin6 in Development of Insulin Resistance andType Diabetes

Thyroid_Cancer

"At present the relationship between hypothyroidism and the risk of breast cancer is still inconclusive Thismetaanalysis was used to systematically assess the relationship between hypothyroidism and breast cancer riskand to assess whether thyroid hormone replacement therapy can increase breast cancer riskMethods The relevant s about hypothyroidism and the risk of breast cancer were obtained on the electronicdatabase platform Relevant data were extracted and odd ratios OR with corresponding confidence intervalsCI were merged using Stata SE softwareResults A total of related studies were included in the metaanalysis including cohort studies and casecontrol studies The results show that hypothyroidism was not related to the risk of breast cancer odd ratios CI “ In the European subgroup we observed that patients with hypothyroidism have a lower risk ofbreast cancerodd ratios CI “ Furthermore no significant correlation was observed betweenthyroid hormone replacement therapy and the risk of breast cancer odd ratios CI “Conclusion Hypothyroidism may reduce the risk of breast cancer in the European population and no significantcorrelation was observed between hypothyroidism and breast cancer risk in nonEuropean populations Due to thelimited number of studies included more largescale highquality longterm prospective cohort studies areneededKeywords Hypothyroidism Thyroid hormone replacement therapy Breast cancer MetaanalysisBackgroundAs a global public health problem breast cancer has anincreasing incidence on a global scale [] According tothe US cancer statistics breast cancer has becomethe most common malignant tumour in women withabout new cases each year accounting for of new malignant tumours in women [] Therefore theidentification of risk factors for breast cancer and the Correspondence Yanhuangdr163com Ruobaolidr163com2Department of Oncology Affiliated Hospital of Weifang Medical UniversityWeifang China3School of Basic Medicine Weifang Medical University Weifang ChinaFull list of author information is available at the end of the adoption of effective early prevention and interventionmeasures are of great significance for patients withbreast cancerThe physiology and pathology of the breast are closelyrelated to the endocrine of the body [] As the largestendocrine an in the human body the thyroid glandhas specific regulation effects on various hormone levelsand cell growth and development in the body whichbrings new enlightenment to the research of breast cancer [“] In Kapdi et alfirst proposed thathypothyroidism maybe increase the risk of breast cancer[] Since then many scholars have studied the relationship between hypothyroidism and the risk of breast The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cWang BMC Cancer Page of [“]cancer However the relationship between the two diseases remains controversial [“] Some studies haveshown that hypothyroidism increases the risk of breastcancerthathypothyroidism reduces the risk of breast cancer []Besides some studies have found no correlation betweenthyroid disease and breast cancer risk [] Thereforewhether hypothyroidism can increase the risk of breastcancer is worthy of further studystudiesshownSomehaveTwo metaanalyses have previously been studied forhypothyroidism and breast cancer risk [ ] Based onprevious research we have included more prospectivestudies and Asian population studies to assess the relationship between hypothyroidism and breast cancer risksystematically Besides the impact of thyroid hormonereplacement therapy on breast cancer risk was exploredin this metaanalysisMethodsSearch strategyRelevant clinical literature was extracted by systematicretrieval of PubMed Medline EMBASE Springer Webof Science and Cochrane Library electronic databasesup to date to October Our search strategy includedorœhypothyroidism or œHT and œthyroid diseases orœbreast cancer or œBC or œbreast neoplasms or œmammarmy cancer and œrisk orœincidence At the sametime we manually screened out the relevant potentialliterature in the references extracteddysfunctionœthyroidtermsforInclusion and exclusion criteria The inclusion criteria Types of studies Published studies exploring therelationship between hypothyroidism and breastcancer risk Subject Female Exposure factors Primary hypothyroidism thediagnosis needs to be based on the detection ofthyroid function Outcome indicators the occurrence of primarybreast cancerThe exclusion criteria Nonprimary hypothyroidism due to other causes Non observational studiesInsufficient information was provided or no fulltext Unable to obtain full text or quality assessment ofthe literature Studies were repeated or publications overlappedData extraction and quality assessmentTwo researchers separately conducted literature screening data extraction and literature quality evaluationand any differences could be resolved through discussionor a third inspector Information secured from the enrolled literature included first author™s surname year ofpublication country ofthe population sample sizefollowup time and data on the relationship betweenhypothyroidism and the risk of breast cancerThe NewcastleOttawa Scale NOS was used to assessthe quality of the study from three aspects cohort selection cohort comparability and outcome evaluation []NOS scores of at least six were considered highqualityliterature Higher NOS scores showed higher literaturequalityStatistical analysisAll data analysis was performed using Stata120 softwareMetaanalysis was performed according to the PRISMAguidelines The OR and 95CI from included studieswere treated with the combined effect size After thatthe heterogeneity test was conducted When P ‰¥ orI2 was performed it mean that there was no apparent heterogeneity and the fixedeffect model shouldbe applied for a merger When P or I2 ‰¥ indicated high heterogeneity the randomeffect model wasapplied Combined effect size if OR indicates thathypothyroidism is an unfavorable factor for breast cancer If OR is the opposite Publication bias Begg funnel plot and Egger test linear regression test were usedto research publication bias detection of the literatureincluded If P indicates obvious publication biasResultsProcess of study selection and description of qualifiedstudiesA total of studies were identified on our online databases After exclusion of duplicate references129 s were considered After screening the andtitle s were excluded After careful review ofthe full texts studies have been excluded because ofthem did not provide relevant data and s didnot have fulltext Nineteen s published between and met the inclusion criteria Fig A total of samples from studies involvingwere enrolled in this metaanalysis [ “ “] Sixcohort studies and casecontrol studies were includedin the study Twelve s were studied in the European population five in the North American populationand two in the Asian population All s are of highquality because of NOS score no less than The chiefcharacteristics of the enrolled materials are detailed inTable 0cWang BMC Cancer Page of Fig Flow chart of search strategy and study selectionRelationship between hypothyroidism and breast cancerriskThere were studies reported the relationship betweenhypothyroidism and breast cancer risk With obviousheterogeneity I p among these studies so a random effect model was used for assessmentThe pooled analysis suggested that was not related tothe risk of breast cancer OR CI “P 0001Fig explorethefurtherrelationshipSubgroup analysis of hypothyroidism and risk of breastcancerTobetweenhypothyroidism and breast cancer risk subgroup analysis was conducted from three aspects study typepopulation distribution and followup time The resultsof subgroup analysis were shown in Table In theEuropean subgroup we observed that patients withhypothyroidism have a lower risk of breast cancer OR CI “ P In the subgroup witha followup date of more than four years patients withhypothyroidism can reduce the risk of breast cancerwith borderline significance OR CI “In otherP found thathypothyroidism was not related to the risk of breastcancersubgroups weRelationship between thyroid hormone replacementtherapy and breast cancer riskA total of studies reported the relationship betweenthe use of thyroid hormone replacement therapy and therisk of breast cancer [ ] Asobvious heterogeneity observed the fixedeffect modelwas usedI p The result suggestedthat patients who received thyroid hormone replacementtherapy was not related to the risk of breast cancerOR CI “109P Fig Publication biasFigure 4a shows the results of publication bias for the relationship between hypothyroidism and breast cancerrisk which were evaluated by funnel plots and Eggerstest The Begg test Pr and the Egger testP were used to detecting publication bias showedthat there was no possibility of publication bias Asshown in Fig 4b there were no publication biases in the 0cWang BMC Cancer Table Main characteristics of the included studies in ouranalysisStudySampleYearRegionAdamiKalacheHoffmanBrintonMosesonSmythSheringTalaminiSimonTurkenKuijpensCristofanilliSandhuHellevikDitschGraniSøgaardWengKimSwedenUKSwedenUSACanadaIrelandIrelandItalyUSAPragueNetherlandsUSACanadaNorwegianGermanyItalyDanishUSAKoreaMedianMean ageyearsNANA ± NANA ± ± ‰¥ ± ‰¥Page of NOSFollowupyearsNANANAStudydesignCasecontrolCasecontrolCohortCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCohortCasecontrolCohortCohortCasecontrolCasecontrolCohortCasecontrolCohortStudyIDAdami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Overall Isquared p ES CIES CI WeightWeightNOTE Weights are from random effects analysisFig Relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Table Stratiedanalysis of the relationship between hypothyroidism and breast cancer riskVariableOR95CINoofstudiesPHeterogeneityI2RegionEur orth AmericaAsiaStudy designCasecontrolCohortFollowup date ‰¤ “ “ “ “ “ “ “ Page of ModelusedFixedRandomedFixedRandomedFixedFixedRandomedPhStudyIDHoffman Kuijpens Sandhu Ditsch Cristofanilli Simon Moseson Brinton Adami Weng ES CIES CI WeightWeightOverall Isquared p NOTE Weights are from random effects analysisFig Relationship between thyroid hormone replacement therapy and breast cancer risk 0cWang BMC Cancer Page of A ]rr[golB ]rh[golBegg's funnel plot with pseudo confidence limitsEgger's publication bias plotse of log[rr]Begg's funnel plot with pseudo confidence limitstceffe idezdradnatstceffi edezdradnatsprecisionEgger's publication bias plotse of log[hr]precisionFig Publication bias assessment a hypothyroidism b thyroid hormone replacement therapy Metaanalysis estimates given named study is omitted Lower CI Limit Estimate Upper CI Limit Adami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Fig Sensitivity analysis for relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Page of s on the study of thyroid hormone replacementtherapy The Egger test was P and the Begg testwas Pr Sensitivity analysisThe results of sensitivity analysis are generally stableand the primary source of heterogeneity is in the research of Cristofanilli []Fig So we excludedthe literature of Cristofanilli and analyzed the otherstudies The results revealed that the hypothyroidismcould reduce the risk of breast cancer was borderlineOR096 95CI092“ P andsignificantthere was no heterogeneityI2 P cohortstudy ofDiscussionMore than years ago Beatson used thyroid extracts to treat patients with metastatic advanced breastcancer The condition was significantly alleviated sparkinginterest in exploring the relationship between thyroid andbreast cancer [] Subsequently a prospective study enrolled women and women with earlier diagnosisof hypothyroidism observed the occurrence of breast cancer during followup showed that low serum free thyroxine levels increased the risk of breast cancer [] In aprospective women withhypothyroidism and hyperthyroidism found thathypothyroidism slightly reduced the risk of breast cancer[] However a prospective cohort study of women with autoimmune hypothyroidism and women with normal thyroid function indicated that autoimmune hypothyroidism was not associated with breastcancer risk [] Besides some animal experiments alsoreflect the relationship between the two [ ] Animalexperiments by López Fontanafound thathypothyroidism mice inhibit the development of breastcancer and promote the apoptosis of breast cancer cellsdue to the low expression of βchain protein and activation of the apoptotic pathway on the tumour cell membrane [] Due to the inconsistency ofthe aboveconclusions we performed a metaanalysis to evaluate therelationship between hypothyroidism and breast cancerrisketalA total of studies were included in this metaanalysis and the results showed that patients withhypothyroidism not related to the risk of breast cancerHowever there was significant heterogeneity among theincluded studies After subgroup analysis and sensitivityanalysis we found that Cristofanilli™s research may causeheterogeneity [] Cristofanilli™s research is a retrospective study and the diagnosis of hypothyroidism patientswas based on the information recorded in the medicalrecords which may lead to the bias risk of misclassification and have a positive impact on the positive results ofthis study [] After excluding Cristofanilli™s researchwe found that patients with hypothyroidism had a lowerrisk of breast cancer with borderline significance [] Theresults of the metaanalysis are inconsistent with the findings of Hardefeldt and Angelousi [ ] Perhaps because our study included more prospective studiesand Asian population cohort study In addition we evaluated the risk of breast cancer in thyroid hormone replacement therapy and show that patients who received thyroidhormone replacement therapy was not related to the riskof breast cancerIn the analysis of the European population the resultsshow that hypothyroidism may reduce the risk of breastcancer We also found that patients with hypothyroidismcan reduce the risk of breast cancer was borderline significance in the subgroup with more longer followupdate However the relationship between the two was notobserved in North American and Asian populationsThe possible reasons for these disparities may be as follows First followup time may be the main contributorsto this difference A longer followup is required to demonstrate the relationship between hypothyroidism andbreast cancer risk In the metaanalysis five studies provided North American population data and two reported Asian population data However only one ofseven nonEuropean studies™ followup time for morethan years Second the differences may be attributedto different ethnicities sharing different genegene andgeneenvironmental backgrounds Third social and environmental factors are another critical cause for thisdifference With these in mind our findings suggest thathypothyroidism may reduce the risk of breast canceronly in the European population and more largescalehighqualitylongterm prospective cohort studies arestill needed to study on different human populationsThe following may explain the potential relationshipbetween hypothyroidism and the risk of breast cancerHealthy mammary epithelial cells can express a largenumber of T3 receptors and breast cancer cells have asimilar ability to bind to T3 [] T3 has an estrogenlike effect that promotes the growth of mammary glandlobes and stimulates normal breast tissue differentiation[ ] Therefore T3 can mimic the effect of estrogenon the proliferation of breast cancer cells When theconcentration of T3 is low in vivo it may inhibit theproliferation of breast cancer cells Hypothyroidism mayreduce the risk of breast cancer by affecting T3concentrationSome basic experiments support this theory In GonzalezSancho studied the relationship betweenT3 and breast cancer [] It was found that there is anoverexpressed T1 gene in human breast cancer cellsand T3 inhibits the proliferation of mammary epithelialcells by inhibiting the expression of cyclin D1 and T1thereby inhibiting the proliferation of breast cancer cells 0cWang BMC Cancer Page of Author details1School of Clinical Medicine Weifang Medical University Weifang China 2Department of Oncology Affiliated Hospital of Weifang MedicalUniversity Weifang China 3School of Basic Medicine WeifangMedical University Weifang ChinaReceived December Accepted July foundthat MartinezIglesias[] Afterthathypothyroidism can inhibit the growth of breast cancercells [] In Tosovic conducted a prospectivestudy of T3 levels associated with breast cancer risk andfound that T3 levels in postmenopausal women werepositively correlated with breast cancer risk in a doseresponse mannerthathypothyroidism through lower levels of T3 could reducethe incidence of breast cancer Our metaanalysis resultsalso confirm the above conjecture[] Therefore we suspectHowever this conclusion needs to be taken with caution as this study has several limitations First the studies that have been included do not adjust for importantrisk factors for breast cancer Second in subgroup analysis for example there are only two s in Asianstudies and we should be cautious about the results ofAsian analysis Third the results of this metaanalysis indicate that there is a large heterogeneity between studiesFourth followup time at different endpoints cannot beuniform Finally publication bias cannot be avoidedentirelyConclusionHypothyroidism may reduce the risk of breast cancer inthe European population and no significant correlationwas observed between hypothyroidism and breast cancerrisk in nonEuropean populations Furthermore therewas no obvious correlation between thyroid hormone replacement therapy and breast cancer risk It is necessaryto conduct a large sample size strictly controlled prospective study of hypothyroidism patients further todemonstrate the relationship between hypothyroidismand breast cancer riskAbbreviationsOR Odd ratios CI Confidence intervals NOS NewcastleOttawa ScaleAcknowledgementsNot applicableAuthors™ contributionsStudy design BW ZL RLYH and TL Data extraction BW ZL TL and YH Dataanalysis BW ZL RLand YH Manuscript writing BW and RL Manuscriptedition RL and YH All authors have read and approved the manuscriptFundingNo sources of funding were used to conduct this study or prepare this letterAvailability of data and materialsAll the published s and data were available onlineEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsNoneReferencesSiegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin “ httpsdoi103322caac21442Praestegaard C Kjaer SK Andersson M StedingJensen M Frederiksen KMellemkjaer L Risk of skin cancer following tamoxifen treatment in morethan breast cancer patients a cohort study Breast cancer “ httpsdoi101007s1228201506605 Mittra I Hayward JL Hypothalamicpituitarythyroid axis in breast cancerLancet “ httpsdoi101016s0140673674903444Adami HO Rimsten A Thoren L Vegelius J Wide L Thyroid disease andfunction in breast cancer patients and nonhospitalized controls evaluatedby determination of TSH T3 rT3 and T4 levels in serum Acta Chir Scand“Dargent M Berger M Lahneche B Thyroid function in patients with Cancerof the breast Acta “ Mustacchi P Greenspan F Thyroid supplementation for hypothyroidism Anlatrogenic cause of breast cancer JAMA “Kapdi CC Wolfe JN Breast cancer Relationship to thyroid supplements forhypothyroidism JAMA “ httpsdoi101001jamaKuijpens JL Nyklictek I Louwman MW Weetman TA Pop VJ Coebergh JWHypothyroidism might be related to breast cancer in postmenopausalwomen Thyroid “ httpsdoi101089thy200515 Weng CH Chen YH Lin CH Luo X Lin TH Thyroid disorders and breastcancer risk in Asian population a nationwide populationbased casecontrolstudy in Taiwan BMJ 201883e020194 httpsdoi101136bmj 2017020194Sogaard M Farkas DK Ehrenstein V Jensen JO Dekkers OM SorensenHT Hypothyroidism and hyperthyroidism and breast cancer risk anationwide cohort study Eur J Endocrinol “ httpsdoi101530EJE150989 Angelousi AG Anagnostou VK Stamatakos MK Geiopoulos GAKontzoglou KC Mechanisms in endocrinology primary HT and risk forbreast cancer a systematic review and metaanalysis Eur J Endocrinol “ httpsdoi101530EJE110838 Hardefeldt PJ Eslick GD Edirimanne S Benign thyroid disease is associatedwith breast cancer a metaanalysis Breast Cancer Res Treat “ httpsdoi101007s1054901220193Stang A Critical evaluation of the NewcastleOttawa scale for theassessment of the quality of nonrandomized studies in metaanalyses Eur JEpidemiol “ httpsdoi101007s106540109491zKalache A Vessey MP McPherson K Thyroid disease and breast cancerfindings in a large casecontrol study Br J Surg “ httpsdoi101002bjs1800690731 Hoffman DA McConahey WM Brinton LA Fraumeni JF Jr Breast cancer inhypothyroid women using thyroid supplements JAMA “ Brinton LA Hoffman DA Hoover R Fraumeni JF Jr Relationship of thyroiddisease and use of thyroid supplements to breast cancer risk J Chronic Dis“ httpsdoi1010160021968184900626 Moseson M Koenig KL Shore RE Pasternack BS The influence of medicalconditions associated with hormones on the risk of breast cancer Int JEpidemiol “ httpsdoi101093ije2261000Shering SG Zbar AP Moriarty M McDermott EW O'Higgins NJ Smyth PPThyroid disorders and breast cancer Eur J Cancer Prevent “Smyth PP Smith DF McDermott EW Murray MJ Geraghty JG O'Higgins NJA direct relationship between thyroid enlargement and breast cancer J ClinEndocrinol Metab “ httpsdoi101210jcem813Talamini R Franceschi S Favero A Negri E Parazzini F La Vecchia CSelected medical conditions and risk of breast cancer Br J Cancer “ httpsdoi101038bjc1997289 0cWang BMC Cancer Page of Simon MS Tang MT Bernstein L Norman SA Weiss L Burkman RT DalingJR Deapen D Folger SG Malone K Marchbanks PA McDonald JA Strom BLWilson HG Spirtas R Do thyroid disorders increase the risk of breast cancerCancer Epidemiol Biomarkers Prevent “Turken O NarIn Y DemIrbas S Onde ME Sayan O KandemIr EG Yaylac IMOzturk A Breast cancer in association with thyroid disorders Breast CancerRes 200355R110“ httpsdoi101186bcr609 Cristofanilli M Yamamura Y Kau SW Bevers T Strom S Patangan M Hsu LKrishnamurthy S Theriault RL Hortobagyi GN Thyroid hormone and breastcarcinoma Primary hypothyroidism is associated with a reduced incidenceof primary breast carcinoma Cancer “ httpsdoi101002cncr20881 Hellevik LR Vierendeels J Kiserud T Stergiopulos N Irgens F Dick ERiemslagh K Verdonck P An assessment of ductus venosus tapering andwave transmission from the fetal heart Biomech Model Mechanobiol “ httpsdoi101007s1023700901554Sandhu MK BrezdenMasley C Lipscombe LL Zagorski B Booth GLAutoimmune hypothyroidism and breast cancer in the elderly BreastCancer Res Treat “ httpsdoi101007s10549008 Ditsch N Liebhardt S Von Koch F Lenhard M Vogeser M Spitzweg CGallwas J Toth B Thyroid function in breast cancer patients Anticancer Res“ Grani G Dicorato P Dainelli M Coletta I Calvanese A Del Sordo M DeCesare A Di Matteo FM D'Andrea V Fumarola A Thyroid diseases inwomen with breast cancer La Clin Terapeut 20121636e401“Kim EY Chang Y Lee KH Yun JS Park YL Park CH Ahn J Shin H Ryu SSerum concentration of thyroid hormones in abnormal and euthyroidranges and breast cancer risk a cohort study Int J Cancer “ httpsdoi101002ijc32283 Beatson GT On The Treatment Of Inoperable Cases Of Carcinoma Of TheMamma Suggestions For A New Method Of Treatment With IllustrativeCases1 Lancet “Lopez Fontana CM Zyla LE Santiano FE Sasso CV CuelloCarrion FDPistone Creydt V Fanelli MA Caron RW Hypothyroidism reduces mammarytumor progression via Betacateninactivated intrinsic apoptotic pathway inrats Histochem Cell Biol “ httpsdoi101007s004180171544x MartinezIglesias O GarciaSilva S Regadera J Aranda A Hypothyroidismenhances tumor invasiveness and metastasis development PLoS One 47e6428 httpsdoi101371journalpone0006428 Nogueira CR Brentani MM Triiodothyronine mimics the effects of estrogenin breast cancer cell lines J Steroid Biochem Mol Biol ““httpsdoi101016s0960076096001173 Alyusuf RH Matouq JA Taha S Wazir JF The pattern of expression and roleof triiodothyronine T3 receptors and type I ²deiodinase in breastcarcinomas benign breast diseases lactational change and normal breastepithelium Appl Immunohistochem Mol Morphol “httpsdoi101097PAI0b013e3182a20917 Pereira B Rosa LF Safi DA Bechara EJ Curi R Control of superoxidedismutase catalase and glutathione peroxidase activities in rat lymphoidans by thyroid hormones J Endocrinol “ httpsdoi101677joe01400073 GonzalezSancho JM Figueroa A LopezBarahona M Lopez E Beug HMunoz A Inhibition of proliferation and expression of T1 and cyclin D1genes by thyroid hormone in mammary epithelial cells Mol Carcinog “ httpsdoi101002mc10046Tosovic A Bondeson AG Bondeson L Ericsson UB Malm J Manjer JProspectively measured triiodothyronine levels are positively associatedwith breast cancer risk in postmenopausal women Breast Cancer Res 123R33 httpsdoi101186bcr2587Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly citedCutaneous metastases are unusual presenting symptoms of lung cancer erefore they are prone to be misdiagnosed and missede report describes a case of a fortynineyearold female with painful zosteriform rashes showing multiple vesiclelike papuleslocalized on the left breast for days e patient had been diagnosed as lung adenocarcinoma at the department of oncology oneyear ago Skin biopsy revealed blue nodular lesions in the dermis composed of clustered heterogeneous tumor cells with glandularformation Immunohistochemical stains confirmed the diagnosis of metastatic lung adenocarcinoma IntroductionLung cancer can metastasize to almost all ans butmore often invades the hilar nodes liver adrenal glandsbones and brain [] e incidence of lung cancer withmetastases to the skin varies between “ [] A lungcancer metastasis is usually classified only as adenocarcinoma squamous cell carcinoma SCC or undiï¬erentiated carcinoma Until the 1980s SCC was reported asthe most common type of lung cancer However adenocarcinoma has replaced SCC as the most common lungcancer subtype especially in women and in neversmokers Sun reported that the type of adenocarcinoma was times more frequent than that of SCC []Skin metastases can appear on any cutaneous surface andthe most common sites are the chest abdomen head andneck [ ] Cutaneous metastases have various manifestations such as single papulesnodules or multiplelesions on anywhere of the skin while other rare formsmay show plaquelike lesions erysipelaslike papuleszosteriform lesions and scars [ ] Case PresentationA fortynineyearold nonsmoker female was admitted toour department with multiple painful papules localizedon the left breast ey appeared eruptively for about days and initially diagnosed as herpes zoster in anotherhospital e patient had been diagnosed as lung adenocarcinoma at the department of oncology one year agoShe was given oxitinib mesylate a targeted drug for thetreatment of nonsmallcell lung cancer In addition thepatient exhibited symptom of pain signs of weight lossanorexia and fatiguePhysical examination showed zosteriform vesiclelikepapules measuring “ cm on the left breast elesions were pink or red firm and tender Figure Excisional biopsy was performed revealing blue nodularlesions ltrating in the dermis composed of clusteredheterogeneous tumor cells with glandular formationSome tumor cells were detected within vessels or lymphatic vessels Some cells were transparent Mitosis wassignificant Figures 2a“2c In immunohistochemistumor cells were positive for cytokeratin CKtrycytokeratin7 CK7thyroid transcription factor1TTF1 and EMA and negative for cytokeratin20CK20 carcinoembryonic antigen CEA and grosscysticGCDFP15Figures 3a“3c Proliferative index as measured byKi67 was approximately “ oftumor cellsAccording to the clinical and pathological features cutaneous metastatic lung adenocarcinoma was madeprotein15diseasefluid 0cCase Reports in Dermatological MedicineFigure Zosteriform vesiclelike papules measuring “ cm on the left breast Pink or red firm and tenderabcFigure Skin biopsy revealed a blue nodular lesions ltrating in the dermis composed of clustered heterogeneous tumor cells withglandular formation HE magnification — b some tumor cells were detected within vessels or lymphatic vessels HE magnification — c some cells were blue and transparent and mitosis was significant HE magnification —abcFigure Immunohistochemical stain highlighting the tumor cells showing a CK7 b EMA and c TTF1 positive DiscussionSkin metastases suggest the progression of primary cancerand portend a poor clinical prognosis Skin metastases fromlung cancer are rare e percentage of patients with lungcancer that develops cutaneous metastases ranges from to percent [] It is seen more often in men than in women[] It does not show any specific presentation It is oftenpainless and less likely to be noticed making it more difficultto be diagnosed correctly which may delay treatment Although described cases show that metastatic nodules arepainless our patient showed severe pain e presence ofzosteriform painful vesiclelike lesions really mimics herpeszoster clinically in our casee mechanisms determining the metastasis of lungcancer in skin remain unknown Pathogenesis is suggested tobe by lymphovascular invasion with poor diï¬erentiationand upper lobe tumors increasing the risk [] Usually skinmetastasis develops after initial diagnosis of the primarymalignancy and late in the course of the disease Occasionally skin lesions that arise from lung cancer may develop before the primary tumor is recognized In our case 0cCase Reports in Dermatological Medicine[] R Koca Y Ustundag E Kargi G Numanoglu andH C Altinyazar œA case with widespread cutaneous metastases of unknown primary origin grave prognostic findingin cancer Dermatology Online Journal vol no p [] N A Babacan S Kilickap S Sene œA case of multifocalskin metastases from lung cancer presenting with vasculitictype cutaneous nodule Indian Journal of Dermatologyvol p skin metastases occurred during the immunotherapy Histology shows most commonly adenocarcinoma and thensquamoussmallcell followed by largecell carcinoma []Immunohistochemical markers are useful for the identification of the primary cancer or when a shorter diï¬erential isdesired AntiTTF is both sensitive and specific for primaryadenocarcinomas bronchioalveolar carcinomas and smallcell carcinomas when thyroid primary is excluded []CK7and CK20ˆ’ are sensitive but not specific for primaryadenocarcinomas and bronchioalveolar carcinomas eCK7CK20ˆ’tumors usually include the lung breast endometrium ovary thyroid salivary gland and mesothelioma [ ]Treatment of a single solitary skin lesion usually includessurgery alone or combined with chemotherapy andor radiation If lesions are more disseminated chemotherapy isthe primary option but may elicit an inadequate response[] Radiation can also be used alone andor in combinationwith chemotherapy andor surgery However despite thecombination of radiotherapy and chemotherapy patientswith lung cancer developing cutaneous metastases have apoor outcome Mean survival is short usually to monthsafter diagnosis of cutaneous metastasis []Conflicts of Intereste authors declare they have no conflicts of interestAcknowledgmentsis work was supported by a grant from the NationalNatural Science Foundation of China References[] T W Mollet C A Garcia and G Koester œSkin metastasesfrom lung cancer Dermatology Online Journal vol no [] S Sun J H Schiller and A F Gazdar œLung cancer in neversmokersa diï¬erent disease Nature Reviews Cancer vol no pp “ [] S Dreizen H M Dhingra D F Chiuten T Umsawasdi andM Valdivieso œCutaneous and subcutaneous metastases oflung cancer Postgraduate Medicine vol no pp “ [] M Khaja D Mundt R A Dudekula œLung cancerpresenting as skin metastasis of the back and hand a caseseries and literature review Case Reports in Oncology vol no pp “ [] W T McSweeney and K Tan œCutaneous metastases as apresenting sign of metastatic NSCLC Journal of Surgical CaseReports vol no [] M H Brownstein and E B Helwig œMetastatic tumors of theskin Cancer vol no pp “ [] R B McGrath S P Flood and R Casey œCutaneous metastases in nonsmall cell lung cancer BMJ Case Reportsvol [] V Jerome Marson J Mazieres O Groussard œExpression of TTF1 and cytokeratins in primary and secondaryepithelial lung tumours correlation with histological type andgrade Histopathology vol no pp “ 0c'

Thyroid_Cancer

"preadipocytes diï¬erentiate into adipocytes During this process the preadipocytes ceaseto proliferate begin to accumulate lipid droplets and develop morphologic and biochemical characteristics of mature adipocytesMesenchymal stem cells MSCs are a type of adult stem cells known for their high plasticity and capacity to generate mesodermaland nonmesodermal tissues Many mature cell types can be generated from MSCs including adipocyte osteocyte and chondrocyteThe diï¬erentiation of stem cells into multiple mature phenotypes is at the basis for tissue regeneration and repair Cancer stem cellsCSCs play a very important role in tumor development and have the potential to diï¬erentiate into multiple cell lineagesAccumulating evidence has shown that cancer cells can be induced to diï¬erentiate into various benign cells such as adipocytesfibrocytes osteoblast by a variety of small molecular compounds which may provide new strategies for cancer treatmentRecent studies have reported that tumor cells undergoing epithelialtomesenchymal transition can be induced to diï¬erentiateinto adipocytes In this review molecular mechanisms signal pathways and the roles of various biological processes in adiposediï¬erentiation are summarized Understanding the molecular mechanism of adipogenesis and adipose diï¬erentiation of cancercells may contribute to cancer treatments that involve inducing diï¬erentiation into benign cells IntroductionAdipogenesis is the process through which mesenchymalstem cells MSCs commit to the adipose lineage and diï¬erentiate into adipocytes During this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs Currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro One isfibroid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells There are two kinds of fibroid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells Anothergroup is fibroblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3T3L1 and 3T3F422A cells [] Cancer cells with tumorinitiation ability designated as cancer stem cells CSCshave the characteristics of tumorigenesis and the expressionof specific stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential [] In addition to CSCs [] cancer cells undergoing epithelialmesenchymaltransformation EMT havebeen reported to be induced to diï¬erentiate into adipocytes[“] Lung cancer NCIH446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro[] The adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of fibroblastderived cancer[] Cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes BRCA mutations can be inducedto diï¬erentiate by poly ADPribose polymerase PARP 0cStem Cells Internationalinhibitors [] The nuclear receptor peroxisome proliferatoractivated receptor Î PPARÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells[] Thyroid cancer cells expressing the PPARÎ fusion proteinPPFP can be induced to diï¬erentiate into adipocytes bypioglitazone [] Adipogenesis can be induced in welldiï¬erentiated liposarcoma WDLPS and dediï¬erentiatedliposarcoma DDLPS cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine IBMX []In this review we highlight some of the crucial transcription factors that induce adipogenesis both in MSCs and inincluding the wellstudied PPARÎ and CCAATCSCsenhancerbinding proteins CEBPs [] as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation We focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes Adipocyte DifferentiationCell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation Theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype [] Adipogenesishas diï¬erent stages Each stage has a specific gene expression pattern [] In general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases The firstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes The preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types In the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespecificproteins []The diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation [] After the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge After reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes Adipocyte precursors exhibit transient mitosiscalled œclonal expansion a process that relies on the actionof induced diï¬erentiation factors Some preadipocyte cellsmouse cell lines 3T3L1 3T3F442A undergo one or tworounds of cell division prior to diï¬erentiation [] whereasother cell lines mouse C3H10T12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion []Whether œmitotic clonal expansion is required for adiposediï¬erentiation remains controversial However it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] When cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets [] Regulatory Pathways inPreadipocytes CommitmentAdipocyte diï¬erentiation is a complex process in which geneexpression is finely regulated The most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported Adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process Figure Wnt Signal Pathway in Adipogenesis Wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo [] The Wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to ‚uence cell fate and development Wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways[] Wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of PPARÎ and CEBPα [] Constitutive Wnt10b expression inhibits adipogenesis Wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes In vivo transgenic expression of Wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development []Wnt10a and Wnt6 have also been identified as determinantsof brown adipocyte development [ ] Wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation [] indicating that preadipocytes integrate inputs fromseveral competing Wnt signals The Hedgehog HH Signaling Pathway MechanismThree vertebrate HH ligands including sonic hedgehogSHHIndian hedgehog IHH and desert hedgehogDHH have been identified and initiated a signaling cascademediated by patched Ptch1 and Ptch2 receptors [ ]HH signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as C3H10T12 KS483 calvaria MSCslines and mouse adiposederived stromal cells [] Thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after HHsignaling was inhibited [] Although it is generally agreedthat HH expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking HH signaling andadipogenesis remain poorly defined [] ERKMAPKPPAR Signal Pathway Extracellularregulated protein kinase ERK is required in the proliferativephase of diï¬erentiation ERK activity blockade in 3T3L1 0cStem Cells InternationalDEX insulin DEMXWNT 10band othersSHHPBC SMOTGF𝛽P SMAD3 SMAD3Testosterone𝛽catentinARIRSPI3KAKTCREBPKAPCREBFOXO1A2TCFLEF GATA23CEBP𝛽MAPKG3K3𝛽P2CEBP𝛽CEBPαPPARá¿«BMPsSMAD1SREBPAdipocytegenesFigure Regulation pathways in preadipocytes commitment BMP and Wnt families are mediators of MSCs commitment to producepreadipocytes Exposure of growtharrested preadipocytes to diï¬erentiation inducers IGF1 glucocorticoid and cAMP triggers DNAreplication leading to adipocyte gene expression due to a transcription factor cascade The dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis Inthe terminal diï¬erentiation phase ERK1 activity leads toPPARÎ phosphorylation which inhibits adipocyte diï¬erentiation This implies that ERK1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedThis reduction is mediated in part by mitogenactivatedprotein kinase MAPK phosphatase1 MKP1 [ ]These extracellular and intracellular regulation factors causeadipocytespecific gene expression and eventually lead toadipocyte formation Adipocyte DifferentiationRegulatory Proteins PPARÎ and Adipocyte Diï¬erentiation PPARÎ is a member of the nuclearreceptor superfamily and is both necessaryand sufficient for adipogenesis [] Forced expression ofPPARÎ is sufficient to induce adipocyte diï¬erentiation broblasts [] Indeedthe proadipogenic CEBPs andKrüppellike factors KLFs have all been shown to induceat least one of the two PPARÎ promoters In contrast antiadipogenic transcription factor GATA functioned in part byrepressing PPARÎ expression [] PPARÎ itself has twoisomers The relative roles of PPARÎ1 and PPARÎ2 in adipogenesis remain an question PPARÎ2 is mainlyexpressed in adipose tissue while PPARÎ1 is expressed inmany other tissues Although both can promote adipocytediï¬erentiation PPARÎ2 could do so eï¬ectively at very lowligand concentration compared with PPARÎ1 [] The twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesisPPARÎ1 can also be expressed in cell types other than adipocytes Ren [] used engineered zincfinger proteins tothe expression ofthe endogenous PPARÎ1 andinhibitPPARÎ2 promoters in 3T3L1 cells Ectopic expression ofPPARÎ2 promotes adipogenesis whereas that of PPARÎ1does not Zhang reported that PPARÎ2 deficiencyimpairs the development of adipose tissue and insulin sensitivity []There are transcriptional cascades between adipocytesgenes including PPARÎ and CEBPα which are the coreadipocyte diï¬erentiation regulators In the early stage of adipocyte diï¬erentiation the expression of CEBP and CEBPδincrease which upregulates CEBPα expressionfurtheractivate PPARÎ PPARÎ activating CEBPα in turn resultsin a positive feedback PPARÎ binding with retinoic acid Xreceptor RXR forms diï¬erent heterodimers The variousdimmers can combine with the PPARÎ response elementPPRE and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes []CEBPs participate in adipogenesis and several CEBPfamily members are expressed in adipocytesincludingCEBPα CEBP CEBPÎ CEBPδ and CEBPhomologous protein CHOP The temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of CEBP and CEBPδ leads toCEBPα expression This notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationCEBP is crucial for adipogenesis in immortalized preadipocyte lines CEBP and CEBPδ promote adipogenesis atleast in part by inducing CEBPα and PPARÎ CEBPαinduces many adipocyte genes directly and plays an important role in adipose tissue development Once CEBPα isexpressed its expression is maintained through autoactivation [] Despite the importance of CEBPs in adipogenesis 0cStem Cells Internationalthese transcription factors clearly cannot function efficientlyin the absence of PPARÎ CEBP cannot induce CEBPαexpression in the absence of PPARÎ which is required torelease histone deacetylase1 HDAC1 from the CEBPαpromoter [] Furthermore ectopic CEBPα expressioncannot induce adipogenesis in PPARΓ“ï¬broblasts []However CEBPα also plays an important role in diï¬erentiated adipocytes Overexpression of exogenous PPARÎ inCEBPαdeficient cells showed that although CEBPα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] Figure Human fibroblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning amplification However PPARÎ exogenousligands need to be added to promote adipocyte diï¬erentiation Therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of PPARÎ [] BMP and Transforming Growth Factor TGF inAdipocyte Diï¬erentiation A variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein BMP []transforminggrowth factor TGF [] insulininsulinlike growthfactor IGF1 [] tumor necrosis factor α and interleukin [] matrix metalloproteinase [] fibroblast growthfactor FGF and FGF2 [] BMP and TGF have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells []The TGF superfamily members BMPs and myostatinregulate the diï¬erentiation of many cell types includingadipocytes [] TGF inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of TGF impairsadipocyte development [] Inhibition of adipogenesis couldbe obtained through blocking of endogenous TGF with adominantnegative TGF receptor or drosophila mothersagainst decapentaplegic protein SMAD inhibitionSMAD3 binds to CEBPs and inhibits their transcriptionalactivity including their ability to transactivate the PPARÎ2promoter [ ] Exposure of multipotent mesenchymalcells to BMP4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion [] Theeï¬ects of BMP2 are more complex and depend on the presence of other signaling molecules BMP2 alone has little eï¬ecton adipogenesis and it interacts with other factors such asTGF and insulin to stimulate adipogenesis of embryonicstem cells [] BMP2 stimulates adipogenesis of multipotentC3H10T12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations [] KLFs in Adipocyte Diï¬erentiation During adipocyte differentiation some KLF family members are overexpressedsuch as KLF4 KLF5 KLF9 and KLF15 while KLF16 expression is reduced [ ] KLF15 is the first KLF family members which were identified to be involved in adipocytediï¬erentiation Its expression increased significantly on thesixth day of 3T3L1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in MSCs andmouse embryonic fibroblasts Inhibition of KLF15 by siRNAor mutation led to a decrease in PPARÎ CEBPα fatty acidbinding protein FABP4 and glucose transporter GLUT4 However overexpression of KLF15 in NIH3T3cells was found to be associated with lipid accumulation aswell as increases in PPARÎ and FABP4 [] Mice with complete absence of KLF5 showed embryonal lethality and micewith singlechromosome KLF5 knockout showed a significant reduction in white fat in adulthood suggesting thatKLF5 plays an important role in adipocyte diï¬erentiationKLF5 can be activated by CEBP or CEBPδ which isinvolved in early adipocyte diï¬erentiation KLF5 can beactivated by CEBP or CEBPδ which is involved in earlyadipocyte diï¬erentiation Direct binding of KLF5 to thePPARÎ2 promoter in combination with CEBPs inducesPPARÎ2 expression [] Transfection of KLF5 dominantnegative mutants in 3T3L1 cells reduced lipid droplet accumulation and inhibited PPARÎ and CEBPα expressionwhereas overexpression of wild KLF5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation Similar to KLF5 KLF9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as PPARÎ CEBPα and FABP4 hence inhibitingadipocyte diï¬erentiation However KLF9 overexpressiondid not upregulate the expression of PPARÎ and CEBPα[] In addition KLF4 can transactivate CEBP by bindingto the region of KB upstream of the CEBP promoter and promote lipid diï¬erentiation [] KLF6 can forma complex with histone deacetylase3 HDAC3 inhibitingpreadipocyte factor1 Pref1 expression and promotinglipid diï¬erentiation [] KLF2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation Overexpressed KLF2 can bind to theCACCC region of PPARÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of PPARÎCEBPα and sterolregulated elementbinding proteinsSREBP by inhibiting the promoter activity [] RNAsequence analysisshowed that KLFl6 expression wasdecreased on the first day of adipocyte diï¬erentiation of3T3L1 cells Adipocyte diï¬erentiation was promoted byKLF16 knockdown but was inhibited by KLF16 overexpression via inhibition of PPARÎ promoter activity [] In addition KLF3 and KLF7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] Signal Transducers and Activators of TranscriptionSTATs and Adipocyte Diï¬erentiation The activated STATprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription In the adipocyte diï¬erentiation of mouse 3T3L1 cells the expression of STAT1 andSTAT5 was significantly increased while that of STAT3and STAT6 was not significantly changed [] In the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells STAT1 expression was significantly decreased[] while the expression of STAT3 and STAT5 wasincreased and STAT6 expression was unchanged [] Therole of STAT1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process Early adipocytediï¬erentiation of 3T3L1 cells was inhibited by STAT1 0cStem Cells InternationalKLF5SREBP1cKLF15KLF2CHOPCEBPá¿«KROX20LigandCEBP𝛽CEBP𝛿GATA23PPARá¿«CEBP𝛼ProadipogenicAntiadipogenicGenes of terminaladipocytedifferentiationFigure A cascade of transcription factors that regulate adipogenesis PPARÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis PPARÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors CEBPα is regulated through a series of inhibitory protein“protein interactions Some transcription factorfamilies include several members that participate in adipogenesis such as the KLFs Black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î Loss of STAT1 in 3T3L1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α [] Other studies have found thatSTAT1 is required for adipose diï¬erentiation and STAT1overexpression in C3H10T12 cells can prevent the inhibition of lipid diï¬erentiation caused by Bcell lymphoma6knockdown [] There was no abnormal adipose tissuein STAT1 knockout mice [] STAT3 not only aï¬ectsthe proliferation of 3T3L1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein [] The FABP4 promoter was used to specificallyknock out STAT3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice [] STAT5A and STAT5B have diï¬erenteï¬ects on adipocyte diï¬erentiation Abnormal adipose tissuewas found in the mice with STAT5A or STAT5B knockout ordouble knockout and the amount of adipose tissue was onlyonefifth of the original adipose tissue in mice withoutknockdown [] Histone Modification in Adipocyte Diï¬erentiation Histone deacetylase sirtuin SIRT plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation [] During the adipocyte diï¬erentiation of C3H1012 cells SIRT1 expressiondecreased [] Overexpression of SIRT1 activated theWnt signal which caused the deacetylation of cateninThe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation SIRT1 knockdown resulted inincreased acetylation of the histones H3K9 and H4K16 inthe secreted frizzledrelated protein sFRP and sFRP2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation [] Forkhead box proteinO FOXO is a member of the transcription factor FOXOfamily It can recruit cyclic AMP response elementbindingprotein CBPhistone acetyltransferase p300 to initiate anacetylation The acetylated FOXO1 can be phosphorylatedby phosphorylated protein kinase B PKBAKT The phosphorylation of FOXO1 by AKT inhibits the transcriptionalactivation of FOXO1 The acetylation of FOXO1 lost the ability of DNAbinding affinity and promoted its shuttling fromnuclei to cytoplasm [] SIRT1 and SIRT2 can deacetylateand active FOXO1 Activated FOXO1 nonphosphorylatednuclear FOXO1 in the nucleus binds to the promoters of target genes encoding p21 p27 and PPARÎ and initiates subsequent transcriptions [] SIRT2 inhibits the acetylation andphosphorylation of FOXO1 thereby induces the accumulation of activated FOXO1 in the nucleus Activated FOXO1could inhibit adipogenesis via PPARÎ [“] Lysinespecific histone demethylase LSD1 expression increasedduring the adipocyte diï¬erentiation of 3T3L1 cells LSD1could reduce the dimethylation levels of histone H3K9 andH3K4 in the CEBPα promoter region thereby promotingadipocyte diï¬erentiation [] SET domaincontaining SETD8 catalyzed the monomethylation of H4K20 andpromoted PPARÎ expression The activation of PPARÎ transcriptional activity leads to the induction of monomethylatedH4K20 and modification of PPARÎ and its targets therebypromoting adipogenesis [] Enhancer of zeste homolog EZH2 is a methyltransferase and can bind methyl groupsto histone H3K27 which is also necessary for lipid diï¬erentiation The absence of EZH2 in brown fat precursors results inreduced levels of the Wnt promoter histone H3K27me3which is also saved by the ectopic EZH2 expression or theuse of a Wntcatenin signal inhibitor [] In addition histone demethylases such as lysinespecific histone demethylase LSDKDM KDM6 and histone lysine demethylasePHF2 are also involved in adipose diï¬erentiation andKDM2B inhibits transcription factor activator protein 2αpromoter via H3K4me3 and H3K36me2 [] Role of microRNA and Long NoncodingRNA in AdipogenesismicroRNA miR can bind and cut target genes or inhibittarget gene translation Endogenous siRNA can be producedby the action of Dicer enzyme and bind to a specific proteinto change its cellular location [] Many kinds of miRsare involved in regulating adipocyte diï¬erentiation The 0cStem Cells Internationalexpression of miR143 increased during the diï¬erentiationof adipose progenitor cells Overexpression of miR143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation Inhibition of miR143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] Additionally miR8 promotes adipocyte diï¬erentiation by inhibiting Wnt signaling [] Moreover miR miR103 miR21 miR519d miR210 miR30miR204211 and miR375 also play a certain role in promoting adipocyte diï¬erentiation while miR130 miR448and let7y inhibit lipid diï¬erentiation [ ] In additionto miRs long noncoding RNA LncRNA is a type of noncoding RNA and is important during epigenetic regulationand can form a doublestranded RNA complex with mRNAcauses protein transcription Lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of PPARÎ2[] As a novel LncRNA HOXAAS3 expression increasedduring the adipose diï¬erentiation of MSCs and HOXAAS3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation [] Zhu et al[] reported that HOXAAS3 interacted with EZH2 toregulate lineage commitment of MSCs HOXA AS3 canregulate the trimethylation level of H3K27 in the Runx2promoter region by binding to EZH2 Therefore HOXAAS3 is considered to be an epigenetic switch regulating MSCslineage specificity [] Adipocyte diï¬erentiationassociatedLncRNA can act as a competitive endogenous RNA of miR in the process of lipid diï¬erentiation thereby promotingthe expression of SIRT1 the target gene of miR204 and thusinhibiting lipid diï¬erentiation [] The LncRNA NEAT1can also regulate adipocyte diï¬erentiation under the ‚uence of miRNA140 [] Other LncRNA including LncRNABlnc1 and Plnc are also involved in regulating adipocytediï¬erentiation [ ] Other Biochemical Response Involved inAdipocyte Differentiation Unfolded Protein Responses in Adipocyte Diï¬erentiationIn the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded RNAdependent proteinkinaselike ER kinase and activating transcription factorATF 6α [] Knockdown of ATF6α aï¬ects the expressionof adipocytes genes and inhibits C3H10T12 adipocyte differentiation [] The inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3T3L1 cells is also due to inducedCHOP and decorin expressions and this inhibitory eï¬ectis ameliorated by CHOP knockout [] In the adipocytediï¬erentiation process of 3T3L1 cells increases in PPARÎand CEBPα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated Eukaryotic translation initiaEIF 2α phosphorylated endoribonucleasetion factorIRE1α ATF4 CHOP and other unfolded protein responsesEndoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationAdditionally EIF2α mutation results in continuous activation or overexpression of CHOP which also inhibits adipocyte diï¬erentiation [] After the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized Exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationFigure Role of Oxidative Stress in Adipogenesis During thedirectional diï¬erentiation of MSCs mitochondrial complexI and III and NADPH oxidase NOX4 are the main sourcesof oxygen species ROS production Currently it is believedthat ROS aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through ‚uencing the signaling pathwaysincluding the Wnt HH and FOXO signaling cascade duringMSCs diï¬erentiation [] The diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which specifically manifests as low ATPcellcontents and a high rate of oxygen consumption The lackof these characteristics indicates stem cell diï¬erentiation[] Adipocyte diï¬erentiation is a highly dependent ROSactivation factor related to mitosis and cell maturation[] Schroder found that exogenous H2O2 could stimulate adipocyte diï¬erentiation of mouse 3T3L1 cells andhuman adipocyte progenitor cells in the absence of insulinH2O2 regulates adipocyte diï¬erentiation of 3T3L1 cells ina dosedependent manner High doses of H2O2 and μM promote adipocyte diï¬erentiation [ ] Tormos found that ROS synthesis increased in humanMSCs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation Byknocking down Rieske ironsulfur protein and ubiquinonebinding protein ROS produced by mitochondrial complexIII was found to be necessary in initiating adipose diï¬erentiation [] However other studies have shown that theexpression levels of adiponectin and PPARÎ were decreasedby using H2O2 “ mM in 3T3L1 cells [] Free radical nitric oxide NO also promotes lipid diï¬erentiationbecause treatment with NO inducer hydroxylamine or NOsynthase NOS substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells NOSinduced adipose diï¬erentiation mainly via eNOS rather thaniNOS [] ROS can induce adipose diï¬erentiation primarily by inhibiting Wnt FOXO and HH signaling pathwaysthat inhibit lipid diï¬erentiation Autophagy in Adipocyte Diï¬erentiation The increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation[] Baerga confirmed that the adipocyte diï¬erentiation efficiency was significantly inhibited in mouse embryonic fibroblasts lacking autophagyrelated gene Atg agene encoding an essential protein required for autophagy[] Knockdown of Atg5 in 3T3L1 cells promotesproteasomedependent degradation of PPARÎ2therebyinhibiting adipocyte diï¬erentiation [] Zhang reportedthat autophagyrelated gene 7Atg7 is also crucial for adipose development Atg7deficient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cStem Cells InternationalCEBP𝛽 geneEBF1 geneKLF4EGR2CEBP𝛽CytosolCEBP𝛿 geneCEBP𝛿KLF5genePPARá¿« geneKLF5NR2F2NFKB11433RELASREBF1A2RXRAPPARá¿«PPARá¿«RXRA heterodimerPPARá¿«RXRAcorepressor complexFABP4Ligands of PPARá¿«FAM120BTHRAP3EP300NCOA2NCOA3HELZ2NCOA1CREBBPEBF1ADIPOQ geneAIDRFCEBP𝛼 geneZNF638ZNF467CEBP𝛼NCOR1HDAC3NCOR2 SLC2A4 geneGLUT4 geneLEP geneFABP4 geneCDK4CCND3PLIN1 genePCK1 geneFABP4CD36 genePPARARXRAcoactivator complexPPARá¿«fatty acidRXRAmediatorcoactivator complexANGPTLgenePPARGC1AMediator complex consensusLPL geneNucleoplasmProteins bind to gene promotersTranscription of genes into proteinsActing on proteins compoundingTGF𝛽1WNT1WNT10BTNF77233ADIPOQGLUT4SLC2A4 tetramerLEPFABP4lipid dropletPLIN1PCK1PaPa Pa4xPalmCCD36PaANGPTL4LPLFigure Regulation of adipocyte diï¬erentiation A regulatory loop exists between PPARÎ and CEBP activation Transcription factor CoeEBF activates CEBPα CEBPα activates EBF1 and EBF1 activates PPARÎ CEBP and CEBPδ act directly on the PPARÎ gene bybinding its promoter and activating transcription CEBPα CEBP and CEBPδ can activate the EBF1 gene and KLF5 The EBF1 and KLF5proteins in turn bind the promoter of PPARÎ which becomes activated Other hormones such as insulin can aï¬ect the expression ofPPARÎ and other transcription factors such as SREBP1c PPARÎ can form a heterodimer with the RXRα In the absence of activatingligands the PPARÎRXRα complex recruits transcription repressors such as nuclear receptor corepressor NCoR NCoR1 andHDAC3 Upon binding with activating ligands PPARÎ causes a rearrangement of adjacent factors Corepressors such as NCoR2 are lostand coactivators such as Transcription intermediary factor TIF2 CBP and p300 are recruited which can result in the expression of CyclicAMPresponsive elementbinding protein CREB followed by PPA

Thyroid_Cancer

RNAs circRNAs are a novel and unique class of noncoding RNAs that are backspliced from premRNAs It hasbeen confirmed that circRNAs are involved in various malignant behaviors of hepatocellular carcinoma HCCHowever the role of circRNA in the regulation of ferroptosis and the underlying mechanism remain unknown HerecIARS hsa_circ_0008367 was found to be the most highly expressed circRNA after sorafenib SF treatment in HCCcells Small interfering RNA against cIARS sicIARS significantly suppressed the cellular sensitivity to SF or Erastinthrough inactivating ferroptosis which may be partially attributed to the inhibition of autophagy and ferritinophagyPrediction analysis and mechanistic identification revealed that cIARS physically interacted with RNA binding proteinRBP ALKBH5 which was a negative regulator of autophagic flux in HCC The dissociation of BCL2BECN1 complexmediated by ALKBH5 silencing was effectively blocked by sicIARS Furthermore the inhibition of ferroptotic eventsautophagic flux and ferritinophagy resulted from sicIARS were significantly rescued by ALKBH5 downregulationOverall cIARS may be an important circRNA positively regulating SFinduced ferroptosis through suppressing theALKBH5mediated autophagy inhibitionIntroductionHCC is a highly refractory and prevalent cancerworldwide with new cases and deathsevery year1 Rapid advances in diagnosis and treatmenthad improved patient outcomes However the survivalgains are stagespecific2 Liver resection transplantationablation ortransarterial chemoembolization benefitspatients in early or intermediate stages3 while treatmentfor latestage HCC has remained challenging The standard firstline systemic drug against advanced HCC issorafenib SF BAY Nexavar which is currentlyCorrespondence Xiaoqing Wei weixqrm163com orJie Li heplijie163com1Department of Hepatobiliary Surgery The First Affiliated Hospital ofShandong First Medical University Jinan Shandong China2Department of Hepatobiliary Surgery Shandong Provincial QianfoshanHospital Shandong University Jinan Shandong ChinaFull list of author information is available at the end of the These authors contributed equally Zhiqian Liu Qi WangEdited by Inna Lavrik The Authors known as a ferroptosis inducer4 exerting only limitedeffects on overall survival and time to tumor progression56 Therefore it is imperative to explore novel andeffective therapeutic target to improve the cellular sensitivity to SFˆ’ was targeted SubsequentlyIn recent years ferroptosis has been confirmed to be anessential mechanism in SF treatment7 It is a kind of finelycontrolled cell death featured with irondependent accumulation of lipid hydroperoxides8 When HCC cells wereexposed to SF the cystineglutamate antiporter systemsystem xcthe cystineuptake was blocked and the biosynthesis of glutathioneGSH was inhibited resulting in accumulation of lipidperoxidation products and eventually inducing ferroptosis Meanwhile SF also induces autophagic flux in cellsWhen autophagy flux is initially induced MTORC1 dissociates from the ULK12ATG13 complex leading todephosphorylation of the complex9 Then phagophorenucleation occursthe ATG14BECN1recruiting Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of PIK3C3PIK3R4 complex10 Nexttwo ubiquitinlikeconjugation systems are involved in phagophore elongation11 When de novo autophagosome formation iscomplete they are delivered to the lysosome initiatinglysosomal degradationActually autophagy is a targetable pathway regulatingcellular sensitivity to ferroptosis12 The crosstalk betweenautophagy and ferroptosis has attracted more and moreattentions in the recent years Autophagy inhibition bybafilomycin A1 or chloroquine or lack of key autophagyassociated gene for example ATG5 ATG7 will partiallysuppressed ferroptotic eventsthrough ferritinophagyinactivation reducing the turnover of ironbinding ferritinand the iron release13“CircRNAs primarily originate from backsplicing eventsof premRNAs The covalently bonded loops are highlystable and abundant16 The deregulation of circRNAs isclosely related to various human diseases including cancer17“ In HCC several studies have revealed that circRNAs are involved in multiple malignant behaviors20However the functions and mechanisms of circRNAs inSF treatment are still not comprehensively understood Inthe current research we found a novel circRNA derivedfrom the IARS gene ID from circBase hsa_circ_0008367named cIARS cIARS was proven to be a promoter offerroptosis in HCC cells after SF treatment which waspartially attributed to the activation of autophagy andferritinophagy The underlying molecular mechanism ofcIARSmediated ferroptosis was also clarified in this studyResultsCircular transcript cIARS hsa_circ_0008367 is significantlyupregulated in SFtreated HCC cellsRNAseq was performed in three pairs of SFtreated anduntreated HCC celllines HepG2 SMMC7721 andHuh7 to screen differentially expressed circRNAs Cellswere treated with μM of SF for h uniquecircRNAs were found Among these circRNAs had already been reported in circBase21 and theother circRNAs were newly discoveredAmong the annotated circRNAs were ofextremely low abundance with fragments per kilobasemillion FPKM values in cellular samples and werethus excluded from this study The other circRNAscomprised two groups presented no significantchanges in expression levels after SF treatment and theother were differentially expressed circRNAs foldchange ‰¥ pvalue The volcano plots demonstrated that there were upregulated and downregulated transcripts Fig 1a The results of hierarchicalclustering are displayed in a heatmap Fig 1b generatedwith Heatmap Illustrator22 and suggest that there weretwo different clusters of transcripts The most highlyexpressed circRNA was cIARS hsa_circ_0008367 andOfficial journal of the Cell Death Differentiation Associationwas marked with black arrow in the volcano plotsAccording to circBase cIARS is an exonic circRNA nt in length originating from the exon and exon of the IARS gene on chr9 “ Fig1c qPCR showed that the relative expression levels ofcIARS were significantly higher in SFtreated cell linesthan in untreated ones Fig 1d Both convergent anddivergent primers of cIARS were applied for amplificationThe band of cIARS was only observed in cDNA samplenot the genomic DNA Fig 1e Sanger sequencing further validated that the sequence around the junction siteabout bp around the site was consistent with theresult of RNAseq and CircInteractome database23 Fig1f In addition cIARS was much more resistant to RNaseR Uμg which degrades linear but not circularthan IARS and GAPDH Fig 1g Whentranscriptsactinomycin D ActD a transcription inhibitor wasadded to HCC cells for the indicated time periods cIARSwas much more stable than its linear counterpart Fig1h These evidences suggested cIARS to be a highlyabundant and stable circular transcript in HCC cellscIARS is found to be a significant regulator of SFinducedferroptosisTo clarify the biological role of cIARS we first knockdown cIARS expression with a junction sitespecificsiRNA vector sicIARS The effects of the sicIARSwas shown in Fig 2a CCK8 assay showed that SFinduced growth inhibition was evidently weakened in sicIARS transfected cells Erastininduced growth inhibitionwas also attenuated by sicIARS Fig 2b c To determinethe underlying mechanism sicIARSintroduced HCCcells were treated with various cell death inhibitorsFerrostatin124 a specific ferroptosissignificantly undermined the therapeutic effects of either SFor Erastin in both sicIARS and NC transfected cellsHowever ZVADFMK an apoptosisinhibitor andNecrosulfonamide a necroptosis inhibitor exerted nosignificant influence on SF or Erastininduced growthinhibition Fig 2c Simultaneously malondialdehydeMDA and the level of Fe2 were significantly reducedwhile intracellular GSH obviously increased in the cIARSsilencing cells following SF or Erastin administration Fig2d These evidences suggested cIARS to be a positiveregulator of ferroptosis in HCC cellsinhibitorcIARS positively regulates SFinduced autophagy andferritinophagycIARS was also found to be an autophagy regulatorWestern blot WB assay showed that cIARS knockdownsignificantly decreased LC3 lipidation and increased p62accumulation Fig 3a Either autophagosomes or autolysosomes were observed via microscopic examinationafter AdmCherryGFPLC3 adenovirustransfection 0cLiu Cell Death Discovery Page of Fig See legend on next pageThis experiment is applied for concurrent observation ofautophagosome and autolysosome The signal of greenfluorescent protein will be quenched during fusion ofautophagosome and lysosome Thus the red signal ofmCherry indicates autolysosome and the merge of greenand red signals yellow puncta indicates autophagosomesicIARS significantly decreased the amount ofredautolysosome and yellow autophagosome puncta percell demonstrating an inhibition of autophagy fluxFig 3b TEM visuallyautophagicsuggested theOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of see figure on previous pageFig Circular transcript cIARS hsa_circ_0008367 is significantly upregulated in SFtreated HCC cells RNAseq was performed in three pairsof SFtreated μM h and untreated HCC cell lines HepG2 SMMC7721 and Huh7 a The differentially expressed circRNAs were visualized withvolcano plots the ˆ’log10 pvalue and the log2 fold change are plotted on the y and x axes respectively The dashed lines signify the filteringcriteria p fold change ‰¥ Upregulated circRNAs are shown in red and downregulated circRNAs are shown in green The most highlyexpressed circRNA was marked by a black arrow b Hierarchical cluster analysis showing the differentially expressed circRNAs The six columnsrepresent the six different cellular samples Each row indicates a circular transcript and the colors represent the abundance of the transcriptsc Schematic diagram of the backsplicing transcript generated from linear IARS Exon and exon are colored red and green respectively dRelative expression of cIARS after SF treatment for h in the HCC cell lines e Divergent and convergent primers for cIARS were applied to amplifyboth cDNA and gDNA Agarose gel electrophoresis visualized the products f The sequence around the junction site was confirmed through Sangersequencing The empty triangle indicates the junction site g RNase R exoribonuclease Uμg °C min was applied to remove the lineartranscripts from cellular extracts leaving circRNAs behind qPCR was applied to assess the resistance of RNAs to RNase R h RNA decay assayevaluating the stability of cIARS and IARS by qPCR after ActD μgml administration The error bars represent the standard deviation SD of at leastthree independent experiments p compartments sicIARS decreased the number of doublemembraned vacuoles to a relatively low level Fig 3cThese results showed that cIARS is a positive autophagyregulator in SFtreated HCC cells Furthermoretheprotein levels of FTH1 and NCOA4 the substrate andcargo receptor of ferritinophagy were determined by WBassay sicIARS resulted in remarkable accumulation ofboth FTH1 and NCOA4 Fig 3d This finding indicatedthatthe ferroptotic events in SFtreated HCC cellspositively regulated by cIARS may be partially associatedwith autophagy and ferritinophagycIARS specifically interacted with RBP ALKBH5 AlkBHomolog RNA demethylaseRecent studies have revealed that circRNA“RBP interaction has important roles in diverse biological processesAccording to CircInteractome database we found six RBPsbearing at least two binding sites matching to cIARSincluding FMRP Fragile X Mental Retardation SFRS1Serine And Arginine Rich Splicing Factor ALKBH5HuR ELAV like RNA binding protein IGF2BP1 InsulinLike Growth Factor MRNA Binding Protein andLIN28A Lin28 Homolog A Fig 4a RBP immunoprecipitation RIP assay in both HepG2 and Huh7 cell linesdemonstrated that the relative levels of cIARS in ALKBH5enriched samples were much higher than in the other fiveRBPs Fig 4b RNA pulldown and RNA EMSA were performed to determine the interaction using an antisenseprobe spanning the junction site of cIARS The presence ofspecific bands demonstrated the physical binding of cIARSand ALKBH5 Fig 4c d Interestingly SF administrationhad no influence on the protein levels of ALKBH5 in bothHepG2 and Huh7 cells Fig 4e but remarkably increasedthe cIARS“ALKBH5 interaction Fig 4f which may be dueto SFinduced expression of cIARScIARS repressed the role of ALKBH5 in the regulation ofautophagyALKBH5 had been previously proven to be an autoprole inin cancer2526 Howeverhagy inhibitoritsOfficial journal of the Cell Death Differentiation AssociationSFtreated HCC cells remains unclear WB assay showedthat siRNA against ALKBH5 siALKBH5 significantlypromoted the transformation of LC3B I to II and degraded p62 Fig 5a This result suggested ALKBH5 to be anegative regulator of autophagy in HCC cells qPCR andWB showed that sicIARS failed to influence ALKBH5mRNA and protein levels Fig 5b c similarly siALKBH5 also had no impact on the relative expressionof cIARS Fig 5d A previous study revealed that siALKBH5 promoted the dissociation of BECN1 and BCL a key step during phagophore nucleation In SFtreated HCC cells we gained similar results via IP assayMore importantly this process can be effectively blockedby cIARS silencing Fig 5e These results demonstratedthat cIARS repressed the biological role of ALKBH5 inautophagycIARS regulates ferroptosis through ALKBH5mediatedautophagyTo explore whether cIARSregulated ferroptosis viaALKBH5 we performed several phenotyperescueexperiments First siALKBH5 successfully rescued theeffects of sicIARS in autophagic flux and ferritinophagyFig 6a b Second ALKBH5 knockdown effectively reintensified the SF cytotoxicity which was remarkablyimpaired by sicIARS Fig 6c Third sicIARSmediateddecrease of MDA Fe2 and increase of GSH can berescued by siALKBH5 Fig 6d“fTaken together a novel circRNA in HCC was revealedin our research We partially clarified its role andmechanism in ferroptosisDiscussionAs a novel class of noncoding RNA circular transcriptshave attracted widespread attention However circRNAregulated ferroptosis in human diseases has not beenwidely investigated A small portion of studies focused onthe circRNAmediated autophagy For instance Chenet al27 reported that circHIPK3 depletion significantlyvia miR1243pSTAT3PRKAAinduced autophagy 0cLiu Cell Death Discovery Page of Fig cIARS is found to be a significant regulator of SFinduced ferroptosis a The expression levels of cIARS after transfection of sicIARS or NCb The evaluation of growth inhibition induced by SF in sicIARS or NC transfected cells at the indicated concentrations for h c The evaluation ofcytotoxicity of SF μM h and Erastin μM h with or without several inhibitors of cell death including ferrostatin1 μM ZVADFMK μM or necrosulfonamide μM d The assessment of MDA Fe2 and GSH during SF treatment μM h p AMPKa axis and there was an antagonistic regulation onautophagy between circHIPK3 and linear HIPK3 Du WWet al28 showed that the oncogenic circDnmt1stimulatedautophagy flux in breast carcinoma via interaction withboth p53 and AUF1 These findings demonstrated thepotential of circRNAsin autophagy regulation andprompted us to explore the role of circRNA in ferroptosiswhich had been identified as an autophagic cell death12research we delineated a mechanism ofcircRNAmediated ferroptosis during SF treatment inHCC cells circRNA cIARS hsa_circ_0008367 wasIn ourOfficial journal of the Cell Death Differentiation Associationscreened from RNAseq analysis Phenotypically cIARSpositively regulated ferroptosis which may be partiallydependent on autophagy and ferritinophagy Mechanistically cIARS physically interacted with RBP ALKBH5and negatively regulated its role in autophagyitis essentialTo comprehensively investigate the complicated cirto deeply evaluate itscRNA networkbinding partners In this study cIARS is found to be aninteractor of RBP ALKBH5 which had previously beenreported to be a N6methyladenosine m6A eraser Itsrole in autophagy regulation is completely different in 0cLiu Cell Death Discovery Page of Fig cIARS positively regulates SFinduced autophagy and ferritinophagy a The expression levels of LC3B and p62 in sicIARSintroduced cellswith or without SF treatment b Microscopic observation of autophagy flux after transfection of AdmCherryGFPLC3 for h mCherryGFPLC3 wasvisualized by fluorescence microscopy The green fluorescent protein is acid sensitive and will be quenched in the acidic condition of lysosome while themCherry is stable at low pH Thus the red puncta represent autolysosomes and the yellow puncta generated from the merge of both green and red signalsrepresent autophagosomes The autophagosomal and autolysosomal abundance was measured by the number of puncta lower density of yellow and redpuncta suggested lower level of autophagic flux c Visualization of autophagic compartments via TEM The red arrowheads indicate doublemembranedvacuoles d The assessment of the protein levels of FTH1 and NCOA4 in sicIARS or NC transfected cells with or without SF treatment μM hlower m6A leveldifferent tissues and diseases252629“ In lung cancer25ALKBH5 upregulation stabilized UBE2C an autophagyinhibitor with maintenance ofInovarian cancer26 ALKBH5 inhibited autophagy throughactivating PI3KAktmTOR signaling pathway stabilizedBCL2 mRNA and promoted the interaction betweenBCL2 and BECN1 Herein cIARS was proven to be apivotal regulator of autophagy ferroptosis and ferritinophagy depending on negatively regulating the biologicalinHCC cellsrole of ALKBH5 an autophagy inhibitibco Carlsbad CA USA and penicillin Umlstreptomycin µgml solution in the medical researchcenter of Shandong Provincial Qianfoshan HospitalShandong First Medical University All of the cell linesmentioned in this research were cultured within tenpassages The transfection experiments were performedaided by Lipofectamine Life Technologies Carlsbad CA USA The RNA oligonucleotides in this workwere designed and constructed by GenePharma Shanghai China including siRNAs against cIARS or ALKBH5and the corresponding negative controls NC and NC™Sequences were shown as follows ²“² sicIARS GACUUU GAG GAG AUC AGA CAC siALKBH5 GGAUAU GCU GCU GAU GAA ATT NCNC² UUC UCCGAA CGU GUC ACG UIn this study the cIARS“ALKBH5 axis was demonstrated to be a key mechanism regulating ferroptosisduring SF treatment Further studies are still needed atthe clinical and mechanistic levelsMaterials and methodsCell culture and transfection assayUnder humidified conditions with CO2 at °C theHCC cell lines HepG2 SMMC7721 and Huh7 boughtfrom the National Infrastructure of Cell Line Resourcewere cultured in Dulbecco™s Modified Eagle™s mediumHyClone Logan UT USA with fetal bovine serumCell Counting Kit8 CCK8 assayThe growth inhibition rate of HepG2 and Huh7 cellswas assessed by the CCK8 Dojindo Laboratories Japanassay The blank or transfected cells × cells perwell were seeded into 96well plates with three replicatewells After the treatment with SF or Erastin or variouscell death inhibitorsFerrostatin1 ZVADFMK orOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig cIARS specifically interacts with ALKBH5 a Prediction analysis of the interaction between cIARS and RBPs through CircInteractome b RIPassay evaluating the physical interaction between cIARS and the six candidate RBPs in HCC cells c d RNA EMSA and RNA pulldown evaluating thephysical interaction between cIARS and ALKBH5 e The protein levels of ALKBH5 in HCC cells in the absence or presence of SF treatment f Theassessment of the physical binding of cIARS and ALKBH5 by RIP assay in the absence or presence of SF treatment p Necrosulfonamidefor h a µl volume CCK8reagent was added to each well Then measuring theabsorbance at nm after incubation with the CCK8solution at °C for hRNA extraction and analysisExpression profiles of genomewide circRNAs in threepairs of HCC cell lines before and after SF treatmentwere explored on an Illumina HiSeq platform byNovogene Beijing China The cDNA from divergentprimers was subjected to Sanger sequencing by Zhonghong Boyuan Biological Technology Jiangxi ChinaAgarose gel electrophoresis was used to detect the qPCRamplification of cDNA and genomic DNA after applyingwith the divergent and convergent primers of cIARSTherelative fold changes in expression were calculated withthe formula ˆ’ΔΔCt The sequences of all the primers werelisted as follows ²“² cIARS F AGC GAT GAC TTTGAG GAG ATC A R CCC AGT AGC ACA GGT CATTG IARS F CAT ATC CAG TTT CTC CAT CGG A RTGG ATT TTC CAG GAG CAA TAC T ALKBH5 FGCA AGG TGA AGA GCG GCA TCC R GTC CACCGT GTG CTC GTT GTA C U6 F CTC GCT TCGGCA GCA CAT A R ATT TGC GTG TCA TCC TTGCG GAPDH F CAG AAC ATC ATC CCT GCC TCTAC R ATG AAG TCA GAG GAG ACC ACC TGRibonuclease R RNase R assayRNase R assay R0301 Uµl Geneseed GuangzhouChina was used for the identification of circRNAAccording to the manufacturer™s guidance µl ×Reaction Buffer and U RNase Rµg RNA were mixedto the total RNA and then added RNaseFree Water toform a µl reaction solution system After digestion withRNase R for min at °C the enzyme then was inactivated at °C for min and then directly performOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig cIARS repressed the role of ALKBH5 in the regulation of autophagy a The expression levels of LC3B and p62 in siALKBH5 or NC²introduced cells with or without SF administration b c The relative expression of ALKBH5 mRNA and protein in sicIARS or NC introduced HCC cellsd The relative expression of cIARS in siALKBH5 or NC² transfected cells e The assessment of the role of sicIARS in ALKBH5mediated interactionbetween BECN1 and BCL2Official journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of see figure on previous pageFig cIARS regulates ferroptosis through ALKBH5 a b The evaluation of the role of siALKBH5 in sicIARSregulated autophagy and ferritinophagyThe WB assay demonstrated that the effect of sicIARS on LC3 lipidation and p62 accumulation can be significantly reversed by siALKBH5 in SF μM h treated HCC cells a the effect of sicIARS on the accumulation of FTH1 and NCOA4 can be evidently rescued by siALKBH5 in either HepG2 or Huh7cells treated by SF μM h b c Identification of the role of ALKBH5 in cIARSregulated growth inhibition by CCK8 The results demonstrated thatsiALKBH5 significantly reversed sicIARSmediated growth inhibition in SF administered HCC cells d“f The rescue experiments evaluating the effects ofsiALKBH5 in the sicIARSmediated ferroptotic events The relative levels of MDA and iron were obviously decreased and the level of GSH increased insicIARS introduced HCC cells these impacts can be effectively reversed by ALKBH5 knockdown The black whiskers indicated the difference between theœsicIARS and œNC groups while the red ones indicated the difference between the œsicIARSNC™ and œsicIARSsiALKBH5 groups p reverse transcription reaction The qPCR assay was usedto determine the relative expression of cIARS IARS andGAPDH compared to the mock groupActinomycin D ActD assayActD assay HY17559 MedChem Express New JerseyUSA was used to detect the stability of RNA μgmlActD reagent was used to treat HepG2 and Huh7 cellsAfter or h of administration the total RNAwas extracted respectively to determine the relativeexpression of cIARS or IARS by qPCR assayWestern blot analysisRadioimmunoprecipitation assay buffer RIPA was appliedto lyse cells Total proteins were then harvested and quantified with bicinchoninic acid assays Beyotime ShanghaiChina The target proteins were separated through SDSPAGE and transferred to polyvinylidene fluoridemembranes Merck Millipore Burlington MA USA Themembranes were blocked with nonfat milk incubated withprimary antibodies and then incubated with secondaryantibody diluted at a ratio of Jackson ImmunoResearch West Grove PA USA The primary antibodieswere antiLC3B Cell Signaling Technology BeverlyMA USA antip62 Cell Signaling TechnologyantiHuR ab28660 Abcam Cambridge MA USA antiSFRS1 ab133689 antiFMRP ab17722 antiALKBH5ab195377antiLIN28Aab46020 antiFTH1 ab65080 antiNCOA4 ab86707antiBCL2 ab32124 antiBECN1 ab62557 and antiGAPDH ab9485 The protein signals were visualized withenhanced chemiluminescence detection reagentsECLMillipore Burlington MA USA and quantified with ImageLab software BioRad Hercules CA USAantiIGF2BP1ab82968based on the 3rd edition of the Guidelines for the Interpretation of Assays for Monitoring Autophagy32ImmunoprecipitationandImmunoprecipitation KitRIP and IP were performed to confirm the RNA“proteinand protein“protein interactions using RIP Kit Milliporeab206996according to the manufacturer™s guidance RIP related detailshad been described in our previous studies3334 The IP assayconsists of four steps including antibody binding beadspreparation bead capture and elution The volume of theantibody binding system was made up to µl with lysisbuffer containing the protease inhibitor cocktail and gentlymixed for h the protein AG sepharose µlreactionwas washed twice with wash buffer centrifuged at × gfor min and aspirated the supernatant between washesRNA pulldown assayPierce„¢ Magnetic RNAProteinPullDown KitThermo was applied to evaluate RNA“protein interaction using biotinlabeled junctionspecific probe and itsnegative control designed and synthesized by ViageneBiotech Jiangsu China Detailed procedure strictly followed the manufacturer™s guide cIARSprobe ²TGTAAA TTA GAG GAG TGT CTG ATC TCC TCA AAGTC3² Negative control ²GCA GCC TGA TCA CGACTG ACT TTA GTG TTT GCA TT3²RNA EMSAThe LightShift Chemiluminescent RNA EMSA KitThermoFisher Scientific was applied to evaluate theinteraction between cIARS and ALKBH5 according to themanufacturer™s guidance The details were described inour as previous research34Observation of autophagy fluxLipid peroxidation assayAdmCherryGFPLC3 adenovirus Servicebio Technology Wuhan China was transfected into HCC cellsAfter h incubation cells were observed and photographed using a fluorescence microscope OlympusFSX100 Tokyo Japan Autophagic compartments werefinely observed through transmission electron microscopyTEM HT7700 HITACHI Tokyo Japan The identification of autophagic vacuoles in TEM images was mainlyThe relative level of MDA was evaluated through Lipidab118970 according to the manuPeroxidation Kitfacturer™s guidanceIron assayThe relative level of intracellular iron was determinedby an Iron Assay Kit ab83366 according to the manufacturer™s instructionsOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Glutathione assayThe relative level of intracellular GSH was assessedthrough a GSH Colorimetric Detection Kit CS0260SigmaAldrich St Louis USA according to the manufacturer™s instructionsStatistical analysisThe statistical analyses in this work were carried outusing Prism The results from at least three independenttests are shown as the mean value ± standard deviationSD The mean values of two groups were compared viaunpaired Student ttests œpvalue  was defined assignificant p p AcknowledgementsThis work was supported by the National Natural Science Foundation of China[Grant No ] Shandong Natural Science Foundation [Grant NoZR201808200282] and Medical and Pharmacological TechnologyDevelopment plan Shandong [Grant Nos 2017WS188 and 2017WS284]Author details1Department of Hepatobiliary Surgery The First Affiliated Hospital ofShandong First Medical University Jinan Shandong China2Department of Hepatobiliary Surgery Shandong Provincial QianfoshanHospital Shandong University Jinan Shandong China 3Department ofReproduction Medicine Jinan Maternal and Child Health Care HospitalAffiliated to Shandong First Medical University Jinan Shandong ChinaAuthor contributionsStudy design ZL XW and JL data collection QW ZX and XW dataanalysis ZL QW and XW cellular experiments QW ZL XW and ZXmanuscript preparation ZL QW ZX and JL figures and manuscriptproofing ZL QW JL and XW project administration XW and JLConflict of interestThe authors declare that they have no conflict of interestPublisher™s noteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsReceived May Revised July Accepted July References Bray F Global cancer statistics GLOBOCAN estimates of incidenceand mortality worldwide for cancers in countries CA Cancer J Clin “ Siegel R L Miller K D Jemal A Cancer statistics CA Cancer J Clin “ Forner A Reig M Bruix J Hepatocellular carcinoma Lancet “ Li J Ferroptosis past present and future Cell Death Dis Llovet J M Sorafenib in advanced hepatocellular carcinoma N Engl JMed “ Cheng A L Efficacy and safety of sorafenib in patients in the AsiaPacificregion with advanced hepatocellular carcinoma a phase III randomiseddoubleblind placebocontrolled trial Lancet Oncol “ Official journal of the Cell Death Differentiation Association Dixon SJ Pharmacologicalinhibition of cystineglutamateexchange induces endoplasmic reticulum stress and ferroptosis Elife e02523 Stockwell B R Ferroptosis a regulated cell death nexus linking metabolism redox biology and disease Cell “ Jung C H ULKAtg13FIP200 complexes mediate mTOR signaling to theautophagy machinery Mol Biol Cell “ Itakura E Mizushima N Characterization of autophagosome formation siteby a hierarchical analysis of mammalian Atg proteins Autophagy “ Geng J Klionsky D J The Atg8 and Atg12 ubiquitinlike conjugation systems in macroautophagy ˜Protein modifications beyond the usual suspects™review series EMBO Rep “ Zhou B Ferroptosis is a type of autophagydependent cell death SeminCancer Biol httpsdoi101016jsemcancer201903002 Torii S An essential role for functional lysosomes in ferroptosis of cancercells Biochem J “ Gao M Ferroptosis is an autophagic cell death process Cell Res “ Hou W Autophagy promotes ferroptosis by degradation of ferritinAutophagy “ Memczak S Circular RNAs are a large class of animal RNAs with regulatory potency Nature “ Bi W CircRNA circRNA_102171 promotes papillary thyroid cancer progression through modulating CTNNBIP1dependent activation of betacateninpathway J Exp Clin Cancer Res Su Y circRIP2 accelerates bladder cancer progression via miR1305Tgfbeta2smad3 pathway Mol Cancer Chen J Circular RNA profile identifies circPVT1 as a proliferative factorand prognostic marker in gastric cancer Cancer Lett “ Wang M Yu F Li P Circular RNAs characteristics function and clinicalsignificance in hepatocellular carcinoma Cancers httpsdoi103390cancers10080258 Glazar P Papavasileiou P Rajewsky N circBase a database for circular RNAsRNA “ Deng W Wang Y Liu Z Cheng H Xue Y HemI a toolkit for illustratingheatmaps PLoS ONE e111988 Dudekula D B CircInteractome A web tool for exploring circular RNAsand their interacting proteins and microRNAs RNA Biol “ Dixon S J Ferroptosis an irondependent form of nonapoptotic celldeath Cell “ Guo J Deregulation of UBE2Cmediated autophagy repression aggravates NSCLC progression Oncogenesis Zhu H A

Thyroid_Cancer

Hepatitis B virus HBV infection has been associated with the risk andprognosis of many malignancies Nevertheless the association between HBV and theprognosis of breast cancer is unclear The objectives of this study were to investigate theprognostic role of hepatitis B surface antigen HBsAg and to integrate HBsAg to establishnomograms for better prognostic prediction of very young patients with breast cancerMethods This analysis was performed retrospectively in a cohort of consecutivevery young ‰ at diagnosis patients who received curative resection for breastcancer The significance of HBsAg in the prognosis of these patients was investigatedUnivariate and multivariate analyses were used to identify independent variables fordiseasefree survival DFS and overall survival OS Nomograms were built based onthose identified variablesResults Overall of the patients were seropositive for HBsAgThe median followup was CI “ months forthe entirepopulation The HBsAgpositive cohort had significantly inferior DFS HR CI “ P and OS HR CI “ P as compared with the HBsAgnegative cohort The rates of 10year DFS andOS were and in the HBsAgpositive group and and in the HBsAgnegative group respectively In multivariable analysis HBsAg statuswas identified as an independent significant unfavorable prognostic factor for DFSP and OS P in very young patients with breast cancer Nomogramswere established and displayed good calibration and acceptable discrimination TheCindex values for DFS and OS were CI “ and CI “ respectively Based on the total prognostic scores TPSof the nomograms different prognosis groups were identified for DFS and OSEdited byImtiaz Ahmad SiddiquiUniversity of Colorado AnschutzMedical Campus United StatesReviewed byAbhinit NagarUMass Memorial Medical CenterUnited StatesNidhi JainBeckman Coulter IndiaCorrespondenceLu YangyanglusysucccnWeiDong Weiweiwdsysucccn These authors have contributedequally to this workSpecialty sectionThis was submitted toCancer Epidemiology and Preventiona section of the journalFrontiers in OncologyReceived March Accepted July Published August CitationLi N Zhong QQ Yang XRWang QC Zhang DT Zheng SYang L and Wei WD Prognostic Value of Hepatitis B VirusInfection in Very Young Patients WithCuratively Resected Breast CancerAnalyses From an Endemic Area inChina Front Oncol 103389fonc202001403Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerConclusions HBsAg is an independent unfavorable prognostic factor for DFS andOS in very young patients with curatively resected breast cancer and nomogramsintegrating HBsAg provide individual survival prediction to benefit prognosis evaluationand individualized therapyKeywords HBV young breast cancer prognosis nomogram survivalINTRODUCTIONGlobally breast cancer is the most common cancer and theleading cause of cancer death for women accounting for of total cancer cases and of total cancer deaths Breast cancer has also been the top one malignancy in termsof incidence in Chinese women constituting of newlydiagnosed cases and of all deaths from breast cancer in theworld Although breast cancer occurs at a lower incidence inChinese women than in western women this disease occurs at ayounger age in China than in highincome countries and China™scontribution to global breast cancer rate is increasing rapidly The disparities between young and old breast cancer include ahigher mortality rate higher risk of recurrence poorer treatmentresponse and more aggressive phenotypes “ Thereforeunderstanding the etiology and identifying novel prognosticfactors are essential for early diagnosis prognosis evaluationearly intervention and personalized therapy in young patientswith breast cancerHepatitis B virus HBV infection is a serious public healthdilemma with ˆ¼ million chronic carriers worldwide China account for about a third of infectionassociated cancerglobally driven by high prevalence of HBV and H pylori infection Although China has made tremendous eï¬orts in controllingHBV over the past years and the prevalence of HBV ininfants and children has remarkably declined the hepatitisB surface antigen HBsAg prevalence is still high in Chineseadults ranging from to “ HBV is the leading causeof hepatocellular carcinoma and cholangiocarcinoma Inaddition there is also accumulating evidence that HBV infectionis associated with many extrahepatic malignancies includingnonHodgkin™s lymphoma pancreatic cancer gastriccancer nasopharyngeal carcinoma lung cancer esophageal cancer and ovarian cancer Thus it seemsreasonable that HBV is an important factor in the developmentof extrahepatic malignancies in endemic areasDespite the facts that HBsAg status is one of the routineexaminations in patients with operable breast cancer and severalstudies have showed that HBV is not associated with therisk of breast cancer the impact of HBV on theclinicopathological characteristics and prognosis of very youngpatients with breast cancer remains to be determined Giventhat both early breast cancer and HBV are endemic in China itis possible that HBV infection is associated with the prognosisof early breast cancer even though the precise mechanismsare yet to be determined It is crucial to address this issuesince HBV has been reported to be found in breast cancertissue We therefore performed this study to investigate theHBsAg prevalence in very young breast cancer and the impactof HBsAg on the survival of these patients and to establishnomograms to better predict prognosis for very young patientswith breast cancerMATERIALS AND METHODSPatient SelectionA retrospective review was conducted in a cohort of consecutive breast tumor women who were aged ‰ years oldand received curative resection for breast cancer at Sun Yatsen University Cancer Center between May and July This study was conducted according to the ethicalstandards of the Declaration of Helsinki Institutional ReviewBoard approval was obtained from the Medical Ethics Committeeof this cancer center All patients were restaged by the eighthinternational classification system for breast cancer Dueto the retrospective nature of this studyinformed consentwas waivedInformation was collected from electronic patient recordsand survival data were obtained from the followup registryofthis center The information collected included HBsAgstatus laterality type of breast surgery type of axillary surgeryhistological type tumor grade tumornodemetastasis TNMstage dates of surgeryrelapsedeath status of estrogen receptorER progesterone receptor PR human epidermal growthfactor receptor HER2 and Ki67 Breast cancers wereclassified as luminal Alike ER PR‰¥ HER2“ andKi6715luminal Blike ER andor PR HER2“HER2enriched ER“ PR“ HER2 or triplenegative ER“PR“ HER2“ subtypesPotentially eligible patients had to have curatively resectedbreast cancer without previous therapy other than neoadjuvanttherapy be aged years old or below and have definiteinformation of HBsAg The main exclusion criteria includedbenign tumor not having surgery having incomplete resectionprevious malignant disease hepatitis viral infections other thanHBV men patients and insufficient data of survival or HBsAgStatistical AnalysisThe main objectives of this study were to compare diseasefreesurvival DFS and overall survival OS between HBsAgpositivepatients and HBsAgnegative patients DFS was defined as theinterval from the date of being diagnosed to the date of diseaserecurrencemetastasis or death from any cause OS was defined asthe interval from the date of being diagnosed to the date of deathfrom any cause Median followup was estimated by KaplanMeier analysis with reversed meaning of status indicator Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerforindependent variablesDFS and OS were estimated by the Kaplan“Meier methodand diï¬erences were compared by the logrank test Univariateand multivariate analyses with a Cox proportional hazardsmodel were used to testforDFS and OS Covariates included laterality left vs righttype of surgery breastconserving surgery vs mastectomytype of axillary surgery sentinellymph node dissection vsaxillary lymph node dissection histological type ductal vsotherstumor grade grade III vs grade III T stageT34 vs T12 N stage N23 vs N1 HER2statustriplenegativeHER2enriched vs luminal All variables reaching asignificance of in univariate analyses were included inmultivariate analysispositive vs negative molecularsubtypesThe nomograms for predicting and 10year DFSand OS were formulated based on the results of multivariateanalysis by the œrms package of R The discriminationofthe nomogram models was estimated by the Harrell™sconcordance index Cindex The value of the Cindex rangesfrom to with implying a random chance and indicating a perfect prediction Calibration curves of thenomogram models for DFS OS were plotted to assess thepredictive value of the model In addition patients weredivided into three diï¬erent risk groups highintermediatelow according to total prognostic scores TPS The totalprognostic scores of patients were transformed into categoricalvariables based on cutoï¬ points which were determined by theminimum Pvalue from logrank × statistics with the Xtileprogram Pearson™s chisquare test was used to compare categoricaldata All Pvalues were twosided and P was consideredstatistically significant Statistical analyses were performed by theSPSS software SPSS Inc version Chicago IL USA and Rfor Windows version httpwwwrprojectData AvailabilityThe authenticity of this has been validated by uploadingthe key raw data onto the Research Data Deposit public platformwwwresearchdatacn with the approval RDD numberas RDDA2020001410 The data that support the findings ofthe study are available from the corresponding authors uponreasonable requestRESULTSPatient CharacteristicsA total of very young ‰ at diagnosis breasttumor patients were screened of whom were excludedbecause of benign tumor n not having surgeryn not having R0 resection n or unknownHBsAg status n With further exclusions forinsufficientfollowup data a total of patients withcurative resection for breast cancer and aged years orbelow were included in this study Figure The patients™FIGURE The process of patient selectionFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerTABLE Patient characteristics by HBsAg statusCharacteristicsHBsAgpositiveN No HBsAgnegativeN No Pvalue FIGURE Number of recurrences by year of entire patientsLateralityLeftRightBilateralType of breast surgeryMastectomyBreastconserving surgeryType of axillary surgeryALNDSLNDHistological typeDuctalInvasive lobularOtherTumor gradeIIIIIIUnknownT StageT1T2T3T4N StageN0N1N2N3HER2 statusPositiveNegativeUnknownMolecular subtypesLuminal ALuminal BHER2enrichedTriple negativeUnknown HBV Hepatitis B Virus ALND axillary lymph node dissection SLND sentinelnode dissectionHER2 human epidermal growth factor receptor2lymphcharacteristics are shown in Table Overall ofthe patients were seropositive for HBsAg HBsAgpositive group and HBsAgnegative group were wellmatchedfor basic characteristics including laterality type of breast andaxillary surgery histological type tumor grade T stage Nstage and molecular subtypes About in patients receivedmastectomy and most patients received axillary lymph nodedissection ALNDAssociations Between the Status of HBsAgand SurvivalThe median followup was CI “ months forthe entire population By the time of analysis December instances of disease recurrence had occurred Thenumber of recurrences in each year of the followup is shown inFigure Of note although HBsAgpositive patients had a higherfrequency of extrahepatic metastasis vs P thanHBsAgnegative patients they had a comparable frequency ofliver metastasis vs P when compared withHBsAgnegative patientsDFS was significantly shorter among those who were HBsAgpositive than among those who were HBsAgnegative HR CI “ P The rates of 10yearDFS were in the HBsAgpositive group and inthe HBsAgnegative group respectively Figure 3A A totalof death events had occurred by the data cutoï¬ HBsAgpositive group had significantly inferior OS compared withHBsAgnegative group HR CI “ P with a 10year OS of and respectivelyFigure 3B The association of HBsAg status and survival ineach molecular subtype was further analyzed As expectedDFS and OS were significantly longer among those in theluminal A subgroup and the HER2enriched and triplenegativegroups had significantly shorter DFS and OS Figures 4ABNotably HBsAgpositive status was associated with shorterDFS P and OS P in the luminalB cohort HBsAgpositive status was also associated with aslightly shorter DFS in the triplenegative cohort and shorterOS in the HER2enriched cohort but statistical significancewas not reached Supplementary Figures There was nosignificant diï¬erence in DFS between HBsAgpositive patientsand HBsAgnegative patients in luminal A and HER2enrichedcohorts and in OS in luminal A and triplenegative cohortsSupplementary Figures In the univariate analysis type of breast surgery tumor gradeT stage N stage molecular subtype and HBsAg status wereFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerindividual patient Figures 6AB The model™s explanatorycovariables consisted of HBsAg status T stage N stage andmolecular subtype Patients with higher scores correspondedto inferior survival The scatter plots for the TPS of DFSand OS and percentage of patient number were presentedin Figures 6CD The Cindex values for DFS and OS were CI “ and CI “ respectively The calibration curves for the probabilityof DFS and OS at or year presented an optimalagreement between the prediction by nomogram and actualobservation Supplementary Figure Next we divided thepatients into the following groups based on the TPS ofthe nomogram modelfor DFS using the Xtile programlowrisk group TPS “ patients intermediateriskgroup TPS “ patients and highrisk group TPS patients The 10year DFS for lowrisk groupintermediaterisk group and highrisk group were and respectively Survival analyses for DFS demonstratedsignificant discrimination between these three groups P Figure 7A Same procedures were performed for OS in theentire population and patients were divided into the following groups based on the TPS of the nomogram model for OS withthe Xtile program lowrisk group TPS “ patientsintermediaterisk group TPS “ patients and highrisk group TPS patients OS diï¬erences were alsoobserved among three subgroups with a 10year OS of and for lowrisk intermediaterisk and highrisk groupsP Figure 7BDISCUSSIONIn this study we investigated the association of HBsAg statusand very young breast cancer and to our knowledge reportfor the firsttime that HBsAgpositive status is associatedwith inferior DFS and OS from a population with a highprevalence of both HBV infection and young breast canceridentified as a significant unfavorableHBsAg status wasprognostic predictor for DFS and OSindependent of anyother clinicopathological features of breast cancer includingT stage N stage and molecular subtype We also integratedHBsAg to build nomograms to better predict prognosis foryoung patients with breast cancer The results of our studydemonstrated that the prevalence of HBsAg in young patientswith breast cancer in southern China was which wasin accordance with the “ reported in the populationof this endemic area This result suggests that unlikecervical cancer young breast cancer is not correlatedwith an increased prevalence of HBV infection Indeed breastcancer patients with HBsAg did not demonstrate a diï¬erentpattern of characteristics It is noteworthy that in our studyHBsAg did not increase the rate of liver metastases for veryyoung patients with breast cancer This results are comparableto those reported in previous studies for esophageal cancerand colorectal cancer which suggested that HBVinfection is associated with decreased risk of liver metastasis inthese malignanciesFIGURE Kaplan“Meier curves for A diseasefree survival and B overallsurvival stratified by HBsAg status in very young patients with breast canceridentified as significant prognostic factors for DFS Figure 5AWhen those variables were further analyzed in the multivariateanalysis we found that T stage P N stage P molecular subtype P and HBsAg status P remained statistically significant indicating that theyare significant independent predictors for DFS Figure 5ABy the same methods for OSthe results showed that Tstage P N stage P molecular subtype and HBsAg status P were independentprognostic factors for OS Figure 5B HBsAgpositive status isan independent negative prognostic factor for survival in veryyoung breast cancerPrognostic Nomograms For Very YoungBreast Cancer PatientsTo better assess the DFS and OS of very young breastcancer patients prognostic nomograms for DFS and OS wereestablished respectively All the independent predictors of DFSand OS in the multivariate analysis were integrated into thenomogram models and and 10year survivals weregraphically computed according to the characteristics of anFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by molecular subtype in very young patients with breast cancerOur study shows that HBsAgpositive status is associatedwith inferior DFS and OS in young patients with curativelyresected breast cancer decreasing the 10year DFS and OS by and respectively The association between HBsAgpositive status and poor prognosis is in keeping with thatdemonstrated in nasopharyngeal carcinoma lung cancer and ovarian cancer However some studies regardingother cancers indicate that HBsAgpositive cancer patientshad a favorable survival as compared with HBsAgnegativepatients This discrepancy can be partly explainedby the diversity and heterogeneity of diï¬erent malignanciesThe genetic or biological mechanisms underlying the inferiorFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Univariate and multivariate analysis for diseasefree survival A and overall survival B for very young patients with breast cancerprognosis remain to be elucidated butthis may largelyrelate to the presence of hepatitis B Xinteracting proteinHBXIP which has been welldocumented to function asan oncoprotein in breast cancer HBXIP can act as atransactivator by activating certain genes including cMyc E2F1STAT4 and Sp1 to play a crucial role in the progression ofbreast cancer HBXIP is associated with controlling cellapoptosis and promoting cell proliferation by mTOC1 activation HBXIP can also act as a modulation factor of cellularoxidative stress by competitively binding KEAP1 to enhancethe progression of breast cancer Previously studies showedthat HBV is not associated with risk of breast cancer These results combined with the data of our study suggestthat HBV is not a risk factor but a prognostic factor forbreast cancerAnother reason for this might relate to the HBV reactivationin HBsAgpositive patients with breast cancer who werereceiving chemotherapy HBV reactivation occurs frequentlyin breast cancer patients who are HBV carriers while receivingcytotoxic chemotherapy HBV reactivation can result inliver failure and interruption of the chemotherapy scheduleOther potential mechanisms underlyingassociationtheFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE AB Nomograms predicting and 10year A diseasefree survival and B overall survival for very young patients with breast cancer CD Thescatter plots of percentage of patient number and groups of C total prognostic score for DFS and D total prognostic score for OSbetween the HBsAg and patient survival include the immunesuppression Chronic HBV infection is characterized by thefailure to elicit an eï¬ective adaptive immune response andthe immune modulation of key innate immune response Chronic HBV infection can lead to immune anergy andimpair the function of the immune system which has longbeen deemed to protect the host against the developmentof nonviral cancerstogether couldpartially explain the positive association between HBsAgpositive status and the poor prognosis in young patients withbreast cancer These reasonsInthisstudy wecharacteristicscombined HBsAgstatus withclinicopathologicaleï¬ectiveprognostic nomogram models of DFS and OS for very youngpatients with breast cancer Both nomograms showed goodcalibration and acceptable discrimination These nomogrammodels can be used for prognosis evaluation at diagnosis forvery young patients with breast cancer and may benefit patientestablishtocounseling and personalized therapy for these patients Weadopted Xtile program to divide these patients into three riskgroups based on TPS from nomograms for DFS and OS Thesurvival curves for DFS and OS separated very well Thusspecial attention should be paid to and active surveillanceshould be conducted over patients with high risk group for DFSand OSThis study nevertheless has certain limitations that shouldbe noted First this study was retrospective in nature andwe cannot rule outthe impact of selection bias Secondthe sample size was relatively small and the sample sizesof the two cohorts were unequal as only patients wereHBsAgpositive The small sample size may be insufficientto allow us to perform subgroup analysis by each molecularsubtype Anotherthat Cantonese constitutemost of our study population The monotonicity ofthestudy population confines the universality of our resultsFurthermore the information was insufficient to perform otherlimitation isFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by risk groups based on total prognostic scores fromnomogram modelsanalysis such as that of hepatic function and HBV DNAcopy number Nevertheless the results of our study providewhat to our knowledge is the first evidence of the impactof HBsAg on the prognosis of very young patients withbreast cancerIn conclusion we have demonstrated in a retrospectivestudy that HBsAg is an independent unfavorable prognosticfactor for patients with very young breast cancer Furtherprospective studies involving varied ethnic populations arewarranted to confirm the prognostic value of HBsAg status invery young breast cancer and simultaneously other potentialclinicopathologic factors for breast cancer and HBV infection arerequired to be taken into account The mechanisms of the impactof HBV infection on the progression of breast cancer also need tobe elucidatedDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the Medical Ethics Committee of SunYatsen University Cancer Center WritteninformedFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerconsentin accordance with the nationalinstitutional requirementsfor participation was not required for this studylegislation and theAUTHOR CONTRIBUTIONSNL QQZ LY and WDW designed the study NL QQZXRY QCW DTZ and SZ collected the data NL QQZ LYand WDW interpreted and analyzed the data All authors wereinvolved in writing the and approved the final versionREFERENCES Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Globalcancer statistics GLOBOCAN estimates of incidence and mortalityworldwide for cancers in countries CA Cancer J Clin “ 103322caac21492 Fan L StrasserWeippl K LiJJ St LouisJ Finkelstein DM Yu 15e279“KD et al Breast cancer 101016S1470204513705679in China Lancet Oncol Colleoni M Rotmensz N Robertson C Orlando L Viale G Renneet al Very young women years with operable breastGcancer“ 101093annoncmdf039at presentation Ann Oncolfeaturesof disease Anders CK Hsu DS Broadwater G Acharya CR Foekens JA Zhang Y et alYoung age at diagnosis correlates with worse prognosis and defines a subsetof breast cancers with shared patterns of gene expression J Clin Oncol “ 101200JCO2007142471 Narod SA Breast cancer in young women Nat Rev Clin Oncol “ 101038nrclinonc2012102 Trepo C Chan HL Lok A Hepatitis B virus infection Lancet “ 101016S0140673614602208 de Martel C Gees D Bray F Ferlay J Cliï¬ord GM Global burden of cancerattributable to infections in a worldwide incidence analysis LancetGlobal Health 8e180“ 101016S2214109X19304887 Lu FM Li T Liu S Zhuang H Epidemiology and prevention of hepatitisJ Viral Hepatitis 17Suppl “B virus infection in China 101111j13652893201001266x Liu J Zhang S Wang Q Shen H Zhang M Zhang Y et al Seroepidemiologyof hepatitis B virus infection in million men aged years in ruralChina a populationbased crosssectional study Lancet Infect Dis “ 101016S1473309915002182 Zhang Y Fang W Fan L Gao X Guo Y Huang W et al HepatitisB surface antigen prevalence among rural women of childbearingage in Hainan Province China a crosssectional study Virol J 1011861743422X1025 Cui Y Jia J Update on epidemiology of hepatitis B and C in China JGastroenterol Hepatol 28Suppl “ 101111jgh12220 Chan SL Wong VW Qin S Chan HL Infection and cancer the case ofHepatitis B J Clin Oncol “ 101200JCO2015615724 Fwu CW Chien YC You SL Nelson KE Kirk GD Kuo HS et al Hepatitis Bvirus infection and risk of intrahepatic cholangiocarcinoma and nonHodgkinlymphoma a cohort study of parous women in Taiwan Hepatology “ 101002hep24150 Kamiza AB Su FH Wang WC Sung FC Chang SN Yeh CC Chronichepatitis infection is associated with extrahepatic cancer developmenta nationwide populationbased study in Taiwan BMC Cancer 101186s1288501629185 Nath A Agarwal R Malhotra P Varma S Prevalence of hepatitis B virusinfection in nonHodgkin lymphoma a systematic review and metaanalysisInternal Med J “ 101111j14455994200902060x Luo G Hao NB Hu CJ Yong X Lu MH Cheng BJ et al HBV infectionincreases the risk of pancreatic cancer a metaanalysis Cancer Causes Control “ 101007s1055201201442FUNDINGThis work was supported by the National Natural ScienceFoundation of China No SUPPLEMENTARY MATERIALThe Supplementary Materialonline202001403fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncat Wei XL Qiu MZ Jin Y Huang YX Wang RY Chen WW et al Hepatitis Bvirus infection is associated with gastric cancer in China an endemic area ofboth diseases Br J Cancer “ 101038bjc2014406 Ye YF Xiang YQ Fang F Gao R Zhang LF Xie SHHepatitis B virusin southern China Cancer Epidemiol Biomarkers Prevent“ 10115810559965EPI150344et alinfection and risk of nasopharyngeal carcinoma Peng JW Liu DY Lin GN Xiao JJ Xia ZJ Hepatitis B virusinfection is associated with poor prognosis in patients with advancednon small cell“ 107314APJCP201516135285lung cancer Asian Pacific J Cancer Prevent Zou J Chen J Xie X Liu Z Cai X Liu Q et al Hepatitis B virus infectionis a prognostic biomarker for better survival in operable esophageal canceranalysis of patients from an endemic area in China Cancer EpidemiolBiomarkers Prevent “ 10115810559965EPI181095 Wong L Cheung TH Yim SF Lao TT Prevalence and impact of hepatitis Bvirus infection in ovarian cancer patients in an endemic areaA retrospectivecohort study J Viral Hepat “ 101111jvh13250 Song C Lv J Liu Y Chen JG Ge Z Zhu J et al Associations between hepatitisB virus infection and risk of all cancer types JAMA Netw Open 2e195718 101001jamanetworkopen20195718 Su FH Chang SN Chen PC Sung FC Su CT Yeh CC Associationrisk abetween chronic viral hepatitisinfection and breast cancernationwide populationbased casecontrol study BMC Cancer Qin B Zhao K Wei J Wang X Xu M Lang J et al Novel evidence indicatesthe presence and replication of hepatitis B virus in breast cancer tissue OncolRep “ 103892or20197393 Te HS Jensen DM Epidemiology of hepatitis B and C viruses a globaloverview Clinics Liver Dis “ vii 101016jcld200911009 Siu SS Cheung TH Chan PK Lin CK Lo KW Patients with malignant or premalignant cervical lesion have increased risk of becoming hepatitis B carrierJ Exp Clin Cancer Res “in patients with colorectal Zhao Y Lin J Peng J Deng Y Zhao R Sui Q et al Hepatitis B virus infectionpredicts better survivalliveronly metastasesundergoing liver resection J Cancer “ 107150jca24544 Liu X Li X Jiang N Lei Y Tang LL Chen L et al Prognostic value ofchronic hepatitis B virus infection in patients with nasopharyngeal carcinomaanalysis of patients from an endemic area in China Cancer “ 101002cncr28377 Zhao Y Li H Zhang Y Li L Fang R Li Y et al Oncoprotein HBXIPmodulates abnormal lipid metabolism and growth of breast cancer cells byactivating the LXRsSREBP1cFAS signaling cascade Cancer Res “ 10115800085472CAN151734 Zhang Y Zhao Y Li H Li Y Cai X Shen Y et al The nuclear import ofoncoprotein hepatitis B Xinteracting protein depends on interacting with cFos and phosphorylation of both proteins in breast cancer cells J Biol Chem “ 101074jbcM113458638 BarPeled L Schweitzer LD Zoncu R Sabatini DM Ragulator is a GEFfor the rag GTPases that signal amino acid levels to mTORC1 Cell “ 101016jcell201207032 Zhou XL Zhu CY Wu ZG Guo X Zou W The oncoproteinHBXIP competitively binds KEAP1 to activate NRF2 and enhanceFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancercancerbreast“ 101038s4138801906985growthcelland metastasis Oncogene Liu Z Jiang L Liang G Song E Jiang W Zheng Y et al Hepatitis B virusreactivation in breast cancer patients undergoing chemotherapy a reviewand metaanalysis of prophylaxis management J Viral Hepat “ 101111jvh12672 Yuen MF Chen DS Dusheiko GM Janssen HLA Lau DTY LocarniniSA et al Hepatitis B virus infection Nat Rev Dis Primers 101038nrdp201835 Dunn GP Old LJ Schreiber RD Theimmunobiology ofimmunosurveillance“ 101016jimmuni200407017immunoeditingandImmunitycancer Giuliano AE Edge SB Hortobagyi GN Eighth edition ofthe AJCCcancer staging manual breast cancer Ann Surg Oncol “ 101245s1043401864866 Schemper MSmith TL A note on quantifyingstudies 101016019724569600075Xfailuretime Control ClinofTrialsfollowup in“ Vickers AJ Elkin EB Decision curve analysis a novel method for “evaluating prediction models Med Decision Making 1011770272989X06295361 Camp RL DolledFilhart M Rimm DL Xtile a new bioinformatics toolfor biomarker assessment and outcomebased cutpoint optimization ClinCancer Res “ 10115810780432CCR040713Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Li Zhong Yang Wang Zhang Zheng Yang and Wei This is anopenaccess distributed under the terms of the Creative Commons AttributionLicense CC BY The use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice No use distribution or reproduction is permitted which does not complywith these termsFrontiers in Oncology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer

breast cancer patients especially those with triple‘negative breast cancer is still grave More effective therapeutic targets are needed to optimize the clinical management of breast cancer Although collagen type VIII alpha chain COL8A1 has been shown to be downregulated in BRIP1‘knockdown breast cancer cells its clinical role in breast cancer remains unknownMethods Gene microarrays and mRNA sequencing data were downloaded and integrated into larger matrices based on various platforms Therefore this is a multi‘centered study which contains breast cancer patients and controls COL8A1 mRNA expression in breast cancer was compared between molecular subtypes In‘house immunohistochemistry staining was used to evaluate the protein expression of COL8A1 in breast cancer A diagnostic test was performed to assess its clinical value Furthermore based on differentially expressed genes DEGs and co‘expressed genes CEGs positively related to COL8A1 functional enrichment analyses were performed to explore the biological function and potential molecular mechanisms of COL8A1 underlying breast cancerResults COL8A1 expression was higher in breast cancer patients than in control samples standardized mean differ‘ence confidence interval [CI] “ Elevated expression was detected in various molecular subtypes of breast cancer An area under a summary receiver operating characteristic curve of CI “ with sensitivity of CI “ and specificity of CI “ showed moderate capacity of COL8A1 in distinguishing breast cancer patients from control samples Worse overall survival was found in the higher than in the lower COL8A1 expression groups Intersected DEGs and CEGs positively related to COL8A1 were significantly clustered in the proteoglycans in cancer and ECM‘receptor interaction pathwaysConclusions Elevated COL8A1 may promote the migration of breast cancer by mediating the ECM‘receptor interac‘tion and synergistically interplaying with DEGs and its positively related CEGs independently of molecular subtypes Several genes clustered in the proteoglycans in cancer pathway are potential targets for developing effective agents for triple‘negative breast cancerKeywords COL8A1 Breast cancer Immunohistochemistry staining MechanismCorrespondence fengzhenbo_gxmu163com chenganggxmueducn Wei Peng and Jian‘Di Li contributed equally as first authors Department of Pathology The First Affiliated Hospital of Guangxi Medical University NO6 Shuangyong Road Nanning Guangxi People™s Republic of ChinaFull list of author information is available at the end of the BackgroundBreast cancer poses a grave threat to female health According to the latest American cancer statistics breast cancer is estimated to be the most common cancer and the second most common cause of cancerassociated The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cPeng a0et a0al Cancer Cell Int Page of deaths in women [] Owing to higher distal metastatic and recurrent rates triplenegative breast cancer TNBC patients exhibit worse overall and diseasefree survival than any other type of breast cancer [“] Etiology investigations suggest that hereditary factors account for nearly onetenth of breast cancer cases Other risk factors such as early or delayed menstruation nulliparity hormone replacement therapy and alcohol consumption also contribute to the prevalence of breast cancer [] Clinical practice guidelines recommend that females aged “ or “ who are at higher risk screen for breast cancer [] Imaging examinations such as bilateral breast Xray imaging positron emission tomography“computed tomography and ultrasound histological findings and especially molecular pathology are the predominant methods of breast cancer diagnosis and assessment [ ] Based on the tumor burden optimal treatments namely breastconserving surgery radiotherapy chemotherapy and endocrine therapy are individually designed for breast cancer patients [] For TNBC patients anthracyclines and taxanes are preferred in the initial treatment and neoadjuvant therapy has been recognized as a standard strategy [“] Unfortunately neither endocrine therapy nor trastuzumab treatment is effective for TNBC Targeted drugs for example vascular endothelial growth factor [VEGF] antibodies epidermal growth factor receptor [EGFR] inhibitors and mammalian target of rapamycin [mTOR] inhibitors are gradually being employed in TNBC treatment even though their therapeutic effects are unsatisfactory [“] Therefore the situation faced by breast cancer”especially TNBC”patients is still grave More effective therapeutic targets are needed to optimize the clinical management of breast cancerMolecular events occurring in breast cancer help us better understand the onset and progression of breast cancer It is generally agreed that chromosome 1q amplification chromosome 16q deletion and PIK3CA mutations are the most common pathways leading to luminal breast cancer [ ] Moreover breast cancer gene BRCA1 and P53 mutations EGFR upregulation and cytokeratin downregulation have been associated with TNBC [ ] It has also been reported that circSEPT9 promotes tumor formation and TNBC progression [] Recently ˆ†Np63 has been found to participate in breast cancer metastasis and dissemination [] On the other hand several genes protect patients from breast cancer progression For example ZNF750 miR5745p and circKDM4C can inhibit breast cancer progression by mediating the epigenetic regulation of prometastatic genes indirectly suppressing SKILTAZCTGF and miR548pPBLD axis regulation [“] Based on these discovered molecular mechanisms some progress has been made in breast cancer treatment Delivering dual microRNA using CD44targeted mesoporous silica nanops proved to be effective in TNBC treatment [] Although many studies provided in a0vitro and in a0vivo a detailed molecular picture of TNBC cancers [“] the underlying cause of breast cancer has not been fully understood Further research is required to elucidate the breast cancer mechanisms and discover effective therapeutic targets for TNBCCollagen type VIII alpha chain COL8A1 also named C3orf7 is located at chromosome and encodes alpha chain in collagen type VIII which is an essential component of extracellular matrix ECM [] Previous studies mainly addressed the relevance between COL8A1 and agerelated macular degeneration ADM as well as cell proliferation [“] Recently limited studies demonstrate the deregulation of COL8A1 in various cancers Elevated COL8A1 expression was found in gastric cancer patients and higher COL8A1 correlated with advanced tumor stages and worse overall survival condition and COL8A1 was selected as a candidate diagnostic biomarker in gastric cancer [“] Additionally upregulation of COL8A1 was also reported in adamantinomatous craniopharyngioma [] Furthermore COL8A1 proved to participate in the progression of colon adenocarcinoma possibly by mediating focal adhesionrelated pathways [] COL8A1 upregulation induced by TGFβ1 was found in renal cell carcinoma carcinogenesis and also correlated with poor prognosis [] Moreover elevated COL8A1 in hepatocellular carcinoma promoted tumor cells proliferation invasion and in a0vivo tumorigenicity [] Thus far only few studies mentioned COL8A1 in breast cancer COL8A1 was one of the key genes restored by epigallocatechin3gallate in a murine breast cancer model [] COL8A1 proved downregulated in both BRIP1knockdown breast cancer cells and MCF10A a0CDH1 noncancer breast cells [ ] However the role of COL8A1 in breast cancer remains unknownConsidering this knowledge gap our study aimed to explore the role of COL8A1 in breast cancer We were focused on investigating the expression of COL8A1 messenger RNA mRNA in various molecular subtypes of breast cancer by analyzing gene microarray and RNA sequencing data sets The COL8A1 protein expression level was validated by immunohistochemistry staining We also aimed to determine prognostic value of COL8A1 in breast cancer to pave the way for future clinical applications Moreover we explored the molecular mechanisms of COL8A1 underlying breast cancer to improve our knowledge of breast cancer carcinogenesis and progression 0cPeng a0et a0al Cancer Cell Int Page of MethodsExpression of a0COL8A1 mRNA in a0breast cancerWe integrated gene microarrays and mRNA sequencing data downloaded from Gene Expression Omnibus The Cancer Genome Atlas TCGA the GenotypeTissue Expression the Sequence Read Archive ArrayExpress and Oncomine The search formula based on MESH terms was as follows Breast OR mammary AND neoplasm OR cancer OR adenoma OR carcinoma OR tumor OR BRCA OR neoplasia OR malignant OR malignancy Studies were screened according to the following criteria i the studied species should be Homo sapiens ii the studied specimens should be tissue dissected from patients or healthy individuals rather than cell lines In the case of duplicated studies or samples the most recent version was retained The exclusion criteria were as follows i expression profiles not including COL8A1 ii breast cancer patients receiving hormone therapy or chemotherapy iii stromal rather than epithelial tumors and iv metastatic rather than primary tumors The included data sets were carefully checked and a log2 transformation was performed if any matrices had not been normalized Additionally the data sets were integrated into larger matrices according to various platforms and batch effects between studies were removed using the limmavoom package in R v361 Subsequently COL8A1 expression values were extracted and grouped according to specimen types Standardized mean difference SMD were calculated to compare the expression of COL8A1 mRNA between breast cancer patients and control samples using STATA v120 Heterogeneity between the included studies was assessed with the I2 statistic Statistical significance was set to an I2 value greater than with a Pvalue less than A random effects model was used in the case of significant heterogeneity Sensitivity analysis was performed to probe the potential source of heterogeneity and a publication bias test was used to evaluate the stability of the SMD results Subgroup analysis was performed to compare the COL8A1 expression levels between molecular subtypes luminal A luminal B human epidermal growth factor receptor 2positive [HER2 ] and TNBCDiagnostic value of a0COL8A1 in a0breast cancerA diagnostic test was performed to assess the clinical significance of COL8A1 in breast cancer Based on the expression value of COL8A1 a receiver operating characteristic ROC curve was plotted to compute the area under the curve AUC using IBM SPSS Statistics v190 AUC values of less than between and and greater than represented weak moderate and strong discriminatory capability of COL8A1 respectively between breast cancer patients and control samples The true positives false positives true negatives and false negatives rates were calculated and the cutoff values were identified A summary receiver operating characteristic sROC curve was drawn using STATA v120 to assess the general discriminatory capability of COL8A1 between breast cancer patients and control samples The significance of the area under the sROC curve was consistent with that of the ROC curve The diagnostic odds ratio DOR sensitivity specificity positive diagnostic likelihood ratio DLR P and negative diagnostic likelihood ratio DLR N were also calculated to precisely determine the accuracy and validity of COL8A1 in distinguishing breast cancer patients from control samplesPrognostic value of a0COL8A1 in a0breast cancerTo explore the relation between COL8A1 mRNA expression and prognosis of breast cancer patients information on clinicopathological parameters was collected The independent samples ttest or oneway analysis of variance was used to identify statistically significant differences in COL8A1 expression between two or more groups A Pvalue of was considered statistically significant Kaplan“Meier curves were used to compare high and low COL8A1 expression groups in terms of survival The logrank test was used to determine whether the prognostic difference was statistically significantInvestigation of a0COL8A1 protein expression in a0breast cancer by a0immunohistochemistry stainingA total of nonspecific invasive breast carcinoma and normal breast tissue specimens were obtained from the First Affiliated Hospital of Guangxi Medical University PRCHINA All patients had previously signed informed consent forms and our research was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University The breast cancer and normal breast tissue specimens were fixed with formalin The two steps immunohistochemistry method was used to determine the protein expression of COL8A1 The primary antibody was polyclonal Antibody to COL8A1 concentrated dilution purchased from Wuhan Pujian COLTD Supervision TM MouseRabbitHRP Broad Spectrum Detection System Product No D300415 was purchased from Shanghai Long Island The experimental procedure conformed to the manufacturer™s instructions The patients™ clinicopathological information was analyzed to determine the relationship between COL8A1 protein expression and prognosisEvaluation of a0genetic alteration and a0mutation landscapesThe cBioPortal for Cancer Genomics httpcbiop ortal proved to be a powerful tool and facilitated our online search and cancer genomics data analysis Using 0cPeng a0et a0al Cancer Cell Int Page of cBioPortal we gained insight into the genetic alterations of COL8A1 in breast cancer patients and obtained information on the association between COL8A1 alterations and breast cancer patient survival We selected the Breast Invasive Carcinoma TCGA Firehose Legacy cohort which contains patients and used a method of mRNA expression zscores relative to diploid samples RNASeqV2 RSEM We also considered the mutation types of COL8A1 in breast cancer patients using the Catalogue of Somatic Mutations in Cancer COSMIC which has been recognized as the most detailed resource for somatic mutations in cancerIdentification of a0differentially expressed genes and a0COL8A1 co‘expressed genes in a0breast cancerTo gain insight into the role of COL8A1 in breast cancer we identified DEGs and COL8A1 CEGs using the limmavoom package The DEG criteria were as follows ilog2FoldChange and ii adjusted Pvalue The CEG criteria were as follows irelation coefficient and ii Pvalue Upregulated DEGs and CEGs positively related to COL8A1 were intersected Similarly downregulated DEGs and CEGs negatively related to COL8A1 were intersectedMolecular mechanisms of a0COL8A1 underlying breast cancerOverlapping genes were used to perform function enrichment to shed light on the potential mechanisms of COL8A1 underlying breast cancer The R clusterProfiler package was used to conduct Gene Ontology GO Kyoto Encyclopedia of Genes and Genomes KEGG Disease Ontology DO and Reactome pathway analyses Proteintoprotein interaction PPI network was constructed using STRING https strin gdb to investigate protein interactions Hub genes and functional modules were identified using Cytoscape v361 Based on breast cancer patients the mutation landscapes of genes clustered in essential pathways were visualized using the TCGAmutations package in R v361ResultsUpregulation of a0COL8A1 mRNA in a0breast cancerAdditional file a0 Figure S1 shows the flow diagram of the study inclusion process A total of studies were included and integrated into larger platform matrices covering breast cancer patients and controls Table a0 COL8A1 was generally upregulated in breast cancer compared to normal breast tissue Thirteen of the twenty platforms showed much higher COL8A1 expression in breast cancer patients than in control samples Additional file a0 Figure S2 Because of significant heterogeneity I2 P a random effects model was used An SMD value of confidence interval [CI] “ showed that COL8A1 expression was significantly higher in breast cancer than in nonbreast cancer tissue Fig a0 Sensitivity analysis indicated that the included studies could not explain the source of heterogeneity Additional file a0 Figure S3a No publication bias existed Additional file a0 Figure S3b Subsequently we compared COL8A1 expression levels between different subtypes of breast cancer COL8A1 expression was universally higher in luminal A luminal B HER2 and TNBC patients than in control samples Additional file a0 Figure S4 Additional file a0 Figure S5 and Additional file a0 Figure S6a Furthermore three platforms showed significantly higher COL8A1 expression in nonTNBC than TNBC while only one showed lower expression in nonTNBC than TNBC Additional file a0 Figure S6b However an SMD of ˆ’ CI ˆ’“ showed no difference in COL8A1 expression between TNBC and nonTNBC patients Additional file a0 Figure S7 Subgroup analysis of four molecular subtypes of breast cancer showed no significant differences in COL8A1 expression levels between them Fig a0Diagnostic and a0prognostic value of a0COL8A1 mRNA in a0breast cancerThe clinical value of COL8A1 in breast cancer was found to be promising Among the thirteen platforms showing high COL8A1 expression twelve platforms indicated the ability of COL8A1 in differentiating breast cancer patients and control samples where four platforms showed strong discriminatory capability of COL8A1 between breast cancer patients and control samples Additional file a0 Figure S8 An area under the sROC curve of CI “ with sensitivity of CI “ and specificity of CI “ displayed moderate capacity in distinguishing breast cancer patients from control samples Fig a03a A DOR of CI “ also highlighted the discriminatory ability of COL8A1 in breast cancer Fig a03b The DLR P and DLR N were CI “ and CI “ respectively Additional file a0 Figure S9 As shown in Additional file a0 Figure S10 and Additional file a0 Table a0S1 elevated COL8A1 expression correlated with race white black molecular subtypes of breast cancer luminal B luminal A TNBC ER PR and HER2 status Moreover Kaplan“Meier curves indicated worse overall survival in high compared to low COL8A1 expression groups Fig a0Expression levels and a0clinical significance of a0COL8A1 protein in a0breast cancerClinical data of breast cancer samples used to perform in immunohistochemistry was summarized 0cPeng a0et a0al Cancer Cell Int Page of ldnaoPESGi abarA iduaSESG ecnarFESG ASUESG inapSESG ecnarFESG ecnarFESG adanaCESGkramneDESGi eropagnSESGl yatIESG ASUESG ynamreGESG inapSESG ASUESGASUESG cil bupeRhcezCESG ldnaerIESGi eropagnSESGASUESG ailartsuAESG iocxeMESGi abarA iduaSESGASUESG ASULPG‘ESGadanaCESG ASUESG adanaCESGASULPG‘ESGASULPG‘ESGadanaCESGianhCESG anhCi ESG ASUESG ASUESGianhCESGl aisyaaMESGi anhCESGadanaCESGi anhCESGASUESGi anhCESGASUESGi eropagnSESGl aisyaaMESGASUESG ESG ESG ESG ESG ESGASUESG ESGASU ESGASU ESGanhCi ESGynamreG ESGASU ESGˆ’‘ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’LPGLPGLPGLPGLPGLPGLPGˆ’LPGLPGLPGLPGLPGLPGLPGxETG‘AGCTESGESGESGESGESGsrebmun latoTseirtnuoC seireSOEG NTNFPFPTleuav‘PfdtlortnoC ACRBnoisseccADSMNDSMN stesataddell a0 orneeht a0fonoitamrofni cisaB elbaT 0cPeng a0et a0al Cancer Cell Int Page of Fig General expression status of COL8A1 in breast cancer BRCA compared to non‘BRCA tissues A standardized mean difference SMD value and 95CI with no overlap of zero indicated COL8A1 was significantly upregulated in BRCA compared to non‘BRCA tissuesAdditional file a0 Table a0 S2 Protein expression confirmed the upregulation of COL8A1 in breast cancer The breast cancer patients whose specimens were analyzed in this study were aged between and mean and their followup durations ranged from to a0 days Immunohistochemistry staining showed varying coloration intensity of COL8A1 in breast cancer and normal breast tissue COL8A1 was negatively weakly moderately or strongly stained in normal breast epithelium Fig a05a“d and breast cancer tissue Fig a0 5e“h According to the staining intensity and color range percentages of breast cancer tissue specimens exhibited low and exhibited high COL8A1 expression whereas of normal breast tissue specimens exhibited low and exhibited high COL8A1 expression A Chi square test confirmed the significantly higher expression of COL8A1 in breast cancer than normal breast tissue χ2 P Moreover elevated COL8A1 expression correlated with estrogennegative ER breast cancer P Genetic alterations and a0mutation kinds of a0COL8A1 in a0breast cancerAlterations and mutations of COL8A1 in breast cancer were relatively frequent Based on cBioPortal COL8A1 was altered in of breast cancer patients Additional file a0 Figure S11 Amplification and high and low mRNA were the main alterations No statistically significant difference in overall and diseasefree survival was found between high and low COL8A1 expression breast cancer groups P Furthermore according to COSMIC substitution missense mutations were the most frequent types Additional file a0 Table a0S3DEGs and a0COL8A1 CEGs in a0breast cancerA total of platform matrices were collected to determine the DEGs The approach has been aforementioned Initially upregulated DEGs downregulated DEGs CEGs positively related to COL8A1 and CEGs negatively related to COL8A1 were identified Additional file a0 Figure S12 shows partial DEGs and CEGs After being intersected 0cPeng a0et a0al Cancer Cell Int Page of Fig Subgroup analysis based on the subtypes of breast cancer The result indicated that the elevated COL8A1 expression shared no significant difference among Luminal A Luminal B HER‘ and Three Negative Breast Cancer TNBC subgroupsthe DEGs and CEGs were divided into two gene sets overlapping upregulated DEGs and CEGs positively related to COL8A1 all genes appeared in no fewer than three data sets and overlapping downregulated DEGs and CEGs negatively related to COL8A1Potential mechanisms of a0COL8A1 underlying breast cancerThe GO KEGG DO and Reactome pathway analyses based on the intersected genes are shown in Additional file a0 Table a0S4 Regarding the overlapping upregulated DEGs and CEGs positively related to COL8A1 the following KEGG pathways were significantly aggregated 0cPeng a0et a0al Cancer Cell Int Page of Fig Diagnostic value of COL8A1 in breast cancer BRCA a Summary receiver operating characteristic sROC curve b Forest plot of diagnostic odd ratio DOR An AUC value and a DOR signified COL8A1 possessed moderate capability in distinguishing BRCA from non‘BRCA patients AUC area under the curve 0cPeng a0et a0al Cancer Cell Int Page of Fig The prognostic value of COL8A1 mRNA in breast cancer tissues a GSE25307 b GSE35629‘GPL1390 c TCGA In the GSE25307 cohort high COL8A1 group possessed poor overall survival condition compared to low COL8A1 group in breast cancer patients 0cPeng a0et a0al Cancer Cell Int Page of See figure on next pageFig Protein expression levels of COL8A1 in breast cancer BRCA and normal breast tissues a‘d normal breast tissues e“h BRCA tissues Magnification × COL8A1 was negatively stained in normal breast epithelium a and BRCA e COL8A1 was weakly stained in normal breast epithelium b and BRCA f COL8A1 was moderately stained in normal breast epithelium c and BRCA g COL8A1 was strongly stained in normal breast epithelium d and BRCA h According to staining intensity and percentage of color range BRCA tissues exhibited low COL8A1 expression and BRCA tissues exhibited high COL8A1 expression While normal breast tissues exhibited low COL8A1 expression and normal breast tissues exhibited high COL8A1 expressionFig a06a proteoglycans in cancer Additional file a0 Figure S13 ECMreceptor interaction Additional file a0 Figure S14 and several cancer pathways such as thyroid cancer colorectal cancer and hepatocellular carcinoma Interestingly genes WNT2 GADD45B FZD2 CDKN1A KRAS LEF1 WNT7B BAK1 BRCA2 CDK4 GRB2 HRAS PIK3R3 and POLK were associated with the breast cancer pathway ID hsa05224 even though is not in the top KEGG pathways Moreover DO analysis indicated that these genes are closely associated with myeloma and bone marrow cancer Fig a06b as well as renal cell carcinoma ovarian cancer renal carcinoma and other types of cancer Furthermore Reactome pathway analysis revealed extracellular matrix anization ECM proteoglycans integrin cell surface interactions and degradation of the extracellular matrix as the top four metabolic pathways Fig a0 6c Regarding GO enrichment extracellular matrix anization extracellular matrix and extracellular matrix structural constituent were the most clustered Biological Process BP Cellular Component CC and Molecular Function MF terms respectively Fig a07a The proteoglycans in cancer and ECMreceptor interaction pathways were selected to construct PPI networks Fig a07b c FN1 and ITGB1 were identified as the hub genes in the two networks respectively The mutation landscapes of the genes in these two important pathways are shown in Fig a0 In particular FN1 was altered in of breast cancer samples where missense mutations accounted for The regulatory networks of COL8A1 and enriched genes in the proteoglycans in cancer and ECMreceptor interaction pathways as well as the top two functional modules are displayed in Additional file a0 Figure S15 On the other hand the enrichment results regarding the overlapping CEGs negatively related to COL8A1 and downregulated DEGs showed no statistical significance these data are therefore not shown Additional file a0 Table a0S5DiscussionThe highlight of this study is that it comprehensively explored the upregulation of COL8A1 mRNA in breast cancer from multiple aspects based on breast cancer patients and controls Our study is multicentered because we collected breast cancer patients from Asia American Europe and Oceania covering different countries This is the first study to investigate the protein expression of COL8A1 in breast cancer using immunohistochemistry staining Moreover this study is the first to assess the clinical prognostic value of COL8A1 in breast cancer Furthermore our study sheds light on the biological function and potential molecular mechanisms of COL8A1 underlying breast cancerThis study demonstrates the upregulation of COL8A1 in breast cancer We found higher COL8A1 expression in breast cancer than normal breast tissue based on large platform matrices integrated from data sets Subgroup analysis based on four molecular subtypes of breast cancer showed that elevated COL8A1 expression is independent of subtypes Further analysis also revealed universally higher COL8A1 expression levels in luminal A luminal B HER2 and TNBC patients than in control samples A comparison of COL8A1 expression between nonTNBC and TNBC patients showed no statistically significant difference Immunohistochemistry staining confirmed the upregulation of COL8A1 protein in breast cancer We thus concluded that COL8A1 expression is higher in breast cancer patients than in control samples and that upregulation is independent of molecular subtypes of breast cancer Though the expression of COL8A1 in tissue cannot reflect the early diagnostic value in breast cancer we assume that if COL8A1 is also differentially expressed in the bodily fluid of patients it will be possible to serve as a potential diagnostic marker for breast cancer Nevertheless no previous studies have demonstrated the expression level of COL8A1 in the bodily fluid of breast cancer patients until nowWe determined the clinical value of COL8A1 in breast cancer for the first time Our diagnostic test showed a moderate discriminatory capability of COL8A1 between breast cancer and normal breast tissue Higher COL8A1 expression levels in breast cancer patients correlated with worse survival outcomes Additionally COL8A1 expression was much higher in patients of the white than of the black race Our TCGA cohort analysis showed lower COL8A1 upregulation levels in TNBC than in the luminal A and B subtypes Furthermore high COL8A1 protein levels were related to ER breast cancer Thus COL8A1 might serve as a prognostic marker for breast cancer 0cPeng a0et a0al Cancer Cell Int Page of 0cPeng a0et a0al Cancer Cell Int Page of Fig Functional enrichment based on overlapping genes of upregulated DEGs and COL8A1 positively related CEGs a Kyoto Encyclopedia of Genes and Genomes b Disease Ontology c Reactcome DEGs differentially expressed genes CEGs co‘expressed genes 0cPeng a0et a0al Cancer Cell Int Page of Fig GO enrichment based on overlapping genes of upregulated DEGs and COL8A1 positively related CEGs a GO analysis b Protein‘to‘protein internet based on KEGG pathway proteoglycans in cancer ID hsa05205 c Protein‘to‘protein internet based on KEGG pathway ECM‘receptor interaction ID hsa04512 GO Gene Ontology DEGs differentially expressed genes CEGs co‘expressed genes KEGG Kyoto Encyclopedia of Genes and GenomesMore importantly our study provides important clues about the role of COL8A1 in breast cancer for the first time The intersected CEGs positively related to COL8A1 and upregulated DEGs were significantly aggregated in several cancer pathways such as thyroid cancer colorectal cancer and hepatocellular carcinoma Interestingly we noticed that genes related to COL8A1 WNT2 GADD45B FZD2 CDKN1A KRAS LEF1 WNT7B 0cPeng a0et a0al Cancer Cell Int Page of Fig Mutation landscapes of COL8A1 and co‘expressed genes clustered in two Kyoto Encyclopedia of Genes and Genomes pathways proteoglycans in cancer and ECM‘receptor interactionBAK1 BRCA2 CDK4 GRB2 HRAS PIK3R3 and POLK were associated with the breast cancer pathway ID hsa05224 even though this pathway is not in

Thyroid_Cancer

"Oral squamous cell carcinoma is one of the most common causes of malignancy‘associated death Early diag‘nosis of oral squamous cell carcinoma OSCC is important in patient treatment and prognostic evaluation Due to the lack of significant therapeutic benefit the ‘year survival rate has not improved Therefore effective novel markers are needed to improve diagnosis To determine novel promising diagnostic biomarkers for OSCC upregulated and downregulated differentially expressed genes were screened from OSCC tissues using an RNA microarray The results suggested that minichro‘mosome maintenance protein MCM5 mRNA was significantly overexpressed in OSCC tissues compared with that in adjacent normal tissues Moreover silencing of MCM5 expression an OSCC cell line SCC‘ significantly impaired proliferation and colony formation Furthermore negative regulation of the mRNA and protein expression of MCM5 and demonstrated that MCM5 served as a cancer‘promoting gene modulating OSCC cell proliferation through induced G2M phase arrest In this process the mRNA expression of cyclin E and cyclin‘dependent kinase was downregulated while p21 expression was upregu‘lated These results suggested that MCM5 may be an important pathogenic factor of OSCC High expression levels of MCM5 may serve as a marker for the early diagnosis of OSCC IntroductionOral squamous cell carcinoma OSCC affects individuals worldwide annually Presently the clinical treatment of OSCC is primarily surgery radiotherapy or chemotherapy Over the past decades the overall survival Correspondence to Dr Xiaofeng Wang Department of Stomatology China‘Japan Union Hospital of Jilin University Xiantai Changchun Jilin PR ChinaE‘mail wangxiaofengjlueducnContributed equallyKey words minichromosome maintenance protein cell cycle oral squamous cell carcinoma biomarker microarrayOS rate of patients with OSCC has not significantly improved with a ‘year survival rate of ‘ Insufficient sensitive and specific biomarkers may lead to the diagnosis of OSCC at advanced stages Therefore it is necessary to identify novel biomarkers for the early diagnosis and treatment of OSCCRecently with the continuous development of sequencing technology researchers can efficiently distinguish differentially expressed genes DEGs by transcriptome sequencing which allows screening of potential tumor markers or therapeutic drug targets For example a number of new potential tumor markers have been found in human malignancies such as breast cancer epithelial ovarian cancer and glioma Minichromosome maintenance protein MCM5 a member of mini‘chromosome maintenance family of proteins plays an important role in cell proliferation and DNA replica‘tion Some studies have confirmed that MCM5 is highly expressed in numerous human malignancies such as renal cell carcinoma pancreatic ductal adenocarcinoma cervical cancer and skin cancer Further studies have found that high expression of MCM5 is closely associ‘ated with the clinicopathological features of specific cancer types For example overexpression of MCM5 is significantly associated with overall survival rate OS in hepatocellular carcinoma Moreover increased expression of MCM5 is positively correlated with larger tumor size positive lymph node metastasis more advanced clinical stage higher histo‘logical grade deeper invasion depth and perineural invasion of OSCC However thus far the expression function and potential mechanisms of MCM5 in OSCC are still unclear Therefore the present study aimed to analyze the DEGs in OSCC using a microarray screen for MCM5 and further eval‘uate the possible functions of MCM5 in OSCC The present results may provide evidence to support the value of MCM5 as a biomarker or a therapeutic target of OSCC Materials and methodsTissue sampling Pairs of OSCC tissues and adjacent normal tissues were obtained from patients undergoing resec‘tion operations at the China‘Japan Union Hospital of Jilin University Changchun China Clinicopathological data were also collected No patient received preoperative treatment including radiotherapy or chemotherapy No other inclu‘sionexclusion criteria were used Matched normal OSCC 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAtissues were obtained from a segment of the resected specimens ‘cm away from the tumor Pathological analysis was used to identify surgically resected specimens Pathological analysis was performed by our group with no specific diagnostic guide‘lines Three paired samples were obtained for transcriptome sequencing Then to confirm the reliability of sequencing data the samples size was increased using the remaining paired tissues and analyzed using quantitative qPCR All compari‘sons between OSCC tissues and adjacent normal tissues were performed simultaneously The Kaplan‘Meier analysis of OS and survival curves were from the Cancer Genome Atlas database TCGA wwwcancergovThe study was approved by the Ethics Committee of China‘Japan Union Hospital of Jilin University Written informed consent was obtained from all patients who participated in this studyTranscriptome sequencing and functional annotation analysis Total RNA extraction RNA library construction and transcriptome sequencing were performed at Sangon Biotech Co Ltd The biological relevance of unique genes in expression profiles of DEGs were screened according to the threshold values of log2fold‘change‰¥ and P005 Then the differentially expressed mRNAs were analyzed by Gene Ontology GO whose annotations were downloaded from Gene Ontology httpgeneontology UniProt sparqluniprot and NCBI wwwncbinlmnihgov Significant GO categories were identified using Fisher's exact test with a P005 which indicated that signifi‘cantly upregulated genes in the set of DEGs were assigned to a specific functional category more often than expected by chance Significant pathways of the DEGs were then analyzed and identified according to the Kyoto Encyclopedia of Genes and Genomes KEGG database wwwkeggjpCell lines The human tongue squamous cell carcinoma SCC‘ and CAL‘ were obtained from the American Type Culture Collection CAL‘ cells were cultured in DMEM medium with fetal bovine serum FBS Gibco Thermo Fisher Scientific Inc Uml penicillin and streptomycin at ˚C in a humidified atmosphere containing CO2 SCC‘ cells were cultured in MEM medium with FBS NEAA Uml penicillin and streptomycin at ˚C in a humidified atmosphere containing CO2MCM5‘specific siRNA and transfection Three MCM5 siRNA sequences were synthesized by Suzhou GenePharma Co Ltd The sequences were as follows '‘' siRNA‘ Forward CCG ACU ACU UGU ACA AGC ATT and reverse UGC UUG UAC AAG UAG UCG GTT siRNA‘ forward CCA AAU GUC UAU GAG GUC ATT and reverse UGA CCU CAU AGA CAU UUG GTT siRNA‘ forward GUC GUC UGU AUU GAC GAG UTT and reverse ACU CGU CAA UAC AGA CGA CTT and scrambled forward UUC UCC GAA CGU GUC ACG UTT and reverse ACG UGA CAC GUU CGG AGA ATT The mock was an untransfected empty vector serving as the controlSCC‘ cells 45x104 cellswell were cultured in ‘well plates overnight at ˚C Then cells were transfected with nM negative control siRNA or MCM5 siRNA using Lipofectamine® Transfection Reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's protocol After h transfection cells were collected and then RNA was extracted by TRIzol® regent Invitrogen Thermo Fisher Scientific Inc for further experiments as indicatedReverse transcription RTqPCR RNA extracted from tissues samples were reverse transcribed into cDNA using a GoScript Reverse Transcription System kit Monad httpwwwmonad‘biotechcom according to the manufacturer's instructions Relative mRNA expressions were quantified by qPCR using the QuantiTect SYBR Green PCR kit Roche Diagnostics and normalized to GAPDH using primers listed in Table I The cycling parameters were cycles of ˚C for sec ˚C for sec and ˚C for sec Relative mRNA levels were assessed by the comparative ‘ΔΔCq method All analyses of the samples were conducted in triplicateFor association of MCM5 expression levels with clinico‘pathologic features of OSCC the relative expression levels of MCM5 were evaluated using qPCR as aforementioned Relative mRNA levels of paired samples adjacent vs cancer tissues were assessed by the comparative ‘ΔΔCq method A ratio was considered to have high MCM5 expression whereas a ratio ‰¤ was considered to have low MCM5 expression Cell proliferation assay To analyze cell proliferation a Cell Counting Kit CCK‘ Dojindo Molecular Technologies Inc was used according to the manufacturer's instructions In total cells were cultured to each well of ‘well plates After h post siRNA transfection cells were incubated for and h CCK‘ reagent was added h prior to detec‘tion The OD was measured at nm using a microplate reader Bio‘Tek The experiment was performed three timesColony formation assay This assay was performed according to a previous study Briefly cells were cultured in ‘well plates at cellswell followed by culture in complete medium DMEM supplemented with FBS Uml peni‘cillin and streptomycin for weeks The colonies were fixed with methanol for min at room temperature and washed with PBS and stained with crystal violet at room tempera‘ture Beyotime Institute of Biotechnology solution for min A cell colony was defied as a group formation of at least cells Finally formed colonies were observed and images were captured under a light microscope at magnification x200 Cell migration analysis using scratching assays SCC‘ cells were cultured in a ‘well plate at 5x105 cellswell overnight at ˚C Then the cells were scratched and scraped with fresh DMEM Cells were observed and images were captured under a light microscope magnification x200 at h The width of the scratch was measured and referred to as Wbefore Then the cells were starved with no FBS and returned to the incubator for h at ˚C The width of the same scratch was measured and referred to as Wafter Migrating distance was calculated by subtracting Wafter from Wbefore The migration of the control was set as Western blot analysis The protein extractions from the cells were isolated using RIPA Lysis Buffer P0013B Beyotime Institute of Biotechnology Then ‘ µg protein was loaded per lane on a gel resolved using SDS‘PAGE and electroblotted onto 0cONCOLOGY LETTERS Table I Primers used for reverse transcription quantitative‘PCRmRNA MCM5 P21 CyclinE CDK2 GAPDH MCM5 minichromosome maintenance protein cyclin‘dependent kinase Forward primer '‘' GATCCTGGCATTTTCTACAG GGAGACTCTCAGGGTCGAAA TTCTTGAGCAACACCCTCTTCTGCAGCC GCTAGCAGACTTTGGACTAGCCAG AGAAGGCTGGGGCTCATTTG Reverse primer '‘'CCCTGTATTTGAAGGTGAAGGGATTAGGGCTTCCTCTTGGTCGCCATATACCGGTCAAAGAAATCTTGTGCCAGCTCGGTACCACAGGGTCAAGGGGCCATCCACAGTCTTCFigure Volcano plots and KEGG pathway analysis of differentially expressed genes between OSCC cancer tissue group and adjacent normal tissue group A Differences in gene expression profiles between OSCC cancer tissue group and adjacent normal tissue group The horizontal line corresponds to a ‘fold log2 scaled change up or down and the vertical line represents P005 The red points on the plot represent the differentially expressed genes with a ‘fold change upregulation while the green points represent downregulation with P005 B Top KEGG enrichment terms of DEG in OSCC The vertical axis represents the pathway category and the horizontal axis represents the enrichment score [‘lgP‘value] of the pathway LgP was the logarithm of P‘value and P005 was considered significant KEGG Kyoto Encyclopedia of Genes and Genomes OSCC oral squamous cell carcinoma DEG different expressed genes OSCC oral squamous cell carcinomaPVDF membranes Roche Diagnostics After blocking at ˚C for h non‘fat milk in PBS plus Tween the blots were incubated with primary antibodies against anti‘MCM5 D220960‘ BBI Life Sciences anti‘p21 Proteintech anti‘cyclin E ProteinTech Group Inc and anti‘β‘actin D16H11 CST Biological Reagents Co Ltd at ˚C for h Western blots were probed with secondary antibodies and detected using the Odyssey infrared imaging system LI‘COR BiosciencesCell cycle analysis SCC‘ cells were harvested and fixed with ethanol on ice for min and then washed with PBS to decant the ethanol solution Then the cells were suspended and stained by PI and RNase A treatment Cell cycle analysis was performed using a flow cytometer FACSARIA…¡ BD Biosciences The data was performed using CXP Analysis software Beckman Coulter Inc Statistical analysis All the data analysis was performed using SPSS version SPSS Inc The results are presented as mean SD Associations between MCM5 mRNA expres‘sion and clinicopathological factors were analyzed using the Pearson's χ2 test or Fisher's exact test The differences in MCM5 mRNA expression between carcinoma and adjacent normal tissues were evaluated by a paired t‘test One‘way ANOVA followed by Tukey's post hoc test was used to determine the differences between groups and unpaired t‘tests for the rest of the data The survival rate was calculated by the Kaplan‘Meier method and compared using the log‘rank test P005 was considered to indicate a statistically significant difference All experiments were performed in triplicateResultsRNA sequencing and functional annotation analysis To explore novel biomarkers for OSCC the RNAs derived from tissue samples by sequencing were detected Three matched primary OSCC tissues and adjacent normal tissues were randomly selected As shown in Fig 1A the aberrant expression of genes was detected in tissue samples To screen 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMATable II Twenty randomly selected differentially expressed genes between oral squamous cell carcinoma and adjacent tissue samples The genes selected randomly instead of listing based on rank or fold‘change in expressionGene ID ENSG00000160182 ENSG00000205592 ENSG00000171195 ENSG00000126549 ENSG00000090382 ENSG00000161798 ENSG00000161055 ENSG00000107562 ENSG00000214711 ENSG00000106066 ENSG00000184330 ENSG00000137745 ENSG00000243207 ENSG00000107159 ENSG00000183072 ENSG00000196611 ENSG00000171217 ENSG00000178445 ENSG00000100297 ENSG00000127564 Log2 fold‘change ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ Gene nameTFF1MUC19MUC7STATHLYZAQP5SCGB3A1CXCL12CAPN14CPVLS100A7AMMP13PPAN‘P2RY11CA9NKX2‘MMP1CLDN20GLDCMCM5PKMYT1P‘value 881x10‘ 468x10‘ 144x10‘ 140x10‘ 209x10‘ 528x10‘ 959x10‘ 604x10‘ 601x10‘ 846x10‘ 234x10‘ 753x10‘ 222x10‘ 161x10‘ 380x10‘ 154x10‘ 705x10‘ 751x10‘ 565x10‘ 240x10‘ Result Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation the candidate biomarkers for diagnosing OSCC the DEGs were selected when changes in RNA expression were fold‘changes As shown in Fig 1A aberrant RNAs were significantly downregulated while RNAs were upregu‘lated In order to represent all differentially expressed genes twenty randomly selected dysregulated genes between OSCC and adjacent tissue samples are summarized in Table II The P‘value and log2 fold‘changes of all aberrant expression genes are shown in Table SI The DEGs were selected randomly instead of listing based on rank or fold‘change in expression To explore the role of differentially expressed RNAs in OSCC KEGG pathway analysis was performed Depending on the P‘value and enrichment signal pathways associ‘ated with OSCC were identified Table SII The top KEGG enrichment terms of DEGs are shown in Fig 1B including ˜cell cycle™ ˜pathways in cancer™ ˜cell cycle‘yeast™ ˜meiosis‘yeast™ and ˜cytokine‘cytokine receptor interac‘tion™ Among these it was reported that some genes such as MCM5 cell division cycle ‘related protein kinase and Cyclin‘dependent kinase homolog were primarily enriched in the ˜cell cycle™ pathway Some genes such as lysozyme C statherin and aquaporin‘ were enriched in the ˜saliva secretion™ pathway MCM5 which participated in cell cycle regulation and had high expression in OSCC was selected for further study and it was hypothesized that MCM5 might be a candidate tumor marker for OSCCValidation of MCM5 using RT‘qPCR To further verify the aforementioned expression profile data MCM5 expression levels were investigated using RT‘qPCR in tumor and Figure Relative expression levels of MCM5 mRNA in paired adjacent normal tissues and oral squamous cell carcinoma tissues using quantita‘tive PCR The relative expression data were analyzed by the ‘ΔΔCq method GAPDH was used as an internal control P005 P001 vs adjacent normal tissue MCM5 minichromosome maintenance proteinadjacent normal tissues As shown in Fig MCM5 mRNA was significantly upregulated in of tumor tissues compared with that in matched normal tissues These results showed that MCM5 was highly expressed in OSCC tissues which was in line with the sequencing data Association of MCM5 expression levels with clinicopathologic features of OSCC and survival analysis The results of the potential association between MCM5 expression and clinicopathological features in patients with OSCC are presented in Table III No significant association with MCM5 expression was found for age sex histological differentiation metastasisrecurrence and survival status P05 0cONCOLOGY LETTERS Value n MCM5 expression‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘Low n3 High n12 Table III Association between expression of MCM5 and clinicopathologic features of patients with oral squamous cell carcinoma Characteristic Age years ‰¥ Sex Male Female Histological differentiation Well and Moderate Poor MetastasisRecurrence Yes No Survival status Death Survival P‘valueA Kaplan‘Meier analysis of OS is shown in Fig Analysis of clinical data from TCGA showed that high MCM5 expres‘sion was no associated with a shorter OS in patients with OSCC log‘rank P062 These results suggested that MCM5 might not be a prognostic biomarker for OSCC Inhibitory effect of MCM5 in OSCC cell lines To determine the functional role of MCM5 first MCM5 expression was analyzed using RT‘qPCR in two OSCC cell lines Notably SCC‘ cells expressed significantly higher levels of MCM5 compared with Cal‘ cells P001 Fig 4A Considering that knockdown of MCM5 in the cell line with high MCM5 expres‘sion may bring about more significant changes the SCC‘ cell line was selected for further investigation of the functional role of MCM5 Three specific siRNA sequences were designed to inhibit MCM5 expression and transfected in SCC‘ cells and the impact on MCM5 expression was determined using RT‘qPCR As shown in Fig 4B siRNA1 siRNA2 and siRNA3 transfection decreased MCM5 expression by 651P001 P001 and P001 respectively compared with the negative control Then the efficiencies were confirmed using western blotting Fig 4C The inhibitory effect of siRNA1 and siRNA2 was significant but not found in siRNA3 The results were consistent with those of RNA expression siRNA1 trans‘fection reduced MCM5 expression significantly in SCC‘ cells Therefore siRNA1 was used for subsequent experiments Effect of MCM5 inhibition on proliferation colony formation and migration To determine whether MCM5 regulates cell cycle and modulates cell proliferation in OSCC the effect of inhibiting MCM5 expression on SCC‘ cell proliferation was investigated As shown in Fig 5A the results showed that downregulation of MCM5 had significant anti‘proliferative Figure Survival curves from The Cancer Genome Atlas datasets n518 containing high and low MCM5 expression levels MCM5 minichromosome maintenance protein HR hazard ratio TPM transcripts per millioneffect compared with the negative control P001 Colony formation assays were performed and the results revealed that compared with the number of colonies in the control group downregulation of MCM5 significantly reduced colony formation P001 Fig 5B and C To estimate the impact of MCM5 on OSCC migration scratching assays were conducted and inhibition of MCM5 showed no significant impact on the migration of SCC‘ cells P005 Fig 5D These results suggested that inhibiting MCM5 expression inhibited cell proliferation and colony formation but had no effect on migration in SCC‘ cells\x0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAFigure Inhibition of MCM5 expression in oral squamous cell lines A MCM5 expression in Cal‘ and SCC‘ cells B Efficiency of siRNA‘MCM5 was confirmed by reverse transcription quantitative‘PCR in SCC‘ cells C The efficiency of siRNA‘MCM5 was confirmed by western blot in SCC‘ cells MCM5 minichromosome maintenance protein si small interfering NC negative controlFigure Effects of MCM5 inhibition on antiproliferation colony formation and migration capacity in SCC‘ cells A Downregulation of MCM5 expression suppressed SCC‘ cell proliferation compared with the corresponding control at different time points B Downregulation of MCM5 expression inhibited SCC‘ cells colony formation compared with the corresponding control C Quantification of MCM5 inhibition on colony formation compared with the corresponding control D Cells scratching wounds observed by microscopy E Downregulation of MCM5 had no effect on the migration capacity of SCC‘ cells P001 vs NC MCM5 minichromosome maintenance protein si small interfering NC negative controlEffect of MCM5 inhibition on cell cycle To determine the potential mechanism for the observed proliferation inhibition of SCC‘ cells by MCM5 inhibition cell cycle analysis was performed using flow cytometry As shown in Fig 6A and B after MCM5 inhibition the number of cells were decreased in the G0G1 phase and the S phase but significantly increased in the G2M phase compared with the negative control These results indicated that MCM5 inhibi‘tion significantly induced G2M phase arrest compared with that in the control group\x0cONCOLOGY LETTERS Figure Effects of MCM5 inhibition on cell cycle regulation in SCC‘ cells A Flow cytometry assays were performed to analysis the cell cycle progres‘sion when SCC‘ cells transfected with siRNA‘MCM5 B The bar chart represented the percentage of cells in G0G1 S or G2M phase as indicated C Expression levels of cell cycle‘regulated genes detected by quantitative PCR and normalized to GAPDH D Expression levels of cell cycle‘regulated proteins determined using western blotting β‘actin was used as the loading control Data are presented as mean ± SD P005 P001 vs control group MCM5 minichromosome maintenance protein si small interfering NC negative control CDK2 cyclin‘dependent kinase To elucidate the mechanism underlying this effect the expressions levels of cyclin E cyclin‘dependent kinase CDK2 and p21 related to cell cycle arrest were deter‘mined using RT‘qPCR As shown in Fig 6D MCM5 inhibition decreased both cyclin E and CDK2 mRNA levels but increased the mRNA expression of p21 significantly Then cyclin E and p21 were selected to detect the protein levels using western blotting As shown in Fig 6C cyclin E levels decreased while p21 levels increased in MCM5‘downregulated SCC‘ cells which was consistent with the RT‘qPCR results DiscussionDespite notable progress in cancer research and treatment the survival rate of patients with OSCC has not significantly improved in the past few decades To date there are no effective tumor‘specific biomarkers for the early detection and prognosis prediction of OSCC Several studies have shown that DEGs serve an important role in the development of tumors in different cancer types ‘ However few studies have reported differentially expressed genes in OSCC The present screened genes that regulate the progression of oral cancer by gene expression profiling and found that genes were dysregulated of which DEGs were upregulated and were downregulated DEGs significantly affected GO terms and KEGG pathways MCM5 did not have one of the highest log2 fold‘change values and log10 qValues however MCM5 is one of the differentially expressed genes in cell cycle signaling pathway which was the most signifi‘cant enrichment of differentially expressed genes Therefore MCM5 which regulates the cell cycle was selected for further investigation However previously published studies on biomarkers in OSCC mainly focused on pathological studies The present study not only verified the over‘expression of MCM5 in OSCC but also confirmed using cell experiments that MCM5 affects cell proliferation by regulating the cell cycle MCM5 is a member of the MCM family of proteins and is a component of the starting complex for DNA synthesis MCM5 has been identified as a cell cycle biomarker of aber‘rant proliferation which is associated with the progression of various cancer types Previously MCM5 has been found to be overexpressed in numerous human malignancies such as esophageal thyroid and ovarian cancer For example increased MCM5 levels in urine sediment cells predicts the presence of bladder cancer Inhibition of transcription factor SOX‘ can inhibit the proliferation of skin melanocytes and MCM5 expression is significantly decreased following downregulation of SOX‘ Moreover high expression of MCM5 is associated with poor prog‘nosis and poor malignant status in patients with cervical adenocarcinoma It is well known that immunohistochemistry and western blot‘ting are necessary methods to evaluate protein expression 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAHowever due to a limited number of tissue samples the present study did not have enough samples for simultaneous qPCR western blotting and immunohistochemistry detection Therefore this is a limitation of the present study However the study did perform qPCR to evaluate the expression of MCM5 at the mRNA level The results demonstrated that of patients with OSCC have high MCM5 expression which is consistent with the results of the aforementioned studies indicating that high MCM5 expression may play an important role in the pathogenesis of OSCC In addition other members of the MCM family of proteins have also served as biological markers of dysplasia and malignancy such as glioma cervical colorectal breast prostate and lung cancer ‘ Therefore some researchers even suggested that changes in MCM5 expression may be a sign of cell cycle disorders It is worth noting that some researchers reported that the high expression of MCM family members may be closely related to tumorigenesis and prognosis For example MCM2 MCM4 and MCM6 are overexpressed in breast cancer of high histological grades MCM7 expression is a potent prognostic marker in non‘small cell lung cancer while MCM5 may be an independent adverse prognostic marker in lung squamous cell carcinoma It is well known that the cell cycle is related to cell proliferation signaling path‘ways In most tumors an increase in the expression level of genes encoding proteins that regulate cell proliferation is observed The abnormal expression of cell‘cycle‘related genes is associated with infinite proliferation of tumors and poor prognosis Thus far only Yu reported the relationship between MCM5 and OSCC The study reported that overexpression of MCM5 in patients with OSCC was significantly associated with tumor site tumor size positive lymph node metastasis later clinical stage higher histological grade deeper infiltration depth and peripheral nerve infiltration However in the present study association between high expression of MCM5 with survival metastasis and poor histologic differentiation was not observed A Kaplan‘Meier analysis of the overall survival rate was not significantly changed in patients with high MCM5 expression compared with patients with low MCM5 expression It was speculated that due to the small number of samples that there were large differences between individuals Therefore in future research a larger sample size should be used to clarify the relationship between high expression of MCM5 and prognosis of OSCC Little is known about the role and potential function of MCM5 in OSCC In the present study a loss‘of‘function analysis was conducted and it was demonstrated that MCM5 participated in regulating cell cycle and cell proliferation in OSCC cells In fact inhibiting the expression of MCM5 in SCC‘ cells resulted in the downregulation of cyclin E and CDK expression and upregulation of p21 expression which ultimately led to G2M phase arrest in oral cancer cells These results further verified that MCM5 is highly expressed in patients with OSCC which promotes the proliferation of OSCC cells and regulates cell cycle In addition it was observed that MCM5 was not only expressed in SCC‘ cells but also expressed in CAL‘ cells Fig 4A Notably according to the ATCC the MCM5 gene had no mutations in either of the two cell lines indicating that the two cell lines selected in this study have similar genetic backgrounds and could be used for the study of MCM5 cell functions Considering MCM5‘knockdown experiments in SCC‘ cells with high MCM5 expression received more significant results SCC‘ cells were selected for follow‘up studies However analyzing the functional effects of MCM5‘knockdown in CAL27 cell lines may provide more information In addition both SCC‘ and CAL‘ cells are transformed cell lines In future investigations untransformed cell lines for multiple comparisons should be used to clarify the role of MCM5 in OSCC Surgical resection is currently the main method to treat OSCC However considering the particularity of the oral struc‘ture surgical resection will lead to a huge impact on patients' quality of life Therefore it is important to find effective diagnostic biomarkers for early detection or to develop targeted drugs for OSCC In the present study it was reported that MCM5 is overexpressed in OSCC and that MCM5 can affect cell proliferation by regulating cell cycle Therefore the results suggested that MCM5 might be one of the important patho‘genic factors of OSCC and is expected to be used as a potential tumor marker for OSCC target drugs The specific mechanism of action of MCM5 is still worthy of further investigationOverall the present study evaluated differentially expressed genes using sequencing patterns in OSCC tumor tissues and further validated MCM5 upregulated expression in OSCC tissues By knocking down MCM5 expression in SCC‘ cells it was revealed that cell proliferation and colony formation was significantly inhibited by inducing G2M phase arrest The results also suggested that during this process cyclin E and cell cycle‘related gene expression levels were decreased while p21 was significantly upregulated Therefore MCM5 may modulate OSCC cell proliferation by regulating the cell cycle MCM5 is an important pathogenic factor and might have important role as a potential diagnostic marker or drug target for OSCCAcknowledgementsNot applicableFunding This study was funded by the National Natural Science Foundation of China grant no the Bethune Project of Jilin University of China grant no 2018B02 the Education Department of Jilin Province grant no JJKH20190074KJ and Department of Science and Technology of Jilin Province grant no 20190103086JH and 20200201398JC Availability of data and materials The datasets used andor analyzed during the present study are available from the corresponding author on reasonable request The other datasets generated andor analyzed during the current study are available in The Cancer Genome Atlas wwwcancergov Gene Ontology httpgeneon‘tology UniProt sparqluniprot NCBI wwwncbinlmnihgov and Kyoto Encyclopedia of Genes and Genomes wwwkeggjp databases\x0cONCOLOGY LETTERS Authors' contributions HW CZ and CL performed the experiments HW MH and XW analyzed the data MH and HW wrote the manuscript MH and XW designed and supervised

Thyroid_Cancer

" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ‚ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11“whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14“ cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classified into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217“The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in specific chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22“ ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and fibroblastgrowth factor receptor FGFR gene fusions30“ Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients benefit from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classification andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aflatoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver flukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difficulty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespecific survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Pacific Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11“ Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14“ Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is afirm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was first described in cHCCICC was not systematicallydescribed until when it was classified into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly defined areas type C mixedtype presents as intimate association without clear boundaries18 In another classification system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identifiable intermediatetransition between HCC and ICC type III fibrolamellar tumors resemblesthe fibrolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classificationis commonly used in which cHCCICC is classified into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a unified classification system greatly adds to the difficulty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdefined cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identified stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA amplifications are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in “of cases Recently some oncogenic genes were identified in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identified as a driver ofHCC45 VEGFA and CCND1FGF19 have also identified in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identified For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22“ and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately “ of cases22“ Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30“ Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in“ of ICC cases32 Fusions amplifications translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS “ is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC “ and ICC “ Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate filament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59“cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difficult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166“ Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efficacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L firstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5fluorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71“ This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current firstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onfirstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand fluoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the firstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe benefit of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] confidence intervalPFS months versus months p This effectiveness wasconfirmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the firstlinetreatment for advanced cholangiocarcinoma[CI] “ and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe firstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77“A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efficacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efficacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased benefit for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095“ Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI “ months CI “ months CI “ and months CI “respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as first line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir firstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the first US Food and Drug Administration FDAapproved firstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPacific have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the first generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have benefitedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1“ FGFR1“ PDGFR RET and KIT In August theFDA approved lenvatinib for firstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the firstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR “ TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade “ adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modifications Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can specifically inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1“IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPacificREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further confirmed the efficacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival benefit observed across all age subgroups and atolerable safety profile supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety profile apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic profile and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a firstinhuman phase I study NCT02508467 of fisogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123“ Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identified in recent years is the inhibitor of the fibroblast growth factorFGF signaling pathway which consists of members labeled FGF1“FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1“ FGF signals regulate cell proliferation in which FGFR2fusions occurred in “ of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the first and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on findings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may benefit from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPfizeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial IdentifierTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi

Thyroid_Cancer

"Immune checkpoint inhibitors ICIs can induce immunerelated adverse events irAEs includingthyroid dysfunction There are only a few reports on Graves™ disease induced by ICIs We report a case of newonsetGraves™ disease after the initiation of nivolumab therapy in a patient receiving gastric cancer treatmentCase presentation The patient was a 66yearold Japanese man who was administered nivolumab mg every weeks as a thirdline therapy for stage IVb gastric cancer His thyroid function was normal before the initiation ofnivolumab therapy However he developed thyrotoxicosis before the third administration of nivolumab Elevatedbilateral and diffuse uptake of radioactive tracer was observed in the 99mTcpertechnetate scintigraphyFurthermore the thyroidstimulating hormone receptor antibody TRAb and thyroidstimulating antibody TSAbtest results which were negative before the first administration of nivolumab were positive after starting thetherapy The patient was diagnosed with Graves™ disease and the treatment with methimazole and potassiumiodide restored thyroid functionConclusions This is the first complete report of a case of newonset Graves™ disease after starting nivolumabtherapy confirmed by diffusely increased thyroid uptake in scintigraphy and the positive conversion of antibodiesagainst thyroidstimulating hormone receptor It is important to perform thyroid scintigraphy and ultrasonographyto accurately diagnose and treat ICIinduced thyrotoxicosis because there are various cases in which Graves™ diseaseis developed with negative and positive TRAb titresKeywords Graves™ disease Nivolumab Thyrotoxicosis Immune checkpoint inhibitor 99mTcpertechnetatescintigraphy Thyroidstimulating hormone receptor antibodyBackgroundImmune checkpoint inhibitors ICIs such as cytotoxicTlymphocyteassociated protein CTLA4 programmed cell death protein1 PD1 and programmeddeath ligand PDL1 inhibitors have been widely usedas a standard cancer treatment during recent yearsimmunerelatedHowever occasionallycauseICIs Correspondence yhiroshinmsacjp1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo JapanFull list of author information is available at the end of the adverse events irAEs which affect different anssuch as the lung gastrointestinal tract liver nervous system skin and endocrine glands The endocrine irAEsinclude hypophysitis thyroid dysfunction adrenal insufficiency and type diabetes While endocrine irAEs dueto CTLA4 inhibitors such as ipilimumab and tremelimumab mainly include pituitary dysfunction those dueto PD1 inhibitors such as nivolumab and pembrolizumab are mainly related to thyroid dysfunction [“]The PD1 inhibitorinduced thyroid dysfunction oftenincludes hypothyroidism rather than hyperthyroidism [ ] Thyrotoxicosis following ICI therapy is caused The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYamada BMC Endocrine Disorders Page of spontaneouslyrecover withmostly by thyroiditis syndrome which has been reportedsubsequenttohypothyroidism in many cases[ ] HoweverGraves™ disease induced by ICI treatment has not beenextensively explored Here we present a case of Graves™disease shortly after the initiation of nivolumab therapyfor gastric cancerthecancerCase presentationA 66yearold man was diagnosed with stage IVbT4bN0M1 human epidermal growth factor receptor HER2positive gastricat Nippon MedicalSchool Chiba Hokusoh Hospital one and a half yearsbefore the onset of thyrotoxicosis After diagnosis hewas not referred for surgery because of liver metastasiswith a portal tumour thrombus rather the patient received cycles of first line chemotherapy with a combination of tegafurgimeraciloteracil S1 cisplatin andtrastuzumab However the patient presented with progressive disease assessed based on the computed tomography CT and oesophagogastroduodenoscopy OGDevaluations following the first line therapy Hence he received a second line chemotherapy with paclitaxel andramucirumab After cycles of the second line chemotherapy although there was a reduction in tumour sizeafter cycles the patient presented with progressivedisease as assessed by CT At this stage nivolumab mg every weeks was started The patient had anormal thyroid function before the first administrationHowever TSH suppression was observed before the second administration and thyrotoxicosis occurred beforethe third administration of the drug hence nivolumabtherapy was discontinued and the patient was referred toour departmentThe patient had complained of fatigue and shortnessof breath during exertion His height was cm bodyweight was kg heart rate was beats per minute and blood pressure was mmHg There wasno evidence of Graves™ orbitopathy or pretibial myxedema He and his family members had no history ofandTgAbantibody ngmL Thyroidthyroid diseases Thethyroidstimulating hormoneTSH free triiodothyronine FT3 and free thyroxineFT4 levels were μIUmL pgmL and ngdL respectively Table The titres of thyroidstimulating hormone receptor antibody TRAb andthyroidstimulating antibody TSAb were positive IUL and respectively whereas those of antithyroglobulinantithyroidperoxidase antibody TPOAb were negative IULrespectively The thyroglobulin Tgand IULlevel wasultrasonographyshowed slight goitre Fig 1a and rich blood flow in theparenchyma Fig 1b 99mTcpertechnetate scintigraphywhich was performed on the first consultation day of thepatient at our department showed elevated bilateraland diffuse uptake of the radioactive tracer Fig Wemeasured antithyroid autoantibodiesin preservedserum samples The titres of TRAb and TSAb werenegative before the first administration of nivolumabwhereas they were positive IUL and respectively before the second administration Thus we diagnosed his thyrotoxicosis as newonset Graves™ diseaseafter the initiation of nivolumab therapy The humanleukocyte antigen HLA typing of the patient showedthe following allelic variants A24022601 B510154 C01021502 DRB104051501 DQA1010203 DQB104010602 DPA10202 and DPB10501We treated the patient with methimazole MMI at adose of mgday and potassium iodide KI at a doseof mgday One month after the initiation of the therapy when the FT3 and FT4 levels of the patient werenormal we discontinued KI Gradually we reduced thedosage of MMI and the continued administration tillthe death of the patient of MMI at a dose of mg everyalternate day stabilised his thyroid function Fig Furthermore as nivolumab was found to be ineffectivebased on the CT and OGD evaluations the patient received irinotecan therapy However after cycles ofchemotherapy the patient was diagnosed with brain metastasis by magnetic resonance imaging MRIforTable TSH FT3 FT4 and Tg levels and TRAb and TSAb titres in our patientTSH μIUmLFT3 pgmLFT4 ngdLTRAb IULTSAb Tg ngmLDay of nivolumab administration NA NANA NANANANANADay first administration of nivolumab Day second administration of nivolumabThe normal range of the thyroid parameters is as follows TSH “ μIUmL FT3 “ pgmL FT4 “ ngdL TRAb IUL TSAb ‰ and Tg ‰ ngmL 0cYamada BMC Endocrine Disorders Page of Fig Thyroid ultrasonography of the patient a Slight swelling in isthmus b Rich blood flow in parenchymawhich he received gamma knife and steroid therapyThe patient died months after his first visit to ourdepartmentDiscussion and conclusionsWe present a case of newonset Graves™ disease after theinitiation of nivolumab therapy in a patient receivinggastric cancer treatment Thyrotoxicosisinduced byICIs is mainly a form of destructive thyroiditis Threecases of newonset Graves™ disease during nivolumabtherapy other than the present case have been reported[“] Table Iadarola [] reported a case ofGraves™ diseaselike hyperthyroidism after the second administration of nivolumab in a patient with left lungIn this case 99mTcpertechnetate scintigcarcinomaraphy in the patient with T3toxicosis showed diffusethyroid uptake of the radionuclide suggesting Graves™diseaselike hyperthyroidism whereas the TRAb testswere consistently negative Thyroid ultrasonographyshowed a multinodular goitre with a normoechoic pattern and normal vascularity ofthe parenchyma []Brancatella [] reported a case similar to that ofIadarola [] with diffuse thyroid uptake and negative TRAb titre In this case ultrasonography showed anenlargement of the thyroid with a hypoechoic patternand mild hypervascularity Kurihara [] reported acase of simultaneous development of Graves™ disease andtype diabetes mellitus during nivolumab therapy InFig 99mTcpertechnetate scintigraphy showing elevated bilateral and diffuse uptake of the radioactive tracer 0cYamada BMC Endocrine Disorders Page of Fig Clinical course of the patient MMI methimazole KI potassium iodide Day first administration of nivolumab Day secondadministration of nivolumabTable Comparison of case reports on newonset Graves™ disease during nivolumab therapyStudyTSHμIUmL FT3pgmLFT4ngdLTRAb IULBeforeNAIadarola []AfterNegativeTSAb BeforeNANAAfterNANANAUSNormalHypervascularNormalRAIU99mTcuptakeHigh99mTcHighRAIUNAHLANANADRB104Brancatella []NANegativeKurihara []NAPositive NAYamada presentcaseUS ultrasonography RAIU radioactive iodine uptake Before before the initiation of nivolumab therapy After after the initiation of nivolumab therapy at theonset of the thyrotoxicosisNegative NegativeHypervascular PositivePositiveHigh99mTcDPB105 0cYamada BMC Endocrine Disorders Page of this case thyrotoxicosis was detected after the sixth administration of nivolumab with positive TRAb titreHowever ultrasonography showed no enlargement ofthe thyroid and a normal vascularisation pattern Thispatient was clinically diagnosed as mild Graves™ diseaseand treated with MMI [] Unlike these cases our caseis important in terms of confirmation of both positiveTRAb titre and diffuse thyroid uptake in scintigraphyMoreover titres of TRAb and TSAb were convertedfrom negative to positive after starting nivolumab therapy It seems reasonable to presume that Graves™ diseasewas induced by nivolumab although there is a possibilityof coincidence Furthermore our patient had HLADPB10501 which has been reported to be associatedwith Japanese Graves™ disease [ ] Although the involvement of HLA cannot be argued based only on asingle case accumulating similar cases might help clarifythe mechanism of development of rare ICIinducedGraves™ diseaseGraves™ disease induced by ICIs other than nivolumabhas been rarely reported Azmat [] reported aipilimumabinduced thyrotoxicosis caused bycase ofGraves™ disease Gan et al[] reported a case oftremelimumabinduced Graves™ hyperthyroidism Yajima [] reported a case of Graves™ disease induced bypembrolizumab a PD1 inhibitor In this case TRAbwas positive after the fifth administration of pembrolizumab and thyroid ultrasonography showed a mild increase in the intrathyroidal blood flow A thyroidscintigraphy was not performed because of the iodinetreatment [] The cases of nivolumabinduced Graves™disease with negative TRAb titre suggest that performingthyroid scintigraphy and ultrasonography can help to accurately diagnose and treat ICIinduced thyrotoxicosisA relationship between thyroid antibodies and PD1inhibitorinduced thyroid dysfunction has not been explained Kimbara [] suggested that patients withpreexisting TgAb and an elevated TSH level at baselineare at a higher risk of thyroid dysfunction induced bynivolumab Osorio [] reported an association between positive thyroid antibodies antithyroglobulin orantimicrosomal antibodies and thyroid dysfunction induced by ICIs In the studies on newonset Graves™ disease during nivolumab therapy it is interesting to notein two caucasian patients with Graves™ diseasethatTRAb was negative [ ] Furthermore TRAb waspositive in two Japanese patients including our patient[] Table However additional evidence is requiredto reveal the role of TRAb in the pathogenesis of ICIinduced hyperthyroidismA limitation of our case was radioactive iodine uptakeRAIU was not performed Because imaging with99mTcpertechnetate reflects both blood flow and uptakevia the symporter and does not assess anificationmalignant nodules may appear hyperfunctioning in pertechnetate imaging but hypofunctioning in 123IimagingIn our study tumours were not detected although a partof 99mTc uptake was strongerIn conclusion we reported a case of Graves™ diseaseshortly after the initiation of nivolumab therapy for gastric cancer Our case presented a typical Graves™ diseasewith both positive TRAb titre and diffuse thyroid uptakein scintigraphy Moreover our case is valuable in termsof confirming the conversion of TRAb and TSAb fromnegative to positive titres after starting the therapy It isimportant to perform thyroid scintigraphy and ultrasonography because there are cases of nivolumabinducedGraves™ disease with negative TRAb titre as previouslyreported To revealthe pathogenesis of ICIinducedGraves™ disease it is necessary to study additional casesof similar natureAbbreviationsCT Computed tomography CTLA4 Cytotoxic Tlymphocyteassociated protein FT3 Free triiodothyronine FT4 Free thyroxine HER2 Humanepidermal growth factor receptor HLA Human leukocyte antigenICI Immune checkpoint inhibitor IrAE Immunerelated adverse eventKI Potassium iodide MMI Methimazole MRI Magnetic resonance imagingOGD Oesophagogastroduodenoscopy PD1 Programmed cell deathprotein1 PDL1 Programmed death ligand RAIU Radioactive iodineuptake Tg Thyroglobulin TgAb Antithyroglobulin antibody TPOAb Antithyroidperoxidase antibody TRAb TSH receptor antibody TSAb Thyroidstimulating antibody TSH Thyroidstimulating hormoneAcknowledgmentsNot applicableAuthors™ contributionsHY FO and NE interpreted the data drafted the manuscript andparticipated in the endocrinological treatment of the patient HS revised themanuscript TO and SF participated in the gastroenterological treatment ofthe patient All authors have read and approved the final version of themanuscript for publicationFundingNot applicableAvailability of data and materialsThe data that support the findings of this study are stored in NipponMedical School Chiba Hokusoh Hospital Inzai Chiba and available from thecorresponding author on reasonable requestEthics approval and consent to participateThis case report was approved by the ethics committee of Nippon MedicalSchool Chiba Hokusoh HospitalConsent for publicationWritten informed consent was obtained from the patient™s next of kin forpublication of this case report and any accompanying images A copy of thewritten consent is available for review by the editor of this journalCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo Japan 2Department of Gastroenterology Nippon Medical SchoolChiba Hokusoh Hospital Kamagari Inzai Chiba Japan 0cYamada BMC Endocrine Disorders Page of Received April Accepted August ReferencesGonzalezRodriguez E RodriguezAbreu D Spanish Group for CancerImmunoBiotherapy GETICA Immune checkpoint inhibitors review andmanagement of endocrine adverse events Oncologist “ Michot JM Bigenwald C Champiat S Collins M Carbonnel F PostelVinay S Immunerelated adverse events with immune checkpoint blockade acomprehensive review Eur J Cancer “Bertrand A Kostine M Barnetche T Truchetet ME Schaeverbeke T Immunerelated adverse events associated with antiCTLA4 antibodies systematicreview and metaanalysis BMC Med Faje A Immunotherapy and hypophysitis clinical presentation treatmentand biologic insights Pituitary “Topalian SL Hodi FS Brahmer JR Gettinger SN Smith DC McDermott DF Safety activity and immune correlates of antiPD1 antibody in cancerN Engl J Med “Robert C Schachter J Long GV Arance A Grob JJ Mortier L et alPembrolizumab versus ipilimumab in advanced melanoma N Engl J Med“Orlov S Salari F Kashat L Walfish PG Induction of painless thyroiditis inpatients receiving programmed death receptor immunotherapy formetastatic malignancies J Clin Endocrinol Metab “Kimbara S Fujiwara Y Iwama S Ohashi K Kuchiba A Arima H et alAssociation of antithyroglobulin antibodies with the development ofthyroid dysfunction induced by nivolumab Cancer Sci “Iadarola C Croce L Quaquarini E Teragni C Pinto S Bernardo A et alNivolumab induced thyroid dysfunction Unusual clinical presentation andchallenging diagnosis Front Endocrinol Lausanne Brancatella A Viola N Brogioni S Montanelli L Sardella C Vitti P et alGraves' disease induced by immune checkpoint inhibitors a case reportand review of the literature Eur Thyroid J “Kurihara S Oikawa Y Nakajima R Satomura A Tanaka R Kagamu H et alSimultaneous development of Graves' disease and type diabetes duringantiprogrammed cell death1 therapy a case report J Diabetes Investig“ Dong RP Kimura A Okubo R Shinagawa H Tamai H Nishimura Y et alHLAA and DPB1 loci confer susceptibility to graves™ disease Hum Immunol“ Ueda S Oryoji D Yamamoto K Noh JY Okamura K Noda M et alIdentification of independent susceptible and protective HLA alleles inJapanese autoimmune thyroid disease and their epistasis J Clin EndocrinolMetab “ Azmat U Liebner D JoehlinPrice A Agrawal A Nabhan F Treatment ofipilimumab induced graves™ disease in a patient with metastatic melanomaCase Rep Endocrinol Gan EH Mitchell AL Plummer R Pearce S Perros P Tremelimumab inducedgraves hyperthyroidism Eur Thyroid J “ Yajima K Akise Y A case report of Graves' disease induced by the antihuman programmed cell death1 monoclonal antibody pembrolizumab ina bladder cancer patient Case Rep Endocrinol Osorio JC Ni A Chaft JE Pollina R Kasler MK Stephens D Antibodymediated thyroid dysfunction during Tcell checkpoint blockade in patientswith nonsmallcell lung cancer Ann Oncol “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Polyphenol induced improvements in glucose metabolism are associated with bile acid signaling to intestinal farnesoid X receptorKevin M Tveter1 Jose A Villa Rodriguez Alrick J Cabales1 Li Zhang2 Fiona G Bawagan1 Rocio M Duran1 Diana E Roopchand To cite Tveter a0KM Villa Rodriguez a0JA Cabales a0AJ et a0al Polyphenol induced improvements in glucose metabolism are associated with bile acid signaling to intestinal farnesoid X receptor BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 –º Additional material is published online only To view please visit the journal online http dx bmjdrc KMT and JAV R contributed equallyReceived March Revised June Accepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Food Science Rutgers The State University of New Jersey New Brunswick New Jersey USA2Key Laboratory of Genomic and Precision Medicine Beijing Institute of Genomics Chinese Academy of Sciences Beijing Branch Beijing ChinaCorrespondence toDr Diana E Roopchand roopchand sebs rutgers eduIntroduction Bile acid BA biotransformation by gut bacteria impacts BA profile and signaling to nuclear receptors such as the farnesoid X receptor FXR regulating glucose metabolism Altered BA FXR signaling was therefore investigated as a potential mechanism linking polyphenol induced gut bacterial changes and improved glucose metabolismResearch design and methods Diabetic dbdb were fed low fat diet LFD or LFD supplemented with a proanthocyanidin rich extract of grape polyphenols LFD GP for weeks Metabolic phenotypes serum BAs gut microbiota composition and gene expression markers relevant to gut barrier and glucose metabolism were assessed Gut anoids were used to investigate effects of individual BAs on ileal FXR activityResults Compared with LFD fed controls GP supplemented dbdb mice showed improved glucose metabolism decreased relative abundance of gut bacteria associated with production of secondary BAs SBAs and depleted serum levels of SBAs taurohyodeoxycholic acid THDCA ωmuricholic acid ωMCA and tauroωmuricholic acid TωMCA Serum levels of primary BAs PBAs increased consistent with higher gene expression of PBA synthesis enzyme Cyp7a1 GP induced BA changes associated with FXR inhibition as evidenced by reduced expression of FXR responsive genes Shp Fgf15 and Fabp6 in ileum tissue as well as hepatic Shp which negatively regulates PBA synthesis GP treatment did not affect expression of hepatic Fxr or expression of Abcb11 Slc51b and Obp2a genes controlling BA transport Ceramide biosynthesis genes Smpd3 Sptlc2 and Cers4 were decreased in liver and intestine suggesting lower tissue ceramides levels may contribute to improved glucose metabolism THDCA ωMCA and TωMCA behaved as FXR agonists in ileal anoid experiments therefore their depletion in serum of GP supplemented dbdb and wild type WT mice was consistent with FXR inhibitionConclusion These data suggest that by altering the gut microbiota GPs modify BA FXR signaling pathways to promote glucoregulationINTRODUCTIONDietary polyphenols in plant based foods contribute to improved glycemic control in Significance of this studyWhat is already known about this subject –º Dietary polyphenols such as proanthocyanidins alter the gut microbial community and are associated with improved metabolic resilience in humans and mice –º Bile acids BAs signal to farnesoid X receptor FXR a nuclear transcription factor that regulates hepatic BA synthesis and glucose metabolismWhat are the new findings –º Grape polyphenol GP supplementation decreased abundances of gut bacterial taxa associated with secondary BA SBA production concomitant with depletion of SBAs and increased primary BAs PBAs in murine serum –º GP supplementation suppressed expression of FXR regulated genes Fgf15 Fabp6 and Shp an inhibitor of PBA synthesis which was consistent with increased serum PBAs –º GP induced FXR inhibition was associated with decreased expression of genes required for biosynthesis of ceramides which impair glucose homeostasis –º The SBAs depleted in GP treated mice were revealed as FXR agonists in ileal gut anoidsHow might these results change the focus of research or clinical practice –º This study highlights BA FXR signaling pathways as an important mechanism for further investigation in human intervention studies of dietary polyphenols and metabolic healthmice and humans1“ Improved glucose metabolism in mice supplemented with berryfruit extracts was related to a proanthocyanidin PAC induced bloom of Akkermansia muciniphila1 a microbe shown to attenuate symptoms of metabolic syndrome MetS and type2 diabetes T2D in obese mice and humans5 We hypothesized that metabolic improvements also result from changes in BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismhost derived bacterial metabolites regulating host energy metabolismBile acids BAs are signaling molecules linking the gut microbiota to host energy metabolism Primary BAs PBAs synthesized in the liver are conjugated with taurine mice or glycine humans7 Bacterial derived bile salt hydrolases deconjugate taurine or glycine from the sterol core of PBAs followed by bacterial transformations such as αdehydroxylation dehydrogenation and epimerization which generate secondary BAs SBAs7 PBAs and SBAs signal to key regulators of energy metabolism such as nuclear transcription factor farnesoid X receptor FXR and Takeda G protein coupled receptor TGR57 Fxrˆ’ˆ’ mice were protected from high fat diet HFD induced obesity and had altered BA and gut microbiota higher Bacteroidetes less Firmicutes profiles compared with wild type WT mice8 Transfer of cecal microbiota from HFD fed Fxrˆ’ˆ’ mice to germ free WT mice resulted in less adiposity and improved glucose metabolism compared with mice that received microbiota from HFD fed WT mice suggesting the altered gut microbial and BA profiles in FXR deficient mice contributed to metabolic improvements8 Studies using tissue specific FXR knockout mice showed that intestinal FXR activity was required to mediate HFD induced metabolic dysfunctions9 Pharmacological inhibition of intestinal FXR using tempol antibiotics metformin or glycinemuricholic acid Gly MCA led to gut microbial remodeling and improved glucose and lipid homeostasis9“In addition to the A muciniphila bloom mice fed HFD supplemented with PAC rich grape polyphenols GPs showed other profound bacterial community changes raising the possibility that altered BA profile and signaling could contribute to observed improvement in glucose homeostasis1 The present study provides compelling evidence in support of this hypothesisRESEARCH DESIGN AND METHODSDietsA complex of grape polyphenols and soy protein isolate GP SPI was prepared as previously described1 Nutritional profiles for SPI and GP SPI are provided in online supplementary table Isocaloric ingredient matched diets Research Diets New Brunswick New Jersey USA used in this study were previously described4 Mice were fed low fat diet LFD containing SPI LFD formulated with GP SPI delivering GP LFD GP HFD containing SPI or HFD formulated with GP SPI delivering GP HFD GP Online supplementary table provides diet formulation detailsAnimalsFour week old dbdb mice B6BKSD LeprdbJ stock no Jackson Laboratory Bar Harbor Maine USA were single housed on a hour lightdark cycle to hours light with ad libitum access to LFD and water in a controlled temperature room °C±°C for week for acclimation Mice were randomized to receive LFD n7 or LFD GP n7 for days Metabolic phenotyping included food intake body weight body composition EchoMRI in1 system Echo Medical Systems Houston Texas USA and oral glucose tolerance tests as previously described1 On day mice were euthanized by CO2 inhalation followed by cardiac puncture and collection of tissues as previously described1 Fecal and cecal samples were collected for microbial community profiling Wild type C57BL6J mice n10 aged weeks were acclimated on LFD for week and randomly divided into two groups and fed HFD or HFD GP n5 per group for weeks after which mice were euthanized by CO2 inhalationTissue gene expression analysisRNA was extracted from ileum jejunum colon and liver “ mg with RNeasy Plus Universal Mini Kit QIAGEN followed by RNA cleanup Machery Nagel RNA purification kit RNA µg was reverse transcribed to cDNA and qPCR was performed using TaqMan primers online supplementary table as previously described4 Data were analyzed using 2ΔCT method using hydroxymethylbilane synthase HMBS as housekeeping geneqPCR of A muciniphila in fecal and cecal samples was performed as previously described1 Briefly gDNA extracted from fecalcecal samples was diluted to ngµL for quantification of A muciniphila abundance relative to total bacteria and archaea by qPCR using A muciniphila AM1 AM2 and universal primer U341F U515R sets1 16S rRNA gene sequencingGenomic DNA was extracted from fecal and cecal samples collected from dbdb mice Illumina protocols were used to prepare V4 amplicons of the 16S rRNA gene for sequencing on a MiSeq system resulting in × reads Denoising and clustering were conducted using DADA2 algorithm to differentiate sequences into amplicon sequence variants ASVs for downstream analysis using QIIME Details are available in online supplementary materialsSerum biochemistrySerum for BA analysis was collected by cardiac puncture BAs were analyzed on a Water™s Alliance e2695 HPLC system Waters Milford Massachusetts USA coupled to a Water™s Acquity QDA mass spectrometer equipped with an electrospray interphase ESI Waters Milford Massachusetts USA and quantified by external calibration curves using pure standards Details are provided in online supplementary materials Serum leptin polypeptide YY PYY interleukin6 IL6 and insulin were determined using a MILLIPLEX MAP Mouse Metabolic Hormone Magnetic Bead kit Millipore with a MagPix instrument Luminex as previously described4anoid experimentsIntestinal crypts were isolated from WT C57BL6J mouse ileum according to established methods16 Crypts were counted and added to Matrigel five crypts per µL BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cCorning growth factor reduced and µL was added per well in well plates and allowed to polymerize °C for “ min followed by addition of mL of complete growth medium CGM see online supplementary materials CGM without Y27632 dihydrochloride monohydrate was replaced every “ days anoids were passaged every “ days ratio Mature anoids days postpassage were treated in triplicate n3 wells with methanol vehicle chenodeoxycholic acid CDCA µM alone or CDCA plus another BA µM in CGM for hours RNA was extracted for qPCR analysis using TaqMan primers as described above4Statistical analysesAnalyses were conducted using Prism GraphPad Software La Jolla California USA Significant differences two groups were assessed with a two tailed unpaired Student™s t test with Welch correction for unequal variance when needed or by one way or two way analysis of variance groups followed by Sidak™s or Tukey™s multiple comparison test Statistical analysis of alpha and beta diversity metrics was calculated using QIIME details in online supplementary materials ADONIS and permutation analysis were conducted using R Studio V342 R Studio Software Boston Massachusetts USA and Python RESULTSGPs improve glucose metabolism in dbdb mice independent of obesityLeptin receptor deficient dbdb mice develop obesity gut barrier dysfunction and hyperglycemia independent of HFD feeding17 Compared with LFD fed controls dbdb mice fed LFD containing PAC rich GPs LFD GP for weeks showed significantly improved oral glucose tolerance figure 1A4 Area under the curve remained stable over time for the LFD GP group but increased in the LFD group figure 1A Mice fed LFD GP initially exhibited a transient decrease in food intake presumably due to taste but at later time points LFD and LFD GP groups consumed similar amounts of food ± and ± gdaymouse respectively p005 online supplementary figure 1A The LFD GP group consumed ± mg of GPs per day Both groups had similar body weight gain body composition and liver weights online supplementary figure 1B“DGPs promote a bloom in A muciniphila without improving markers of metabolic endotoxemiaCompared with controls GP supplemented dbdb mice had decreased αdiversity as evidenced by richness Shannon index and Faith™s phylogenetic diversity index figure 2A Principal coordinate analysis showed that GPs significantly altered fecal community structure within days online supplementary figure As previously observed1 GPs promoted increased cecal mass online supplementary figure Metabolism1E a phenotype common to antibiotic treated and germ free mice and consistent with reported antibacterial properties of PACs18 GP supplemented mice had an increased relative abundance of phylum Verrucomicrobia at the expense of Firmicutes in fecal days “ and ceca samples day figure 2BC online supplementary figure 3AB Ceca of GP supplemented mice had higher relative abundance of Bacteroidetes 25LFD although except for day fecal Bacteroidetes remained similar between groups figure 2B online supplementary figure 3C GP supplementation did not consistently alter levels of Proteobacteria or Actinobacteria figure 2B online supplementary figure 3DE Consistent with increased Verrucomicrobia quantitative qPCR analyses confirmed that GPs promoted a bloom in Akkermansia muciniphila figure 2C at the expense of other taxa figure 2DReduced abundance of A muciniphila and metabolic endotoxemia characterized by gut dysbiosis compromised gut barrier integrity lipopolysaccharide LPS leakage and intestinal inflammation was associated with impaired glucose metabolism in obese mice and humans5 Oral administration of A muciniphila in obese diabetic mice and humans resulted in improved glucose homoeostasis and attenuated metabolic endotoxemia through improved gut barrier integrity5 GP induced improvement in glucose metabolism in HFD fed mice has therefore been considered a consequence of the A muciniphila bloom leading to reduced inflammation and increased gut barrier integrity1 Reduced metabolic endotoxemia could not however explain the improved glucose tolerance in GP supplemented dbdb mice despite increased A muciniphila in feces day and cecum figure 2C Relative to control GP supplementation did not change intestinal gene expression of markers of inflammation Tnf Il6 iNOS gut barrier integrity Tjp1 Ocln Muc2 peripheral lipid deposition Fiaf or glucose transport Glut2 online supplementary figure 4A“C GP supplemented dbdb mice had less Muc3 expression in jejunum and ileum online supplementary figure 4AB suggesting lower mucus secretion There were no differences in serum insulin IL6 or glucoregulatory hormones PYY and leptin online supplementary table These data suggested that other mechanisms were driving improved glucose metabolismGPs reduced gut bacterial taxa associated with production of SBAsAlthough total bacterial and archaeal abundance was not significantly different between LFD and LFD GP groups figure 2E GPs induced profound gut microbial changes online supplementary figure that would be expected to alter BA diversity abundance and signaling Targeted liquid chromatography mass spectrometry LC MS analysis revealed that GP supplemented dbdb mice had higher serum concentrations of BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure GPs improve glycemic control in obese dbdb mice A Oral glucose tolerance tests were performed after mice consumed LFD squares n7 or LFD GP closed squares n7 for and weeks Blood glucose concentrations mgdL expressed as mean±SD were measured at the indicated time points “ min following administration of glucose gkg Between group difference was determined by unpaired two tailed t test with Welch™s correction p01 p001 B Scatter plot of blood glucose AUC determined for individual mice at weeks and where mean AUC±SD are shown as horizontal and vertical bars Difference was determined using two way ANOVA followed by Tukey™s intragroup posthoc test different letters indicate significant difference p005 or by Sidak™s intergroup posthoc test p001 ANOVA analysis of variance AUC area under the curve GP grape polyphenol LFD low fat dietPBAs driven by increased cholic acid CA and taurocholic acid TCA figure 3A and C online supplementary figure 6A Concentrations of muricholic acid MCA tauroMCA TMCA tauroαMCA TαMCA taurochenodeoxycholic acid TCDCA were similar between groups αMCA was not detected in LFD GP group figure 3A online supplementary figure 6A Increased PBA pool correlated with an overall reduction in SBAs figure 3C where ωMCA TωMCA and taurohyodeoxycholic acid THDCA were undetectable in dbdb mice fed LFD GP although deoxycholic acid DCA was increased figure 3A Total serum BAs were similar between groups figure 3BThe GP induced depletion of SBAs was unrelated to the leptin receptor mutation in dbdb mice Compared with HFD fed controls wild type C57BL6J mice fed HFD supplemented with GP HFD GP for weeks also showed serum depletion of SBAs THDCA ωMCA and TωMCA reduced overall levels of serum SBAs and no difference in concentration of total serum BAs online supplementary figures 6B and Unlike dbdb mice GP supplemented WT mice had decreased tauro deoxycholic acid TDCA and no significant difference in CA or overall PBA pool although TCA concentration was increased online supplementary figures 6B and In dbdb mice multiple correlation analyses were performed to associate GP induced changes in serum BAs figure to changes in fecalcecal gut bacteria online supplementary figure The GP induced increase of CA TCA PBAs and DCA SBA was positively and significantly associated with increased abundance of Akkermansia Blautia Clostridium ASV59 and S247 figure 3EF Blautia and Clostridium possess BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Supplementation of GPs remodeled gut microbial composition and diversity A Microbial αdiversity metrics of fecal samples collected from mice after consuming LFD squares n7 or LFD GP closed squares n7 for indicated number of days “ and of cecal Cec samples collected at endpoint day Difference was determined by two way ANOVA followed by Sidak™s test B Per cent relative abundance of main bacterial phyla based on Naïve Bayes taxonomic classifier non rarified data Phyla present at relative abundance were classified as ˜Other™ Percentage of C A muciniphila DNA and D nonA muciniphila DNA relative to total bacterial and archaeal DNA in fecal and cecal samples where relative abundance was determined by qPCR using primers specific for A muciniphila AM1AM2 and universal V4 primers 515F806R E Total 16S bacterial and archaeal gene countsng of gDNA extracted from fecal or cecal samples Group mean±SD at each time point are illustrated by horizontal and vertical lines Difference between diet groups over time panels C“E was determined using two way ANOVA followed by Sidak™s posthoc test intergroup comparison or Tukey™s posthoc test intragroup comparison Different letters a b and c indicate significant difference within diet groups p005 while the same letter indicates no difference Between group differences panels A C D E p005 p001 p0001 p00001 ANOVA analysis of variance ASV amplicon sequence variants AUC area under the curve GP grape polyphenol LFD low fat diet PD phylogenetic diversity7αdehydroxylating activity therefore taxa within these genera may be responsible for DCA production via dehydroxylation of CA21 GP treated mice had lower levels of taxa within the Clostridiales order ASV50 and Ruminococcaceae and Lachnospiraceae families ie ASV56 and and Clostridium genus ASV74 online supplementary figure which are reported to possess 7αdehydroxylating activity required for conversion of PBAs to SBAs21“ In agreement with evidence from mice and humans the GP induced decrease in these bacterial taxa encoding 7αdehydroxylation activity was highly correlated to the GP associated depletion of SBAs ωMCA and TωMCA figure 3EF21 Finally positive and significant associations were found between reduced αMCA TωMCA andor ωMCA and reductions in taxa belonging to RF39 Anaeroplasma Ruminococcus Butyricicoccus Dorea Dehalobacterium Christensenellaceae Lactococcus Streptococcus and Oscillospira figure 3EFGPassociated BA changes promote inhibition of FXR signaling and upregulation of classical BA synthesis pathwayTo investigate the consequences of GP induced serum BA changes gene expression of FXR TGR5 and their downstream targets were analyzed in tissues GP supplementation did not change ileal Fxr gene expression however FXR transcriptional activity was decreased as expression of its target genes fibroblast growth factor Fgf15 small heterodimer partner Shp and ileal BA binding protein I BABP gene Fabp6 were suppressed in ileum figure 4A Intestinal FXR signaling negatively regulates hepatic PBA synthesis through interaction of Fgf15 with hepatic fibroblast growth factor receptor 4Klotho receptor complex or by regulating BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure GPs increased PBAs and reduced SBAs in serum in association with depletion of bacterial ASVs related to SBA production A LC MS and pure standards were used to determine the mean concentration of individual PBAs and SBAs mean±SD in serum samples n7 samples collected from individual mice fed LFD white bars or LFD GP purple bars B Total serum BA concentration mean±SD was determined based on sum of individual PBA and SBA concentrations shown in panel A quantified for each mouse fed LFD n7 or LFD GP n7 C Serum PBA and SBA concentrations mean±SD in LFD versus LFD GP diet groups were calculated by summing the individual PBAs or SBAs shown in panel A D Using data from panels B and C pie charts illustrate pooled PBAs green and pooled SBA gray as a percentage of total serum BA concentration quantified for LFD and LFD GP groups For panels A“C significant difference was determined using unpaired two tailed t test followed by Welch™s correction p005 p001 Heatmap representation of the Spearman™s r correlation coefficient between serum BA profile and significantly changed bacterial ASVs at genera or family level of taxonomy in GP treated mice relative to control diet group from E day27 fecal samples or F day29 cecal samples Shades of red indicate serum BA and bacterial taxa are positively correlated to while shades of blue indicate a negative correlation to ˆ’ Significant positive or negative correlations are shown p005 p001 p0001 ASV amplicon sequence variant CA cholic acid DCA deoxycholic acid GP grape polyphenol LC MS liquid chromatography mass spectrometry LFD low fat diet MCA muricholic acid PBA primary bile acid SBA secondary bile acid TCA taurocholic acid TCDCA taurochenodeoxycholic acid TDCA tauro deoxycholic acid THCDA taurohyodeoxycholic acid TαMCA tauroαMCA TMCA tauroMCA TωMCA tauroωMCA TUDCA tauro ursodeoxycholic acidBMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Expression of genes involved in FXR signaling ceramide synthesis and glucose metabolism in response to GP supplementation Scatter plot of relative mRNA levels of indicated genes expressed in A ileum and B liver tissues collected from individual mice fed LFD squares or LFD GP closed squares Group mean±SD n7 samples group is illustrated by horizontal and vertical lines Data represent qPCR of technical duplicates analyzed by ˆ’ΔCT method Between group difference was determined by unpaired two tailed t test with or without Welch™s correction for unequal variance p005 p001 p0001 FXR farnesoid X receptor GP grape polyphenol LFD low fat dietthe hepatic expression and gene repressive function of Shp via liver receptor homologue1 and hepatocyte nuclear factor4α26“In agreement with reduced ileal Fgf15 hepatic Shp expression was significantly decreased figure 4B Fgf15 and Shp negatively regulate BA synthesis therefore their reduced expression was consistent with hepatic upregulation of cytochrome P450 family subfamily A member Cyp7a1 the rate limiting enzyme in the classical BA synthesis pathway7 and a trending increase in downstream enzyme Cyp8b1 p006 figure 4B Gene expressions of Cyp27a1 and Cyp7b1 online BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismsupplementary figure 4E involved in the alternative BA synthesis pathway were unaffected by GP treatment To determine if reduced hepatic Shp expression altered hepatic FXR activity we examined FXR target genes Abcb11 encoding bile salt export pump Bsep Slc51b encoding anic solute transporter Ost30 and Obp2a encoding lipocalin Lpn13 an acute phase protein32 Compared with control GP treatment did not affect hepatic expression of Fxr Abcb11 Slc51b or Obp2a figure 4B These data suggest that GP mediated effects on FXR activity are restricted to intestine resulting in suppression of ileal Fgf15 and Shp transcription and increased Cyp7a1 activity and PBA synthesis figure FXR activity regulates Tgr5 expression therefore we measured Tgr5 mRNA levels and downstream targets33 On activation by BAs TGR5 signals the release of incretin glucagon like peptide1 Glp1 to promote euglycemia33 Glp1 is produced when proglucagon protein Gcg is cleaved by prohormone convertase PC13 encoded by Pcsk134 GP supplemented mice showed reduced ileal expression of Tgr5 and no changes in Gcg or Pcsk1 figure 4A GPs did not change expression of Fxr Fgf15 or Tgr5 in colon tissue GPs induced an increase in colonic Gcg however Pcsk1 was unchanged indicating Glp1 levels were unaffected online supplementary figure 4D These data further support the idea that GPs inhibit ileal FXRGPmediated FXR inhibition is associated with downregulation of ceramide synthesis genes and improved markers of hepatic energy metabolismTissue accumulation of ceramides is linked to insulin resistance and diabetes which can be ameliorated by pharmacological or genetic inhibition of ceramide biosynthesis35 FXR activity positively upregulates genes required for ceramide synthesis in ileum which leads to impaired glucose metabolism and hepatic steatosis in mouse models of MetST2D10 Synthesis and accumulation of ceramides in liver contributes to hepatic insulin resistance steatohepatitis and metabolic disease35 We found that GP induced FXR inhibition was associated with lower expression of de novo ceramide synthesis genes specifically sphingomyelin phosphodiesterase Smpd3 in ileum figure 4A and serine palmitoyltransferase long chain base subunit Sptlc2 and ceramide synthase Cers4 in liver figure 4B Consistent with lower expression of ceramide biosynthesis genes GP supplemented mice showed improvements in markers of hepatic energy metabolism evidenced by lower hepatic expression of carbohydrate response element binding protein Chrebp a transcription factor that activates key enzymes of de novo lipogenesis38 glucose phosphatase G6Pase which catalyzes the final step in hepatic glucose production and Idh3a a subunit of the IDH3 isocitrate dehydrogenase heterotetramer complex that regulates fatty acid metabolism and whose inhibition is associated with hepatic glycogen synthesis figure 4B39 GP supplemented mice had reduced expression of Lbp encoding LPS binding protein suggesting decreased liver inflammation and insulin resistance40 Hepatic gene expression of gluconeogenesis enzyme phosphoenolpyruvate carboxykinase Pck1 and CEBP homologous protein Chop which are normally upregulated during hepatic endoplasmic reticulum stress41 were similar between groups online supplementary figure 4ESBAs depleted in GPsupplemented mice are FXR agonists that promote expression of ceramide synthesis genesThe majority of secreted BAs are reabsorbed in ileum and returned to the liver via the portal vein7 Cecum colon and feces have similar BA profiles while the serum BA profile is most closely related to that of ileum and portal vein as a minor fraction of reabsorbed BAs enter systemic circulation25 We sought to investigate how individual BAs altered by GP supplementation might affect ileal FXR signaling Gut anoids were cultured from ileal crypts isolated from WT mice figure 5A and treated with individual PBAs or SBAs that were differentially detected in serum of GP supplemented mice figure 5B To validate the system anoids were treated with CDCA a potent FXR agonist or CDCA in combination with the FXR antagonist TMCA As expected CDCA increased the expression of Fxr Fgf15 and Shp compared with vehicle treated anoids and this effect was attenuated by addition of TMCA figure 5B When anoids were treated with individual BA alone ie µM in the absence of CDCA activator FXR activity was unaffected as Fxr Fgf15 and Shp mRNA expression remained similar to untreated anoids online supplementary figure We therefore investigated individual BAs in the presence of FXR activator CDCA as previously reported11 to reveal agonistic or antagonistic effects on FXR signaling anoid cultures treated with CDCA in combination with ωMCA TωMCA THDCA or DCA increased CDCA induced expression of Fxr Fgf15 and Shp indicating that these SBAs are FXR agonists figure 5B In agreement with FXR agonistic activity anoids treated with CDCA in combination with TωMCA resulted in increased expression of Smpd3 Cers4 and Sptlc2 ceramide synthesis genes Cotreatment with CDCA and ωMCA THDCA or DCA only upregulated Sptlc2 figure 5B CA a PBA reported to be a weak FXR agonist and detected at higher concentration in GP supplemented dbdb but not WT mice increased CDCA induced activation of Fxr Fgf15 and Shp and increased expression of Cers4 and Sptlc243 TCA a PBA detected at higher concentration in GP supplemented dbdb and WT mice attenuated CDCA induced gene expression of Fxr and Fgf15 but not Shp and reduced CDCA induced gene expression of Cers4 figure 5B These anoid data suggest that GPs led to the depletion of FXR activators ωMCA TωMCA and THDCA and increase of an FXR antagonist TCA Consistent with in vivo data the net effect BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Ileal anoids treated with BAs revealed agonistic or antagonistic effects on FXR and ceramide pathway genes A Ileal crypts were isolated and cultured in Matrigel medium detailed in methods Spheroids representative day photo matured into anoids representative day photo B Scatter plot of relative mRNA levels of indicated genes expressed in anoids after hours of treatment with vehicle methanol black diamonds µM CDCA closed red circles a combination of µM CDCA and known FXR inhibitor TMCA µM a combination of µM CDCA and indicated PBA µM red circles a combination of µM CDCA and indicated SBA µM blue squares Data shown were combined from two independent experiments and for each experiment three wells containing mature anoids were treated with indicated BAs Data represent qPCR of technical duplicates analyzed by ˆ’ΔCT method Group mean±SD n6 wells total per treatment group is illustrated by horizont

Thyroid_Cancer

thyroid cancer THCAprognosis and construct a polygene risk prediction model for prognosis predictionand improvementMethods The HTSeqCounts data of THCA were accessed from TCGA databaseincluding cancer samples and normal tissue samples œedgeR package wasutilized to perform differential analysis and weighted gene coexpression networkanalysis WGCNA was applied to screen the differential coexpression genes associatedwith THCA tissue types Univariant Cox regression analysis was further used for theselection of survivalrelated genes Then LASSO regression model was constructed toanalyze the genes and an optimal prognostic model was developed as well as evaluatedby KaplanMeier and ROC curvesResults Three thousand two hundred seven differentially expressed genes DEGs wereobtained by differential analysis and coexpression genes COR P were gained after WGCNA analysis In addition eight genes significantly related to THCAsurvival were screened by univariant Cox regression analysis and an optimal prognostic3gene risk prediction model was constructed after genes were analyzed by the LASSOregression model Based on this model patients were grouped into the highrisk groupand lowrisk group KaplanMeier curve showed that patients in the lowrisk group hadmuch better survival than those in the highrisk group Moreover great accuracy of the3gene model was revealed by ROC curve and the remarkable correlation between themodel and patients™ prognosis was verified using the multivariant Cox regression analysisConclusion The prognostic 3gene model composed by GHR GPR125 and ATP2C2three genes can be used as an independent prognostic factor and has better predictionfor the survival of THCA patientsKeywords THCA WGCNA prognostic 3gene risk prediction model prediction prognosisINTRODUCTIONThyroid cancer THCA derived from parafollicular cells or thyroid follicular cells is the mostcommon endocrine malignancy accounting for about of all kinds of human cancers Papillary PTC follicular anaplastic and medullary thyroid carcinomas are the four subtypes ofTHCA among which papillary and follicular carcinomas are common and have better prognosisEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byTrevor Edmund AngellUniversity of Southern CaliforniaUnited StatesRoberto VitaUniversity of Messina ItalyCorrespondenceHaixing Fanghaixing01231163comSpecialty sectionThis was submitted toThyroid Endocrinologya section of the journalFrontiers in EndocrinologyReceived November Accepted June Published August CitationZhao H Zhang S Shao S and Fang H Identification of a Prognostic3Gene Risk Prediction Model forThyroid CancerFront Endocrinol 103389fendo202000510Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancer while anaplastic carcinoma is rare to be seen with extremelypoor prognosis Therefore it™s very important to find eï¬ectiveapproaches for the improvement of the overall THCA prognosisAt present the conventional prognostic model of THCA inclinical practice is constructed according to predictive factorslike age tumor size and lymph nodule metastasis Withthe development of highthroughput sequencing technologymRNA expression profiles of specific cancers are easy to obtainwhich helps us better find more robust prognostic signals For instance microarraybased gene expression analysis enablesus to identify the important genes during tumor progressionand helps to define and diagnose prognostic characteristics In this way many THCA prognostic biomarkers have beenverified However these markers are almost single genes and havenot been widely accepted Polygenic combination has beenreported to possess better predictive ability for cancer prognosisthan single genes Therefore recent studies have involvedin the identification of the biomarkers for THCA prognosis However restricted by research methods novel biologicalalgorithm needs to be explored to construct more accuratediagnostic or prognosis modelsIn the present study a large number of mRNA expressionprofiles of THCA patients were accessed from TCGA databaseand modules associated with THCA were identified by WGCNAA 3gene risk prediction model was constructed using Cox andLASSO regression models which could help us better predictTHCA prognosisMATERIALS AND METHODSData ResourceExpression profiles of THCA mRNA and corresponding clinicaldata were accessed from TCGA database cancergenomenihgovsamples and normaltissue samples The study was in line with the guidelinesreleased by TCGA httpcancergenomenihgovpublicationspublicationguidelinesincluding cancerIdentification and Confirmation ofTHCAAssociated GenesœedgeR packagebioconductorpackagesreleasebiochtmledgeRhtml was used to perform diï¬erential analysisbetween cancer tissues and normal tissues Genes met thecriteria logFC and P were considered to havesignificant diï¬erencesModule Selection With WGCNAThe mechanism of WGCNA is the research for coexpressionmodules and the exploration of the correlation between the genenetwork and the phenotypes which is motivated by the analysesof scalefree clustering and dynamic tree cut on expressionprofiles In the present study modules that were most relatedto THCA tissue types in the coexpression network constructedby WGCNA package cranrprojectwebpackagesWGCNAindexhtml were selected and genes meeting P and COR were extracted for further studyConstruction of the Prognostic RiskPrediction ModelTHCA prognosisassociated genes werescreened usingunivariant Cox regression analysis Then a prognostic modelwas constructed using the least absolute shrinkage and selectionoperator LASSO According to this model risk score of eachsample was calculated and patients were divided into thehighrisk group and lowrisk group with the median risk scoreas the threshold KaplanMeier was used to evaluate the survivalof the two groups The ROC curve was drawn for the evaluationof the prognosis performance of the model and the area underthe curve AUC was calculated Furthermore multivariantCox regression analysis was performed to assess the correlationbetween the risk score and patients™ prognosis KaplanMeierand ROC curves of each gene in this model were plotted to makea comparison with those curves of the modelStatistical AnalysisUnivariant and multivariant Cox regression analyses wereboth performed in TCGA dataset œglmnet package of theR software wwwrproject was used for LASSOstatistic algorithm IBM SPSS statistical software IBMCorp Armonk NY USA was applied for statistical analysis P was considered statistically significantRESULTSIdentification of THCAAssociatedModulesAs shown in A a total of DEGs were identifiedlogFC P WGCNA was used to screen THCArelated modules and appropriate adjacency matrix weightparameter power was selected to ensure the scalefreedistribution of the coexpression network as possible In therange of ‰ ‰ log k and log Pk were calculated for linearmodels™ construction respectively is the squared value of thecoefficient R As shown in B the soft threshold poweris higher with the elevated R2 suggesting that the network closelyapproaches to scalefree distribution In the present study R2 for the first time was selected to ensure the realizationof scalefree distribution as possible and make the values on thecurve approach to the minimum threshold When themean connectivity of RNA in the network was Cwhich was consistent with the smallworld network in the scalefree one Then cluster dendrogram was constructed Dand dynamic tree cut was performed deep split Modulesobtained were merged with the minimum size of and modules were eventually developedThe correlation and significance between the modulecharacteristics and sample phenotypes were calculated Amongthe modules genes in blue brown pink and turquoisemodules were verified to be most associated with THCAprognosis E THCA tissue typeassociated geneswere obtained from the four modules taking the P andCOR as the threshold FFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE Identification of the THCA tissue typeassociated RNA functional modules A Volcano plot of DEGs B Analysis of scaleindependence index for varioussoft threshold powers Horizontal axis is the soft threshold power and vertical axis is the scalefree topology fitting indices R2 The red line refers to the standardcorresponding to the R2 of C Analysis of the mean connectivity under different soft threshold powers D Cluster dendrogram of all DEGs clustered based on adissimilarity measure E Distribution of average gene significance and errors in the modules associated with the progression of THCA F Venn diagram of the genesin the four modules for coexpression genes selectionFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerConstruction of a Prognostic 3Gene RiskPrediction Model for THCAUnivariant Cox regression was performed for analysis ofthe coexpression genes suggesting that eight genes weresignificantly correlated with survival as shown in Table LASSO regression model was constructed to analyze thegenes and an optimal prognostic risk prediction modelwasScore — GHR —GPR125 — Atp2c2 Risk prediction wasperformed according to this model and patients were rangedFigure 2A RiskeventuallydevelopedTABLE Basic information of the eight prognostic genesidHRHR95LHR95HPvaluebased on the risk scores Figure 2B The median risk score wasused as the critical value to group the patients into the highriskgroup n and lowrisk group n As shown inthe KaplanMeier curve in Figure 2C patients in the highriskgroup had worse overall survival OS than those in the lowriskgroup ROC curve was plotted to predict the 3year survival andthe results showed in Figure 2D revealed that AUC of the 3genemodel was which indicated the good performance of therisk score in survival prediction Multivariant Cox proportionalhazards regression analysis was then performed combined withclinical factors and the correlation between the risk score andprognosis of patients was verified Figure 2E From the heatmaps of the expression profiles of these three genes Figure 2Fthe expression levels of GHR GPR125 and Atp2c2 were found tobe positively correlated with the risk score and all of them wereregarded as highrisk genesAtp2c2GPR125GHRCLMNCYTH3PLA2R1RYR2C8orf88Evaluation of the 3Gene Risk PredictionModelKaplanMeier curves of the three genes were drawn using thelog rank test As shown in Figures 3A“C THCA patients withlow expression of GHR GPR125 and Atp2c2 had longer survivaltime indicting that these three genes were highrisk genes whichwas in agreement with the results predicted by univariant Coxregression analysis Furthermore ROC curves Figures 3D“FFIGURE Construction of a 3gene risk prediction model A LASSO regression model B 3gene based distribution of risk scores C Survival analysis of realhub genes in the TCGATHCA dataset D ROC curve of real hub genes in the TCGATHCA dataset E The correlation between the risk score and patients™prognosis F Heatmap of the genes expression profilesFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE The evaluation of the 3gene risk prediction model A“C Survival analyses of GHR GPR125 and Atp2c2 in the TCGATHCA dataset D“F ROCanalyses of GHR GPR125 and Atp2c2 in the TCGATHCA datasetrevealed that the AUC of GHR GPR125 and Atp2c2 was and respectively all of which were smaller than thatof the 3gene risk prediction model Findings above demonstratethat risk score is a good indicator for prognosis and the 3genemodel has a higher accuracyDISCUSSIONWith the development of the microarray and RNA sequencingtechnologies new era of large data on biology is coming It hasbeen reported that microarraybased gene expression analysiscould achieve characterization in human cancers identificationof the important genes during tumorigenesis and the definitionas well as the diagnosis of prognostic features However therole of genes as prognosis factors has been few investigated In the present study a large amount of RNAseq profiles andclinical prognosis data of THCA patients were accessed fromTCGA database and coexpression gene modules were screenedusing WGCNA Studies have shown that gene modules are muchreliable in cancer prognosis than biomarkers While there arefew studies on the crosstalk among the modules and someimportant modules might be ignored Therefore in ourstudy gene coexpression network was constructed via WGCNAand was used to identify THCA tissue typeassociated genemodules including blue brown pink and turquoise Twentythree common genes were obtained from the four modulesand an optimal prognostic 3gene risk prediction model wasthen constructed by univariant Cox and LASSO regressionanalyses Along with the LASSO model all independent variablescan be processed simultaneously verifying the more accurateperformance than the stepwise regression model GHRGPR125 and Atp2C2 were the three genes in this model GHR isa kind of proteincoding gene coding transmembrane receptorsof the growth hormone In prior studies GHR has been verifiedto be a oncogene in some cancers such as breast cancer pancreatic ductal carcinoma and melanoma but therole in THCA prognosis is firstly reported GPR125 a 57KDafactor for transmembrane signal transduction is considered toplay a key role in cell adhesion and signal transduction It™sreported that GPR125 is upregulated in human cerebral cancertissues and promotes cell adhesion as well as the formationof myelosarcoma In our study GHR and GPR125 wereverified as highrisk genes in THCA which was consistent withthe previous studies Moreover we found that these two genescould be used as independent risk predictive factors but theaccuracy was lower than that of the 3gene risk prediction modelwhich was further verified by ROC and KaplanMeier curvesAs the expression profiles of THCA and clinical informationare just from one dataset of TCGA the samples for analyzingthe prognostic 3gene model are limited In addition the modelconstructed in this study might be not available when it comesto other databases and it™s necessary to improve the model withFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancermore datasets In a word a 3gene model is constructed to be anindependent predictor in this study which provides novel viewand approach for the prognosis of THCA patientsDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in thesupplementary materialAUTHOR CONTRIBUTIONSHZ contributed to the study design and gave the finalapproval of the version to be submitted SZ conducted theliterature search and performed data analysis and draftedSS acquired the data and revised the HF wrote the All authors contributed to the and approved thesubmitted versionREFERENCES Hedayati M Zarif Yeganeh M Sheikholeslami S Afsari F Diversityof mutations in the RET protooncogene and its oncogenic mechanismin medullary thyroid cancer Crit Rev Clin Lab Sci “ Carling T Udelsman R Thyroid cancer Annu Rev Med “ 101146annurevmed061512105739 Dralle H Machens A Basa J Fatourechi V Franceschi S Hay IDet al Follicular cellderived thyroid cancer Nat Rev Dis Primers 101038nrdp201577 SmallridgeRCCoplandand emergingAnaplasticJAtherapies Clin Oncolthyroidcarcinoma “pathogenesis 101016jclon201003013 Shaha AR Implications of prognostic factors and risk groups in themanagement of diï¬erentiated thyroid cancer Laryngoscope “ Zhao QJ Zhang J Xu L Liu FF Identification of a fivelong noncoding RNA signature to improve the prognosis prediction for patientswith hepatocellular carcinoma World J Gastroenterol “ 103748wjgv24i303426et Hebrant A Dom G Dewaele M Andry G Tr©sallet C LeteurtreEthyroidcarcinoma molecular anatomy of a killing switch PLoS ONE 7e37807 101371journalpone0037807al mRNA expression in papillaryand anaplastic Brennan K Holsinger C Dosiou C Sunwoo JB Akatsu H Haile R et alDevelopment of prognostic signatures for intermediaterisk papillary thyroidcancer BMC Cancer 101186s1288501627716 ZuoS Dai G Ren XIdentification ofa6genepredicting prognosis 101186s1293501807247for colorectal cancer Cancer CellsignatureInt Cui ZJ Zhou XH Zhang HY DNA methylation module networkcancer Genestyping ofbased prognosisand molecular 103390genes10080571 Gu JX Zhang X Miao RC Xiang XH Fu YN Zhang JY et alrecurrencefreein hepatocellular carcinoma World J GastroenterolSixlongsurvival“ 103748wjgv25i2220noncodingsignaturepredictsRNA Arumugam A Subramani R Nandy SB Terreros D Dwivedi AK Saltzstein Eet al Silencing growth hormone receptor inhibits estrogen receptor negativebreast cancer through ATPbinding cassette subfamily G member Exp MolMed 101038s1227601801978 Subramani R LopezValdez R Salcido A Boopalan T Arumugam A NandyS et al Growth hormone receptor inhibition decreases the growth andmetastasis of pancreatic ductal adenocarcinoma Exp Mol Med 46e117 101038emm201461 Proudfoot NJ Gil A Whitelaw E Studies on messenger RNA ™ endformation in globin genes a transcriptional interference model for globin geneswitching Prog Clin Biol Res “ Wu Y Chen W Gong L Ke C Wang H Cai Y Elevated Gprotein receptor GPR125 expression predicts good outcomes in colorectal cancer andinhibits Wntbetacatenin signaling pathway Med Sci Monit “ 1012659MSM910105 Pickering C Hgglund M SzmydyngerChodobska J Marques F Palha JAWaller L et al The adhesion GPCR GPR125 is specifically expressed in thechoroid plexus and is upregulated following brain injury BMC Neurosci Fu JF Yen TH Chen Y Huang YJ Hsu CL Liang DC et alInvolvement of Gpr125 in the myeloid sarcoma formation inducedby cooperating MLLAF10OMLZ and oncogenic KRAS in a mousebone marrow transplantation model “ 101002ijc28195J CancerInt Li X Dai D Wang H Wu B Wang R Identification of prognostic signaturesassociated with longterm overall survival of thyroid cancer patients basedon a competing endogenous RNA network Genomics “ 101016jygeno201907005 Li J Yu X Liu Q Ou S Li K Kong Y et al Screening of importantadenocarcinomalncRNAsbased on integrated bioinformatics analysis Mol Med Rep“ 103892mmr201910061associated with the prognosis oflung Tavares C Melo M CameselleTeijeiro JM Soares P Sobrinhothyroid cancer 174R117“ 101530EJESimoes M Endocrine tumours genetic predictors ofoutcome EurJ EndocrinolConflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Zhao Zhang Shao and Fang This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer

High burden of depression among cancerpatients on chemotherapy in University ofGondar comprehensive hospital and FelegeHiwot referral hospital Northwest EthiopiaAdhanom Gebreegziabher BarakiID1 Getahun Mengistu Tessema2 EyayawAdisu Demeke3 Department of Epidemiology and Biostatistics College of Medicine and Health Sciences Institute of PublicHealth University of Gondar Gondar Ethiopia Department of Internal Medicine College of Medicine andHealth Sciences School of Medicine University of Gondar Gondar Ethiopia Department ofPhysiotherapy Bahirdar University Bahirdar Ethiopia adsh04gmailcomAbstracta1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Baraki AG Tessema GM Demeke EA High burden of depression among cancerpatients on chemotherapy in University of Gondarcomprehensive hospital and Felege Hiwot referralhospital Northwest Ethiopia e0237837 101371journalpone0237837Editor Nu¨lu¨fer Erbil Ordu University TURKEYReceived September Accepted August Published August Copyright Baraki This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data containspotentially identifying characteristics as well assensitive patient information eg HIV statusTherefore please send all data requests to theDirector of School of Medicine Dr MezgebuSilamsew at msilamsawgmailcom orPostgraduate committee Mr Getasew Amare atgetasewa23gmailcomFunding The authors received no specificfunding for this workIntroductionCancer the most stressful event a person may experience often triggers depressionDepression among these groups of people in turn affects chemotherapy adherence lengthof hospitalization quality of life and cancer treatment outcome Even though the problem isenormous studies that address it are limited Therefore this study was conducted to determine the prevalence of depression and associated factors among cancer patients on chemotherapy in FelegeHiwot referral hospital and University of Gondar referral hospitalNorthwest EthiopiaMethodsAn institutionbased crosssectional study was conducted from April to May A total of cancer patients on chemotherapy were included Depression was assessed using thepatient health questionnaire PHQ9 Binary logistic regression was used to select variablesand determine Crude Odds Ratio COR Variables with P value were entered into multivariable logistic regression Adjusted Odds Ratio AOR with confidence intervals forvariables with Pvalue was estimated to show factors affecting depression amongcancer patients The fitness of the model was checked by using the HosmerLemeshowgoodnessoffit testResultsThe prevalence of depression among cancer patients on chemotherapy was CI Educational status of college and above AOR CI Jobless AOR CI UnderweightAOR CI PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaCompeting interests The authors have declaredthat no competing interests existAbbreviations AOR Adjusted Odds Ratio CICrude Odds Ratio COR Crude Odds Ratio PHQPatient Health Questionnaire SD StandardDeviation UoGCSH University of GondarComprehensive Specialized Hospitalchemotherapy duration � months or more AOR CI were notablyassociated with depressionConclusionThe burden of depression among cancer patients in this study was high We recommendconcerned bodies working to curve the problem to intervene based on the identified risk factors Improving educational status reducing work stress and maintaining normal weightwould reduce depressionIntroductionThe global burden of cancer has risen to million new cases and million deaths in Worldwide the total number of people who are alive within years of a cancer diagnosis isestimated to be million []Depression is a common mental disorder characterized by persistent sadness and a loss ofinterest in activities that one normally enjoys accompanied by an inability to carry out dailyactivities for at least two weeks More than million people are now living with depressionan increase of more than between and [] The national prevalence of depression among the general population in Ethiopia was []Cancer the most stressful event that a person may experience often triggers depression [] The prevalence of depression among cancer exceeds that observed in the general population [] and it ranges from to [ “] Depression among cancer patientsaffects treatment since they have to take medications for both cancer and depression [] affectacceptance of adjuvant cancer treatment [] adherence [] extend hospitalization reducesthe quality of life [ ] and increases the risk of suicide [] Depression also predicts cancerprogression and mortality [ ]Several factors affect depression among cancer patients these include age sex marital status educational status occupation pain type of cancer phase of treatment [ ] andsocial support [ ]Even though routine screening of distress is recommended internationally for good cancercare [] less emphasis is given in the study area and most of the care focuses on cancer Studies on the magnitude and the contributing factors are also limited Therefore this study wasconducted to fill this information gap by determining the prevalence of depression among cancer patients and factors affecting itMethodsStudy design and periodAn institutionbased crosssectional study was conducted among cancer patients from Aprilto May Study areaThis study was conducted on cancer patients who are getting treatment and have followed upat the oncology unit of the University of Gondar comprehensive specialized hospitalUoGCSH and FelegeHiwot referral hospital FHRH The two hospitals are found in theAmhara region northwest Ethiopia km and km away from the capital Addis AbabaPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiarespectively The oncology unit of UoGCSH currently has beds for the management of cancer patients whereas the oncology unit of FHRH has currently beds for inpatient treatmentof cancer patientsParticipantsThe source populations were all adult cancer patients visiting the oncology unit and treatedwith chemotherapy in these hospitals All adults with any type of cancer patients under chemotherapy treatment and follow up during the study period were included in the studySample size and sampling procedureA final sample size of was found by using single population proportion formula with population correction for total cancer patients of in the two hospitals using the followingassumptions the prevalence of depression Za2 for confidence interval andmargin of error of The final sample size was proportionally allocated to the two hospitals for UoGCSHand 176FHRH A systematic random sampling method was employed to select every 3rdpatients who were coming to the oncology unit during the data collection period and full fillthe inclusion criteria We had a plan to randomly select and replace the nonresponders butno study participant refused to participateVariablesThe dependent variable of depression was measured using the widely used Patient HealthQuestionnaire PHQ9 The Amharic Local language version of the scale has been validatedin Ethiopia sensitivity and specificity [] We have used a cutoff point of toclassify patients as having depression or notIndependent variables like Age sex marital status average monthly income educationallevel smoking habit alcoholic habit physical activity were collected by intervieweradministered questionnaire whereas variables like Body Mass Index Type of cancer clinicalstagetype of chemotherapy Duration of chemotherapy and comorbidities like Hypertension DMHIV and Anemia were collected from patient charts The smoking and Alcohol use habitswere assessed by asking the patients if they ever smoke cigarette or drink alcohol and socialsupport was evaluated using the Oslo item social support scale with scores ranging from to poor “ moderate “ and strong “ []Data collection procedure and quality assuranceThe data was collected by interviewing the participants using a structured pretested questionnaire and chart review The data was collected by three nurses working in each oncology unitData collectors were trained for one day about the objective of the study and ethical considerations Data collectors were supervised by the principal investigator Data was reviewed andchecked for completeness accuracy and consistency after each day of data collectionData processing and analysisData were entered into Epiinfo version and STATA version was used for analysis Frequencies and percentages were computed for all variables Data were presented in tables andgraphs Binary logistic regression was used to select variables and to determine Crude OddsRatio COR Variables with P value were entered into multivariable logistic regressionAdjusted Odds Ratio AOR with confidence intervals for variables with Pvalue PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiawas estimated to show factors affecting depression among cancer patients The fitness of themodel was checked by using the HosmerLemeshow goodnessoffit testEthics statementEthical approval to conduct the study was received from the University of Gondar ethicalreview board School of Medicine Reference number SOM12372019 A permission letterwas received from the two hospitals To keep the privacy of participants™ name and other personal identifiers were not collected Consent to participate in the study was also orally takenfrom patients Patients with depression were also linked to the psychiatry clinicResultsSociodemographic characteristic of study participantsA total of patients participated in the study From the total respondents the majority ofthem were females were married and were housewivesRegarding to age distribution the mean and standard deviation of participant™s age was SD Table Behavioral and comorbidity characteristicsFour participants were declared that they were smoking cigarettes daily and were alcohol consumers Most of the participants were physically active Most ofthe participants were in normal weight category based on their BMI whereasTable Baseline characteristics of cancer patients in UoGCSH and FHRH FrequencyPercentageVariablesSexMarital statusMaleFemaleSingleMarriedDivorcedWidowedEducational levelOccupationNo educationPrimary educationSecondary educationCollege and aboveGovernment employeeMonthly Income ETBFarmerMerchantUnemployed ““ 101371journalpone0237837t001PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Behavioral factors and comorbidities among study participant at UoGCSH and FHRH VariablesSmoking statusNoYesAlcohol drinkingNoYesPhysical activityNoYesBody mass index “ComorbidityNoYesDiabetesNoYesHypertensionNoYesAnemiaNoYesHIVAIDSNoYesFrequencyPercentage101371journalpone0237837t002 and were underweight and overweight respectively Ninetytwo participants had additional comorbidity Table Type of cancer and treatmentrelated characteristicsBreast cancer was the commonest cancer Whereas cervical cancer colorectal cancer35 and lung cancer are ranked second to fourth Most of thepatients were diagnosed with the disease in the past six months prior to the studyRegarding the clinical stage of the disease the third stage accounts for patients Atotal of participants have taken chemotherapy for less than three months Table Prevalence of depression among cancer patientsIn this study patients had depression making the prevalence CI The prevalence of depression among male cancer patients was CI whereas it was CI among female patients From the totalpatients with depression and had moderate moderatelysevere and severe depression respectively The magnitude of depression has also shown thedifference among different types of cancer Fig PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Type of cancer and treatmentrelated characteristics of study participant at UoGCSH and FHRH VariablesFrequencyPercentageType of cancerBreast cancerLung cancerColorectal cancerGastric cancerCervical cancerHead and Neck cancerEsophageal cancerBlood cancerSkin cancerThyroid cancerBladder cancerLymphomaLiver cancerSarcomaTesticular cancerClinical stageStage Stage Stage Stage UnknownDuration since diagnosis months“ months monthsDuration since start of chemotherapy“ months“ months months101371journalpone0237837t003Factors associated with depression among cancer patientsIn bivariable logistic regression age sex marital status educational status occupation BMISocial support and duration of chemotherapy were found to have Pvalue 02subsequentlythese variables were subjected to multivariable analysis and educational level occupational status BMI status and duration of chemotherapy were statistically associated with depressionamong cancer patientsThe odds of depression among patients who attended college and above was significantlyreduced when compared to those with no education AOR CI Whencompared to government employees patients who are unemployed had less risk of depressionAOR CI Underweight patients had 239AOR CI times higher odds of depression as compared to those with normal body mass indexPatients who took chemotherapy for six months or more had AOR CI times higher odds of depression as compared to their counterparts Table PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaFig Prevalence of depression among cancer patients on chemotherapy in UoGCSH and FHRH northwest Ethiopia101371journalpone0237837g001DiscussionIn this study we have assessed the magnitude of depression among cancer patients and the factors affecting it We have found prevalence and occupation educational status bodymass index and duration of chemotherapy were found to be independent predictors ofdepressionThe magnitude of depression in this study was consistent with other studies conductedamong Chinese cancer patients [] whereas this figure was higher than a study conducted in Addis Ababa [] Iran [] and metaanalysis done by Krebber [] This discrepancy could be attributable to the difference in the study populations in terms of types ofcancer the tool used for screening or other sociodemographic variations and severity ofdepression consideredThe odds of depression was significantly reduced in patients who are unemployed whencompared to government employees This piece of evidence is supported by another multicenter study [] This could be related to workrelated stress which worsens feelings of inadequate control over one™s work frustrated hopes and expectations leading to depression []The odds of depression among patient who attended college and above was reduced whencompared to those who have no education This finding is supported by a study from China[] Atlanta [] and Greece [] The possible reason could be these patients may have a betterunderstanding of the disease and have early screening which increases their recovery A higherproportion of educated people are in the first or second clinical stage of cancer ascompared to of patients without educationUnderweight cancer patients had more than double odds of depression as compared tothose who have a normal body mass index This finding is supported by several single studies[ ] and systematic review and metaanalysis [] showing underweight people at higherrisk of depression This shows malnutrition has a significant role in the mental health of peopleand maintaining a healthy weight is essential to improve health in general and mental healthin particularEven though the chemotherapy duration has shown no significant association with depression in few pieces of literature [ ] The odds of depression among cancer patients whoPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Factors affecting depression among cancer patientsat UoGCSH and FHRH VariablesDepressionCOR CIAOR95 CIYesNoAge mean sd SexMarital statusMale Female Single Married Divorced Widowed Educational levelNo education Primary Secondary College OccupationGovernment Farmer Merchant Unemployed Body mass indexUnderweightNormalOver weight Social supportPoorModerateStrongDuration of chemotherapy� months months� Pvalue � � � �101371journalpone0237837t004took chemotherapy for more than six months was higher than their counterparts in this studyThis could be a side effect of chemotherapy [] or it could be also associated with the staggering cost of chemotherapy which makes these patients stress to buy it for an extended durationThis study assessed the frequently ignored aspect of cancer comorbidity depression Butthe study has some limitations as it was a crosssectional study The causeeffect relationshipsare not guaranteed in these studies therefore we recommend a prospective study Even thoughwe have used a validated tool some of the symptoms used in PHQ“ like weight loss andtiredness might be related to cancer itself and may overestimate depressionConclusionThe burden of depression among cancer patients in this study was high Occupation educational status body mass index and duration of chemotherapy were found to be independentlyassociated to depression We recommend concerned bodies working to curve the problem toPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiaintervene based on the identified risk factors Improving educational status reducing workstress and maintaining normal weight would reduce depression Clinicians shall also provideintegrated care of mental health and cancer treatmentAcknowledgmentsWe would like to thank the University of Gondar Oncology unit staff of FelegeHiwot referralhospital and University of Gondar comprehensive specialized hospitals and all data collectorsAuthor ContributionsConceptualization Adhanom Gebreegziabher Baraki Getahun Mengistu Tessema EyayawAdisu DemekeData curation Eyayaw Adisu DemekeFormal analysis Adhanom Gebreegziabher Baraki Eyayaw Adisu DemekeInvestigation Adhanom Gebreegziabher BarakiMethodology Adhanom Gebreegziabher Baraki Getahun Mengistu TessemaProject administration Eyayaw Adisu DemekeSoftware Adhanom Gebreegziabher BarakiValidation Getahun Mengistu TessemaVisualization Getahun Mengistu TessemaWriting “ original draft Adhanom Gebreegziabher BarakiWriting “ review editing Adhanom Gebreegziabher Baraki Getahun Mengistu TessemaEyayaw Adisu DemekeReferences Bray F Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA a cancer journal for clinicians p “ World Health anization Mental Health [cited August ] wwwwhointmental_healthmanagementdepressionen Hailemariam S The prevalence of depression and associated factors in Ethiopia findings fromthe National Health Survey International journal of mental health systems p 10118617524458623 PMID Nikbakhsh N Prevalence of depression and anxiety among cancer patients Caspian journal ofinternal medicine p “ PMID AlShakhli H Harcourt D and Kenealy J Psychological distress surrounding diagnosis of malignantand nonmalignant skin lesions at a pigmented lesion clinic Journal of Plastic Reconstructive Aesthetic Surgery p “ Sotelo JL Musselman D and Nemeroff C The biology of depression in cancer and the relationshipbetween depression and cancer progression Int Rev Psychiatry p “ PMID Krebber A Prevalence of depression in cancer patients a metaanalysis of diagnostic interviewsand selfreport instruments PsychoOncology p “ 101002pon PMID Hong JS and Tian J Prevalence of anxiety and depression and their risk factors in Chinese cancerpatients Supportive care in cancer p “ 101007s005200131997y PMID Mitchell AJ Prevalence of depression anxiety and adjustment disorder in oncological haematological and palliativecare settings a metaanalysis of interviewbased studies The lancet oncology p “ 101016S147020451170002X PMID PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaLinden W Anxiety and depression after cancer diagnosis prevalence rates by cancer type gender and age J Affect Disord “ p “ 101016jjad201203025PMID JimenezFonseca P Factors associated with anxiety and depression in cancer patients prior toinitiating adjuvant therapy Clin Transl Oncol p “ 101007s1209401818739 PMID Alemayehu M Deyessa N Medihin G Fekadu A A descriptive analysis of depression and pain complaints among patients with cancer in a low income country PloS one Colleoni M Depression and degree of acceptance of adjuvant cytotoxic drugs Lancet p “ 101016S014067360002821X PMID Pitman A Depression and anxiety in patients with cancer Bmj p k1415 101136bmjk1415 PMID Prieto JM Psychiatric morbidity and impact on hospital length of stay among hematologic cancerpatients receiving stemcell transplantation J Clin Oncol p “ 101200JCO200207101 PMID Tian J Chen ZC and Hang LF The effects of psychological status of the patients with digestive systemcancers on prognosis of the disease Europe PMC p “ Yousaf U Suicides among Danish cancer patients “ British journal of cancer p “ 101038sjbjc6602424 PMID Spiegel D and GieseDavis J Depression and cancer mechanisms and disease progression Biologicalpsychiatry p “ 101016s0006322303005663 PMID Ng CG Anxiety depression perceived social support and quality of life in Malaysian breast cancer patients a 1year prospective study Health and quality of life outcomes p Burgess C Depression and anxiety in women with early breast cancer five year observationalcohort study Bmj p 101136bmj38343670868D3 PMID Grassi L Screening for distress in cancer patients a multicenter nationwide study in Italy Cancer p “ 101002cncr27902 PMID Gelaye B Williams MA Lemma S Deyessa N Bahretibeb Y Shibre T Validity of the patienthealth questionnaire9 for depression screening and diagnosis in East Africa Psychiatry research Dec “ 101016jpsychres201307015 PMID Dalgard OS Dowrick C Lehtinen V VazquezBarquero JL Casey P Wilkinson G Negative lifeevents social support and gender difference in depression Social psychiatry and psychiatric epidemiology Jun “ 101007s0012700600515 PMID Iacovides A The relationship between job stress burnout and clinical depression Journal ofaffective disorders p “ 101016s0165032702001015 PMIDTorres MA Predictors of depression in breast cancer patients treated with radiation role of priorchemotherapy and nuclear factor kappa B Cancer p “ 101002cncr28003 PMID Polikandrioti M EVALUATION OF DEPRESSION IN PATIENTS UNDERGOING CHEMOTHERAPY Health Science Journal De Wit LM Depression and body mass index a ushaped association BMC public health p MartinRodriguez E Relationship between body mass index and depression in women a 7yearprospective cohort study The APNA study European Psychiatry p “ 101016jeurpsy201511003 PMID Jung SJ Association between body size weight change and depression systematic review andmetaanalysis The British Journal of Psychiatry p “ 101192bjpbp116186726 PMID Ciaramella A and Poli P Assessment of depression among cancer patients the role of pain cancertype and treatment PsychoOncology Journal of the Psychological Social and Behavioral Dimensionsof Cancer p “ Atag E Prevalence of depressive symptoms in elderly cancer patients receiving chemotherapyand influencing factors Psychogeriatrics p “ 101111psyg12329PMID Thornton LM Delayed emotional recovery after taxanebased chemotherapy Cancer 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Thyroid_Cancer

thyroid stimulating hormone highlightspleiotropic effects and inverse association withthyroid cancerWei Zhouet alThyroid stimulating hormone TSH is critical for normal development and metabolism Tobetter understand the genetic contribution to TSH levels we conduct a GWAS metaanalysisat million genetic markers in up to individuals and identify genomewidesignificant loci for TSH of which are previously unreported Functional experiments showthat the thyroglobulin proteinaltering variants P118L and G67S impact thyroglobulin secretion Phenomewide association analysis in the UK Biobank demonstrates the pleiotropiceffects of TSHassociated variants and a polygenic score for higher TSH levels is associatedwith a reduced risk of thyroid cancer in the UK Biobank and three other independent studiesTwosample Mendelian randomization using TSH index variants as instrumental variablessuggests a protective effect of higher TSH levels indicating lower thyroid function on risk ofthyroid cancer and goiter Our findings highlight the pleiotropic effects of TSHassociatedvariants on thyroid function and growth of malignant and benign thyroid tumorsA list of authors and their affiliations appears at the end of the paperNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zNormal thyroid function is essential for proper growth anddevelopment and for metabolic functions Approximately million people in the United States are affected bythyroid disorders and of the population is expected todevelop thyroid conditions over their life span1 Thyroidstimulating hormone TSH is secreted by the pituitary glandand stimulates the growth of the thyroid gland and its synthesisand secretion of thyroid hormones These include thyroxine T4most of which is converted to its more bioactive form ²triiodothyronine T3 TSH levels are negatively regulated by T3and T4 and lower or higher levels than the reference rangerespectively usually suggest that the thyroid gland is overactive asin primary hyperthyroidism or underactive as in primary hypothyroidism The complex inverse relationship between TSH andthyroid hormones means TSH is a more sensitive marker ofthyroid status a feature that has been used to identify individualswith thyroid dysfunction2Thyroid disorders affect multiple ans and are associatedwith a range of clinical consequences including an increased riskof metabolic disorders and cardiovascular mortality3“ Over thepast few decades a steady increase of the incidence rates of nonmedullary thyroid cancer henceforth referred as thyroid cancerhas been observed in most areas of the world including in Europe7 Previous studies have led to inconsistent s on therelationship between TSH levels and thyroid cancer risk8 Severalstudies have observed an association between low TSH levelswhich can occasionally occur as a consequence of autonomousthyroid nodules and an increased risk of thyroid cancer8“ Incontrast several studies have indicated that TSH promotes thegrowth of thyroid cancers814“ which has led to the recommendation to lower TSH levels among people with thyroid cancerto reduce the risk of cancer recurrence Two initial genomewideassociation studies GWAS identified five significant loci forthyroid cancer in Europeans and the risk alleles of all five locihave been associated with decreased TSH levels1718 In contrast amore recent GWAS identified five additional loci associated withthyroid cancer none of which were even nominally associatedwith TSH19 A recenttwosample Mendelian randomizationstudy suggested a causal inverse association between TSH levelsand overall cancer risk including thyroid cancer20 Additionalstudies are needed to clarify the role of TSH and TSHassociatedvariants in thyroid cancerTwin studies have shown TSH levels are moderately heritablewith estimates up to Previous TSH GWAS studies haveidentified independent TSHassociated loci172223 accountingfor of TSH variance thus leaving a large proportion of theTSH heritability unexplained2324 With the goal of identifying themissing genetic components for TSH to further understand itsunderlying genetic architecture and impact on thyroid cancer weperform a GWAS metaanalysisfor TSH levels on thepopulationbased NordTr¸ndelag Health Study HUNT studyN Michigan Genomics Initiative26 MGI N consortium up to N and the ThyroidOmics samples23To investigate the genetic relationship between TSH andthyroid cancer and other human diseases we examine phenomewide associations in the UK Biobank UKBB27 for TSHassociated index variants We also conduct phenomewide association tests for the polygenic scores PGS of TSH in the UKBBand the FinnGen study We observe an association between highTSH PGS and low thyroid cancer risk and replicate that observation in two other study populations from Columbus USA andIceland19 To evaluate the potential causality of TSH on thyroidcancer we perform a twosample Mendelian Randomizationanalysis using the TSHassociated top association signals asinstrumental variables and the thyroid cancer GWAS results on individuals cases and controls from ametaanalysis of UKBB2728 MGI26 and results from a previousmetaanalysis for thyroid cancer based on a Icelandic data setfrom deCODE referred to as deCODE in this manuscript aswell as four other case“control data sets with European ancestryas reported in Gudmundsson et al19ResultsDiscovery of genetic loci for TSH We identified loci associated with TSH Table Supplementary Data and andSupplementary Fig in our metaanalysis of the HUNT studyN the MGI biobank N and the ThyroidOmics consortium23 up to N Twentyeight of the loci have not been previously reported for TSH172223Table To identify secondary independent association signalswe performed stepwise conditional analysis within each locususing GCTACOJO29 based on GWAS summary statistics fromthe metaanalysis of HUNT MGI and ThyroidOmics and thelinkage disequilibrium LD correlation between variants estimated in HUNT We observed additional associations in novelTSH locus B4GALNT3 and previously known TSH lociTable and Supplementary Data In total independent topvariants have been identified at the loci explaining ofthe variance of TSH levelsDespite having only moderate effect sizes top variants inseveral novel TSH loci point to nearby genes with a known orsuspected link to thyroid function Table and SupplementaryFig An intronic variant rs10186921 in the thyroid adenomaassociated gene THADA was identified to be associated with TSHTHADA has been identified as a somatic mutatedrearrangedgene in papillary thyroid cancer30 and observed to be truncated inthyroid adenoma31 Although THADA is known to play a role incold adaptation obesity and type diabetes its role in thyroidvariantfunction remainsrs145153320 in gene B4GALNT3 is associated with TSHminor allele frequency in HUNT MAFHUNT effectsizeHUNT standard deviation SD confidence intervalCI “ SD PvalueHUNT — ˆ’ and is times more frequent in the Norwegian HUNT samples than inother nonFinnish Europeans34 The WNK1B4GALNT3 genefusion has been identified in papillary thyroid carcinoma35elusive3233 A rare missenseTwo novel independent rare coding variants with effect sizeslarger than one SD were identified in the known TSH locus TSHRwhich encodes the TSH receptor Both variants were only observedin HUNT The rare missense variant TSHR pR609Q rs139352934MAFHUNT effect sizeHUNT SD CI “SD is the most significant variant in the locus PvalueHUNT — ˆ’ followed by pA553T rs121908872 MAFHUNT CI “ SDPvalueHUNT — ˆ’ TSHR pR609Q rs139352934is times more frequent in HUNT than in other nonFinnishEuropeans34 TSHR pR609Q has been reported to aggregate in afamily with nonautoimmune isolated hyperthyrotropinemia36 andTSHR pA553T has been previously detected in a family withcongenital hypothyroidism37sizeHUNT SDeffectAs singlevariant association tests may lack power for rarevariants MAF ‰ and to search for genes with multiple rareproteinaltering variants we performed exomewide genebasedSKATO38 tests as implemented in SAIGEGENE39 to identifyrare coding variants associated with TSH We grouped missenseand stopgain variants with MAF ‰ and imputation qualityscore ‰¥ within each gene and tested genes with at leasttwo variants This analysis identified two genes TSHR andB4GALNT3 as significantly associated with TSH Pvalue —ˆ’ Supplementary Table and Supplementary Fig RareNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zeulavPytienegoreteHcnoitceriDNPESbtceffEˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’E“E“Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’aqerFIRMCNKDCIFRMfroCPAHTKOBADAHTLXSASRECCFGEVDEDPPPLSNTCOLNDLCGARPCOMSTCADXNSSRFSTAEYTNLAGBTNLAGBCELOCCDCCKENSAHPDFNZBHSBARHOPAGNGCNILCSACGRDCNILPBPSHRPPPGNGIRMTNWmaertsnwoDicnegretniicnegretnIiicnortn_ANRcnicnortnIicnegretnIicnortnIicnortnIicnortnIicnegretnIicnortnIicnortnIicnortnIicnegretnIicnegretnIicnortnIicnortnIicnortnIicnortnIsuomynonysnoNicnegretnIsuomynonysnoNicnortnIsuomynonysnoNicnortnIiicnortn_ANRcnicnegretnIicnortnIicnegretnIicnegretnIGAAACTGGGAGCAATCGGATCCCTAGCGTCAGGGACTAAGCTGCATACGCTTTCGCTACTsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsisylanaatemscimOdioryhTIGMTNUHseneGtseraeNyrogetaCtlAfeRDIsrdliubnoitisoPemosomorhCxednIsucoLscimOdoryhTidnaIGMTNUHfosisylanaatemehtnidefiitnediHSThtiwdetaicossaicoltnednepednilevoninhtiwstnairavdaeLelbaTtesatadigndnopserrocehtnignissimsitnairavehtfisadetoNlyevitcepseriscmOdoryhTidnaIGMTNUHstesatadliaudvdniinieelllaetanretlaehtfoHSTnonoitceridslevelHSTfoDSfotinuehtnieelllaetanretlaehtottcepserhtiwdetropererasezistceffEbtceffEctesatadlsisyanaatemdenbmociehtnieelllaetanretlaehtottcepserhtiwdetropereraisecneuqerFaNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zvariants in both genes associated with TSH were also identifiedfrom singlevariant analysis After conditioning on the two rarevariants in TSHR that were genomewide significant in the singlevariantandrs139352934 the gene TSHR was still exomewide significantwith Pvalue — ˆ’ while B4GALNT3 was no longersignificantly associated with TSH with Pvalue afterconditioning on the top variant rs145153320Pvalue — ˆ’rs121908872analysisFinemapping for potentially causal variants among TSH lociTo identify potentially causal variants at TSH loci we conductedfinemapping using SuSiE40 which estimates the number ofcausal variants and obtains credible sets of variants with cumulative posterior probability through Iterative BayesianStepwise Selection41 Supplementary Data The LD matrixused in SuSiE was calculated based on HUNT We identified eightindependent causal variants at the TSHR locus by finemappingusing SuSiE40 and seven independent association signals by thestepwise conditional analysis Supplementary Data suggestingallelic heterogeneity at the TSHR locusIn addition finemapping by SuSiE40 and stepwise conditionalanalysis identified two association signals in the locus of thethyroglobulin gene TG TG encodes a highly specializedhomodimeric multidomain glycoprotein for thyroid hormonebiosynthesis27 it is the most highly expressed gene in the thyroidgland and its protein product represents roughly half the proteinof the entire thyroid gland4243 The TG locus has been reportedin a recent TSH GWAS23 The credible set for each causalassociation contains one missense variant that is in strong LDwith the most strongly associated intronic variant Supplementary Table and Supplementary Fig In the HUNT study themissense variant TG pG67S rs116340633 MAF effectsize SD CI “ SD Pvalue — ˆ’is in strong LD r2 with the most strongly associatedvariant rs117074997 intronic At the other association signalmissense variant TG pP118L rs114322847 MAF effectsize SD CI “ SD Pvalue — ˆ’is in strong LD r2 with the most strongly associatedvariant rs118039499 intronic Supplementary Table andSupplementary Fig TG pP118L has been previously detectedamong familial cases with congenital hypothyroidism44 TG pP118L rs114233847 is significantly associated with nontoxicnodular goiter odds ratio OR CI “Pvalue — ˆ’ in the UKBB2728 while the association ofTG pG67S rs116340633 with nontoxic nodular goiter isless significant OR CI Pvalue —ˆ’ TG pP118L has been previously detected in patients withsporadic congenital hypothyroidism in a Finnish cohort44Functional followup of missense variants in the gene TG Weperformed sitedirected mutagenesis studies to investigate theimpact on the protein expression of TG of the two independentmissense variants both located in the highly conserved Tg1domain of unclear function The protein encoded by the humanTG is conserved in mice with nearly perfect conservation of allcritical amino acid residues including those that maintain theprotein structure and hormone synthesis45 A cDNA encodingwildtype mouse Tg mTgWT expressed in 293T cells hasnormal synthesis and secretion of thyroid hormones46 We thenintroduced the observed human TG variants rs116340633 andrs114322847 into the mTg cDNA 293T cells were eitheruntransfected or transfected with pcDNA31 in which a cytomegalovirus promoter drives expression of mTgWT or the pP118L or pG67S Tg variants mature Tg numbering Then weexamined the intracellular vs secreted levels of the mTgWT andthese two human Tg variants TgpP118L and TgpG67STransfected cells were incubated overnight and the culturemedium and cell lysates were analyzed by SDSpolyacrylamide gelelectrophoresis PAGE and immunoblotting with antiTg antibody The experiment was independently repeated three timesand the results analyzed in a manner that is independent oftransfection efficiency On average of the total expressedWT form of mTg was recovered in the media and extracellular intracellular MC ratio of mTg was as expected between and the TgP118L variant showed a significant reduction in the MC ratio Pvalue and the TgG67S variant also showed asignificant reduction in the MC ratio Pvalue Fig Compared with the WTPrioritization of TSH genes pathways and tissues To furtherunderstand the biology underlying TSH associations we prioritized associated genes tissues and cell types in which TSH genesare likely to be highly expressed using Datadriven ExpressionPrioritized Integration for Complex Traits DEPICT47 based on loci with TSH association Pvalue cutoff — ˆ’ andclumped based on LD in HUNT As expected the membranesand thyroid gland are the most strongly associated tissues followed by tissues from the digestive system ileum gastrointestinaltract pancreas and colon respiratory system lung and accessory ans for eyes conjunctiva eyelids and anterior eyealthough none of the tissues reached the Bonferroni significantthreshold Pvalue or have false discovery rate FDR Supplementary Data Based on functional similarity toother genes among TSH loci genes at the TSHassociated lociwere prioritized by DEPICT with FDR ‰ SupplementaryData among which the prioritized genes ZFP36L2B4GALNT3 PPP1R3B FAM109A GNG12 GADD45A BMP2VEGFC LPP and MAL2 were at the novel TSH loci identified inour metaanalysis Table In addition among reconstituted gene sets gene sets were enriched among TSH lociwith FDR The most significantly enriched one is the CTSDPPI subnetwork followed by gene sets for regulation of phosphorylation Supplementary Data Pleiotropic effects of TSH loci To explore the pleiotropic effectsof the TSH loci we examined associations of the nonhumanleukocyte antigen HLA independent TSH top variants with human diseases PheCodes272848 and continuoustraits httpwwwnealelabisukbiobank in the UKBB variants and rs121908872 are not available in the UKBB Dueto the strong associations between HLA variants and autoimmune diseases49 we excluded two HLA variants associatedwith TSH rs1265091 and rs3104389 in the analysis for pleioPvalue — ˆ’tropic effects We identified significantpleiotropic association for out of nonHLA variants across disease phenotypes Supplementary Fig 5a and Supplementary Data including thyroid disorders diabetes cardiovasculardisease digestive system disorders asthma and cataractsIn addition nonHLA variants were significantly associatedPvalue — ˆ’ with one or more quantitativetraitsincluding body mass index lung function measurements metricsof bone density spherical powermeridian measurements andblood cell counts Supplementary Fig 5b and SupplementaryData TSHincreasing alleles at one or more loci were associated with an increased risk of cardiovascular disease smallerbody size reduced bone mineral density decreased lung functionand an increased risk of hypothyroidism and a decreased risk ofgoiter These results are generally consistent with previous studies23 We also examined the associations between the TSH indexin the ThyroidOmicsvariantsand freethyroxinelevelsNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zaTg kDabcUntransfectedmTgWTmTgP118LmTgG67SMCMCMCMCMCMCMCTg kDaTg kDaddnab nillubogoryhTeUA ytisnetniCellMediaWTG67SP118Loitar CMFig Both the TGP118L and TGG67S point mutants exhibit a secretion defect a“c Three independent replicate experiments Western blotting ofTG in 293T cells that were either untransfected a no detectable bands or transfected with constructs encoding mouse TG wild type WT or P118L 67S point mutants in the pcDNA31 background in which the CMV promoter drives the respective cDNA expression Serumfree media M werecollected overnight and the cells C were lysed Equal volumes of media and cells were analyzed by SDSPAGE electrotransfer to nitrocellulose andimmunoblotting with antiTgspecific antibodies Full scans of western blotting are presented in Supplementary Fig From scanning densitometryd shows the content of thyroglobulin and its variants intracellularly and in the secretion e The extracellular intracellular MC ratio of each constructd e Three independent replicate experiments All boxplots in d and e indicate median center line 25th and 75th percentiles bounds of box andminimum and maximum whiskersWTG67SP118Lconsortium23 Out of TSHassociated variantsfor whichassociation results with free thyroxine were available have TSHlowering alleles associated with higher free thyroxinelevels Supplementary Data Pvaluebinomial — ˆ’We further examined the association with thyroid cancer forTSH index variants We metaanalyzed UKBB2728 and a previousmetaanalysis of deCODE and four other case“control data sets19for thyroid cancer in thyroid cancer cases and controls and examined out of TSH nonHLA index variantsthat are available in the metaanalysis for thyroid cancerSupplementary Data The TSHincreasing alleles of outof TSHassociated variants were associated withreduced thyroid cancerrisk Supplementary Data andSupplementary Fig 6a and 6b Pvaluebinomial — ˆ’Eighteen out of the TSHassociated variants tested wereat least nominally associated with thyroid cancer P Pvaluebinomial — ˆ’ For out of the TSHassociatedvariants the TSHincreasing alleles were associated with reducedthyroid cancer risk Pvaluebinomial — ˆ’ SupplementaryData and Supplementary Fig 6c d Moreover when weexamined alleles that predisposed to thyroid cancer17“ out of had a consistent direction of effect towards lower TSH Pvaluebinomial Ofriskalleles that were at least nominally associated with TSH levelP all six variants were associated with lower TSHPvaluebinomial Supplementary Data and Supplementary Fig thyroidcancerthesixAssociations of polygenic scores of TSH with other phenotypesAlthough individual TSH variants may exhibit pleiotropic effectsit is also possible that the cumulative effects of TSHmodifyinggenetic variants may lead to disease Therefore we constructedcodesICDPGS from the independent nonHLA TSH top variantsrs1265091 and rs3104389 are HLA variants and rs121908872and were not in UKBB and examined their association with the human diseases constructed from International Classification of Diseasesin theUKBB272848 As in the pleiotropy analysis we excluded the twoHLA variants in the PGS calculation to study the cumulativegenetic effects of TSHassociated variants in nonHLA regionswith human diseases The TSH PGS was significantly associatedwith phenotypes Pvalue — ˆ’ Bonferroni correctionfor phenotypes including an increased hypothyroidism riskand decreased risk of goiter thyrotoxicosis and hyperhidrosisSupplementary Data and Supplementary Fig We alsoevaluated the phenotypic variance Nagelkerke™s r250 explainedby TSH PGS for phenotypes in the UKBB that have at least cases in unrelated white British samples Supplementary Data The phenotypes with highest r2 were nontoxicnodular goiter r2 secondary hypothyroidism r2 and thyrotoxicosis with or without goiter r2 InFinnGen we also observed that high TSH PGS was associatedwith high risk of hypothyroidism and low risk of goiter HighTSH PGS in FinnGen was marginally associated with an increasein risk of depression OR per SD of TSH PGS CI “ Pvalue — ˆ’ and a reduced risk of pregnancyhypertension OR per SD of TSH PGS CI “ Pvalue — ˆ’ The phenomewide associationresults for the TSH PGS in FinnGen are shown in SupplementaryData and Supplementary Fig Depressive symptomsand hypertension during pregnancy have been observed to beclinically associated with hypothyroidism and thyroid dysfunction51“ respectively However their genetic associations havenot been extensively studiedNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zaUKBBrecnac doryhit f oecneaverPlQuintiles of TSH PGSbdeCODEirecnac doryht fo ecneaverPlQuintiles of TSH PGSrecnac doryhit fo oitar sdrecnac doryht fo oitar sddOQuintiles of TSH PGSQuintiles of TSH PGScFinnGenirecnac doryht fo ecneaverPlirecnac doryht fo oitar sddOQuintiles of TSH PGSQuintiles of TSH PGSFig The risk of thyroid cancer is lower for individuals with genetically predicted higher TSH levels Plots of thyroid cancer prevalence by quintiles ofTSH PGS left and odds ratio of thyroid cancer in relation to the lowest quintile right in data sets UKBB a N case N control deCODEb N case N control and FinnGen c N case N control N sample size N case sample size of cases N controlsample size of controls Error bars represent confidence intervalsthyroid cancer CI “In the UKBB TSH PGS was significantly associated with aOR per SD ofdecreased risk ofPvalue — ˆ’TSH PGSSupplementary Data and Fig Compared with the rest ofthethyroid cancer ofindividuals with TSH PGS in the lowest quintile was “ and the OR for thyroid cancer of individuals withTSH PGS in the highest quintile was “ suggestingthe OR CIsamplesforthe protective effects of TSHincreasing genetic variants onthyroid cancer riskWe successfully replicated the association between high TSHPGS and low thyroid cancer risk in study populations fromColumbus USA19 OR CI “ Pvalue — ˆ’ and deCODE19 OR CI “Pvalue — ˆ’ We also observed the association inFinnGen OR CI “ Pvalue — ˆ’NATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zalthough the evidence was much less strong The Columbusstudy19 is a case“control study of thyroid cancer cases and controls with much higher thyroid cancer prevalence thanthe three populationbased biobanks UKBB3031 cases and controls FinnGen cases and controls anddeCode19 cases and controls In Fig theprevalence of thyroid cancer left panel and OR of thyroid cancerright panel are plotted against the TSH PGS for the threepopulationbased cohorts results for Columbus are provided inSupplementary Fig Similar plots of hypothyroidism andgoiter are plotted for UKBB and FinnGen in SupplementaryFigs and Mendelian randomization for TSH thyroid cancer and goiterWe investigated a possible causal effect of TSH on thyroid cancerusing twosample Mendelian randomization Ninetyfour nonHLA genetic variants for TSH identified by our metaanalysis ofHUNT MGI and ThyroidOmics were used as instrumentalvariables Fstatistic for all single nucleotide polymorphismsSNPs rs1265091 and rs3104389 are HLA variants andthe summary statistics for thyroid cancer were not available forrs121908872 rs4571283 and To avoid sampleoverlap for the TSH and thyroid cancer GWASs we used effectson TSH estimated by metaanalyzing HUNT and ThyroidOmicsto construct the instrumental variable for TSH levels and wemetaanalyzed MGI deCODE and UKBB for thyroid cancer Wefound that a one SD increase in TSH SD mUL wasassociated with a decreased risk of thyroid cancer inversevariance weighted OR CI “ MREgger intercept Pvalue Sensitivity analyses using the penalizedweighted median method the weighted median method and theweighted mode method including all variants are presented inFig 3a and Supplementary Data Similar results were observedbetween methods with the exception of the weighted modewhich was strongly attenuated To reduce the possibility that theresults were influenced by occult thyroid dysfunction typicallyoccurring in older age we repeated the analysis using SNPTSHeffect estimates obtained among those younger than years ofage at the time of TSH measurement Supplementary Data Similar results were observed except for the weighted modewhich was again attenuated towards the null OR CI“ Supplementary Data Furthermorethere wasstrong evidence of heterogeneity suggesting some instrumentswere invalid Nevertheless when repeating the main analysisusing MRPRESSO which excluded nine variants due to thedetection of specific horizontal pleiotropic outlier variants54 thecausal association was similar MRPRESSO outlier corrected OR CIs “ Fig 3a and Supplementary Data Finally using only the proteincoding nonsynonymous variant pP118L in the TG gene Fstatistic we observed a protective effect of increased TSH on thyroid cancer Wald ratio OR CI “ To investigate if TSH may also influencethe risk of benign thyroid growth disorders we similarly performed atwosample Mendelian Randomization analysisbetween TSH and goiter The effects on TSH were estimated by ametaanalysis of HUNT and ThyroidOmics SupplementaryData and and the GWAS results for goiter from the UKBBwere used2728 A SD increase in TSH SD mULwas associated with a decreased risk of goiter inversevariance weighted OR CI “ MREgger interceptPvalue Fig 3b and Supplementary Data DiscussionMetaanalysis of the HUNT study the MGI biobank and theThyroidOmics consortium for TSH on up to individualsidentified TSH loci of which are previously unreported AllTSH loci reported by previous GWAS studies172223 are replicated in our metaanalysis Several novel loci pointed to nearbygenes with a known or suspected link to thyroid functionAdditional independent signals were identified among several locibased on GWAS results in the metaanalysis and LD informationin the HUNT study including two rare variants rs546738875 andrs145153320 at the B4GALNT3 locus and two rare missensevariants TSHR pA553T rs121908872 and TSHR pR609Qrs139352934 which have been observed to be associated withcongenital hypothyroidism in previous family studies3637 TSHRpR609Q rs139352934 is the most strongly associated with TSHin the TSH receptor gene TSHR with an effect size greater thanone standard deviation of TSH mUL As these rare variants were only imputed in HUNT not in MGI or ThyroidOmicsfurther followup to verify the associations is needed As individual GWAS was conducted on inversenormal transformed TSHlevels before metaanalysis it is challenging to convert the effectsizes reported by our metaanalysis to actual scales of TSH levelsFinemapping for potential causal variants among TSH locidetected two independent missense variants in the TG gene TGpG67S and pP118L The two variants have a similar frequency but pP118L shows stronger evidence for an associationwith goiter and with thyroid cancer Functional experimentsdemonstrated each of these defects in the TG gene pP118L andpG67S respectively causes defective secretion of TG Furtherstudies are needed to investigate how the proteinaltering variantsimpact TSH levelsAs expected membranes and thyroid gland were identified asthe tissue in which genes from TSHassociated loci are most likelyto be highly expressed Genes ZFP36L2 B4GALNT3 PPP1R3BFAM109A GNG12 GADD45A BMP2 VEGFC LPP and MAL2were prioritized as functional candidates among the novel TSHassociated loci based on functional similarity to other genes at allTSH loci using DEPICT47A PheWAS ofthe TSHassociated variants in the UKBBdemonstrated that TSHincreasing alleles are associated with anincreased risk of cardiovascular disease smaller body sizereduced bone mineral density decreased lung function and anincreased risk of hypothyroidism but were favorably associatedwith a decreased risk of goiter Our results suggest that thesevariants have pleiotropic effects although they tend to affect TSHthrough actions in the thyroid glandPhenomewide association tests in the UKBB and FinnGen forthe TSH PGS showed that genetically predicted increased TSH isassociated with a high risk of hypothyroidism and a low risk ofgoiter thyrotoxicosis hyperhidrosis and thyroid cancer Twosample Mendelian randomization analyses suggested that lowerTSH causes an increased risk of thyroid cancer and goiter This isan unexpected direction given that TSH promotes the growth ofthyroid cancers814“ Nonethelessit has previously beenspeculated that lower TSH levels may lead to less differentiationof the thyroid epithelium which could predispose to malignanttransformation17 Alternatively our genetic instrument for TSHmay represent a thyroid gland phenotype that influences bothTSH through the negative feedback of thyroid hormones on thepituitary gland and thyroid growth increasing the risk of thyroidcancer and goiter Supplementary Fig In that scenario theeffect on thyroid cancer would not be downstream of TSH andaltering TSH levels eg by medication would not be expected toalter thyroid cancer risk Tissue enrichment analyses of genes atTSHassociated loci and the observation that TSHloweringalleles were generally associated with higher free thyroxine levelsSupplementary Data suggest that most TSHassociated variants act primarily on the thyroid gland where effects on boththyroid function and growth have previously been described forNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0carecnac doryhit no tceff ePNSbretiog no tceffe PNSNATURE COMMUNICATIONS 101038s4146702017718z““““MethodInverse variance weightedMR EggerMRPRESSOPenalized weighted medianWeighted medianWeighted modeSNP effect on TSHMethodInverse variance weightedMR EggerMRˆ’PRESSOPenalized weighted medianWeighted medianWeighted modeSNP effect on TSHFig Twosample Mendelian randomization analysis for casual associations between TSH and thyroid cancer and between TSH and goiter a Twosample MR between TSH and thyroid cancer based on summary statistics from the metaanalysis of HUNT and ThyroidOmics n for TSH andfrom the metaanalysis of UKBB2728 MGI26 deCODE and four other case“control data sets with European ancestry as reported in Gudmundsson et al19for thyroid cancer association casescontrols b TSH and goiter based on summary statistics from the same TSH metaanalysis asabove and from the GWAS of UKBB2728 for goiter association N case N control The crosshairs on the plots represent the confidence intervals for each SNPTSH or SNPoutcome association The variant on the top left corner is the rare nonsynonymous variant B4GALNT3 pR724W rs145153320 N sample size N case sample size of cases N control sample size of controlsTSHR mutations55 Nonetheless the Mendelian randomizationfindings were robust to most of the sensitivity analyses performedto detect and correctfor pleiotropy Restricting the geneticinstrument to TSHlowering variants associated with lower freethyroxine levels could have helped resolve the causal question butthose variants were too few to yield meaningful estimat

Thyroid_Cancer

Purpose Pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition Encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer LAPC prevents surgical resection This study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationMethods In this phase III trial patients with LAPC were randomised to gemcitabinenab paclitaxel plus Arm A n24 or minus Arm B n13 pamrevlumab Those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria Resection rates progression free and overall survival were evaluatedResults Eighteen patients in Arm A and seven in Arm B completed six cycles of therapy with similar toxicity patterns In Arms A and B carbohydrate antigen “ response as defined by ‰¥ decline from baseline occurred in and respectively Sixteen per cent of patients were radiographically downstaged by National Comprehensive Cancer Network criteria in Arm A and in Arm B Positron emission tomography normalised in vs of patients in Arm A vs Arm B respectively and correlated with surgical exploration Eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in Arm A vs Arm B p01193 respectively Postoperative complication rates were not different between armsConclusions Neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with LAPC without added toxicity This combination merits evaluation in a larger patient cohortIntRoduCtIonPancreatic cancer is currently the third leading cause of cancer death in the USA1 and by it will likely become the second leading cause of cancer related death after Key questionsWhat is already known about this subject –º Pamrevlumab is anti CTGF1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseaseWhat does this study add –º This study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerHow might this impact clinical practice –º This study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged OS curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 Surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 However only of patients have disease amenable to upfront curative resection at the time of diagnosis4 Approximately “ of patients are diagnosed with locally advanced disease5 determined surgically unresectable per National Comprehensive Cancer Network NCCN guidelines6 Patients with locally advanced pancreatic cancer LAPC have a prognosis similar to those with metastatic disease with a historical median overall survival OS of Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access“ months with recent trials demonstrating median OS of months7 Recent single institution retrospective studies have reported the potential for resection of LAPC with neoadjuvant therapy irrespective of imaging findings with promising results8 However these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation Current prospective trials have documented resection rates of LAPC in the range of to therefore novel approaches are needed to improve patient outcomesThe tumour biology inherent to pancreatic ductal adenocarcinoma PDAC significantly contributes to the poor outcomes seen in this disease Notably PDAC exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor CTGF expression12 CTGF appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition This leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 This stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours Executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticsPamrevlumab is a human monoclonal antibody that targets CTGF Preclinical studies showed that CTGF overexpression is associated with both desmoplasia and gemcitabine resistance in the KPC pancreatic cancer mouse model14 When pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of XIAP an antiapoptotic protein15 When tested in patients with advanced pancreatic cancer Stage IV and locally advanced Stage III treated with gemcitabine and erlotinib in a phase III study n75 pamrevlumab displayed multiple favourable outcomes16We hypothesised that through inhibition of the downstream effects of CTGF overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of PDAC tumours With this in mind this novel phase III randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in LAPC with special emphasis on surgical eligibility and safetyMetHodsstudy designThis was a phase III randomised trial of safety and efficacy in patients with LAPC who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy The randomisation was preplanned and blinded to the investigator The study was approved by individual institutional review boards at nine US institutions and conducted according to the Declaration of Helsinki The trial was registered at clinicaltrials gov as NCT eligibilityKey protocol eligibility requirements included biopsy proven diagnosis of PDAC radiographic staging consistent with locally advanced unresectable disease as defined NCCN guidelines V2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per Response Evaluation Criteria in Solid Tumors RECIST V11 Eastern Cooperative Oncology Group ECOG performance status of or adequate haematological renal and hepatic function no prior therapy for PDAC and no concomitant cancer diagnosis within the past yearsstudy schemaEligible patients were randomised to Arm A or Arm B to receive a total of six treatment cycles “ weeks of therapy figure Patients in Arm A received pamrevlumab mgkg by intravenous infusion on Days and of each day cycle with an additional dose given on Day in the first cycle Patients in both Arms A and B received gemcitabine mgm2 by intravenous infusion on Days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on Days and of each day cycle Doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care SOC15 Patients remained on therapy for six treatment cycles “ weeks unless they had disease progression an intolerable adverse event AE or toxicity withdrew consent or were withdrawn at the investigator™s discretion All patients were followed for drug toxicity until days after the last drug dose Patients undergoing surgery were followed for days following hospital discharge for surgical complications CTGF levels were obtained prior to treatment from all patients Plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug After all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy Postoperative complications including day readmissions and day mortality were notedResponse assessmentPatients were evaluated for response by the following measures carbohydrate antigen CA “ measured at baseline first day of each cycle and end of treatment EOT RECIST V11 read based on full body CT imaging high resolution dual phase fine cut CT imaging at baseline and every weeks thereafter fluorodeoxyglucose FDG positron emission tomography PET imaging and NCCN V2 resectability criteria at baseline and EOTPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessFigure Patient flow and surgery outcomes In Arm A four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery In Arm A four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive SMA encasement In Arm B one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement SMA superior mesenteric arterysurgical assessmentSubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol PP defined criteria Given that patients included in the trial were determined to be initially unresectable by radiographic imaging and NCCN criteria objective criteria were developed to standardise attempts at surgical resectionPatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma CA “ level by ‰¥ at EOT compared with baseline reduction in FDG PET maximum standardised uptake value SUVmax by ‰¥ at EOT compared with baseline radiological tumour response per RECIST of partial response PR or complete response CR at EOT or met the definition of resectable or borderline resectable per NCCN guidelines Subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on CT scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein PVsplenic vein thrombosis pancreatitis or decline in performance status to a Karnofsky score ‰¤ or Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes The final decision regarding whether resection was to be performed was made by the treating surgeonendpointsSafety endpoints included serious adverse events SAE during neoadjuvant therapy and surgical complications postresection The efficacy endpoints included surgical eligibility R0 resection R0R1 resection median OS progression free survival PFS and year survival rate All patients were followed and data analysis was stratified by PP population and intention to treat ITT cohortstatistical considerationsThe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the χ² test Exact CIs for the point estimates as well as the treatment difference were obtained from the SAS PROC FREQ procedure with the EXACT option The two treatment arms were compared using the Cochran Mantel Haentzel test controlling for baseline factors TNM stage ECOG CA “ PET SUVmax 0c accesssuperior mesenteric artery SMA involvement coeliac abutment and so on as prespecified in the protocol All cause mortality was used in determining OS which was analysed by the Kaplan Meier method Survival status was updated within month before the data cut off date Data from patients who were alive at the cut off date were censored for survival analysis All statistical tests were performed at the significance level of α005 using two sided testsResultsPatient characteristics and dispositionThirty seven patients were randomised to study treatment to Arm A pamrevlumabgemcitabinenab paclitaxel and to Arm B gemcitabinenab paclitaxel alone Patient characteristics at baseline are summarised in table All patients enrolled were unresectable by NCCN criteria patients had tumour arterial involvement SMA encasement ° coeliac abutment Table Patient characteristicsBaseline demographics “ years “ years ‰¥ years Median Male FemaleAge group Sex BMI kgm2 Mean SD Median Min maxECOG Grade Grade TNM stage T3 N0 M0 T3 N1 M0 T4 N0 M0 T4 N1 M0 T4 NX M0Location of the tumour in the pancreas Non resectability per NCCN criterion Head Body Tail Median tumour size mm ° SMA encasement Any coeliac abutment Inferior vena cava invasion or encasement Unreconstructible SMVportal occlusion Aortic invasion and encasementArm AGNPPN24 Arm BGNPN13 TotalN37 to to to · OK as isNot mutually exclusiveBMI body mass index ECOG Eastern Cooperative Oncology Group G gemcitabine n number of subjects NCCN National Comprehensive Cancer Network NP nab paclitaxel P pamrevlumab PV portal vein SMA superior mesenteric artery SMV superior mesenteric veinPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible PVsuperior mesenteric vein SMV occlusion A higher percentage of patients with SMA encasement ° were randomised to Arm A vs Arm B Patient disposition is summarised in figure Twenty four patients in Arm A received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease three patients AEs two patients or physician decision one patient Thirteen patients in Arm B received gemcitabinenab paclitaxel patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease two patients AEs two patients or patientphysician decision two patientssafetySAEs are summarised in table Forty one per cent of patients had a treatment emergent SAE Arm A Arm B No individual toxicity category occurred with frequency except systemic infection patients There was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapyTable Summary of treatment emergent serious adverse eventsSystem organ classpreferred term Ascites Nausea Pancreatitis Vomiting Device occlusion Drug withdrawal syndrome FeverNo of patients with any treatment emergent SAEBlood and lymphatic disorders Haemolytic uremic syndrome LymphadenopathyCardiac disorders Cardiac failure Supraventricular tachycardiaGastrointestinal disorders General disorders and administrative site conditions Hepatobiliary disorders Infections Sepsis Cellulitis Urinary tract infectionInjury poisoning and procedural complications Respiratory thoracic and mediastinal disorders Skin and subcutaneous disorders Cholangitis Hyperbilirubinaemia Craniocerebral injury Pneumonitis Pulmonary embolism RashArm An24n Arm Bn13n Overalln37n · OK as isPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessResponse to therapyIn Arm A had ‰¥ CA “ decline at EOT response by RECIST PR ‰¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria During the treatment period the median CA “ decline was patients were non secretors Seven out of patients had best objective RECIST response CRPR Some patients had ˜exceptional™ responses defined as normalisation or ‰¥ decline of CA “ patients or normalisation PET SUVmax in In Arm B had ‰¥ CA “ decline at EOT response by RECIST PR ‰¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria Four out of patients had best objective RECIST response CR PR In Arm B of patients had an œexceptional CA “ response and had an ˜exceptional™ PET response as defined by either ‰¥ normalized Ca response normalized SUV max andorradiographic downstaging post therapy completion surgical evaluationOverall of the total study patients were eligible for surgical exploration using protocol defined criteria Arm A Arm B p00019 Resection was completed in of the patients Arm A Arm B p01193 Details of the nine resected patients are shown in table In Arm A of the patients were eligible for surgical exploration in the ITT population and of the patients were eligible in the PP population patients who completed six cycles of treatment In Arm A out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined Eight out of patients in Arm A were resected R0 R1 The remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively In Arm B of the patients were eligible for surgical exploration in the ITT population and were eligible in the PP population Of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionPredictors of resectionHigh CA “ response ‰¥ decline andor normalisation was contributive to surgical eligibility vs p03 Normalisation versus non normalisation of PET SUVmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 Combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection All nine successful resections were identified by one or both of these criteria Table Summary of resected patientsSitesubject IDTreatmentarmResponse to treatmentNCCNbaselineNCCNend of treatmentResection status“““““““““AAAAAAAAB UnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacUnresectablecoeliacUnresectableSMVUnresectableSMAUnresectableSMA SMV coeliacUnresectableSMAUnresectablecoeliacUnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacBorderline resectableUnresectableSMVUnresectableSMAUnresectablecoeliacUnresectableSMAUnresectablecoeliacR0R1R0R0R1R1R1R0R0Protocol defined criteria CA “ decrease FDG PET SUVmax decrease ‰¥ RECIST V11 response PR or CR NCCN resectable or borderline resectable criteriaCA carbohydrate antigen CR complete response FDG fluorodeoxyglucose NCCN National Comprehensive Cancer Network PET positron emission tomography PR partial response RECIST Response Evaluation Criteria in Solid Tumors SMA superior mesenteric artery SMV superior mesenteric vein SUVmax maximum standardised uptake valuePicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cConversely radiographic features of response did not correlate with operative potential Neither RECIST response nor radiographic downstaging per NCCN criteria statistically correlated with completed resectionsurgical complicationsPostoperative complications were summarised according to the Clavien Dindo classification posthoc analysis Ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in Arm A Grade II There was one episode of clinically significant pancreatic leak in each arm Grade IIIA no reoperations and no day or day surgical mortality were noted One patient in Arm B had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively Grade IIIB No wound complications or superficial site infections were noted in either group Four out of patients and out of patients in Arm A and B respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalAs of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months PFS was months CI to and months CI to in Arm A and Arm B respectively One year survival and median OS were and months CI accessto in Arm A and and months CI NR in Arm B The median OS for all patients who were eligible for surgical exploration Arm A Arm B vs ineligible Arm A Arm B was months CI NR vs months CI to p00766 The median OS for resected Arm A Arm B vs non resected patients Arm A Arm B was not reached CI NR vs months CI to p00141 figure dIsCussIonThe treatment of LAPC with neoadjuvant therapy remains challenging and there is no established SOC Several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 The combination of more active regimens delivered over an extended period and surgeons™ comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates A meta analysis of studies using FOLFIRINOX has demonstrated resection rates ranging from to in LAPC17 One of the larger studies including patients with LAPC reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen FOLFIRINOX or gemcitabine based18 Recently a single institution and single arm prospective study of neoadjuvant FOLFIRINOX and losartan with selective use of radiation in patients with LAPC reported an R0 resection rate of Figure Overall survival Resected vs Non resected patientsPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access However the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease These retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of LAPCthe anti CTGF mechanism of action With respect to gemcitabine based therapy a recent large scale prospective trial of patients with LAPC treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 Furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation More recently the LA PACT trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with LAPC were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigator™s choice11 Last although FOLFIRINOX has been the most studied induction combination chemotherapy regimen in this population recent randomised data from European patients who received neoadjuvant FOLFIRINOX versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to R0R1 to resection rate vs p0135 or OS vs months p0268Given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates Previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a ˜softening™ of tumours by endoscopic ultrasound elastography21 This stromal depletion also translated into a decrease of SUV uptake on PET22 In the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsThe protocol specified therapeutic response criteria CA “ PET SUVmax RECIST and NCCN criteria were used as criteria to determine eligibility for surgical exploration in LAPC This is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards NCCN criteria For example by NCCN conversion alone ie converted from unresectable to borderline resectable only of patients in Arm A would have been eligible for surgical exploration However by protocol criteria of patients in Arm A were eligible for surgical exploration A higher percentage of patients were eligible for surgical exploration by the above criteria in Arm A vs Arm B vs respectivelyOverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size Of the nine subjects that were successfully resected in this trial only one was converted by NCCN criteria to borderline resectable prior to surgical exploration Despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti CTGF mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients This hypothesis needs to be confirmed and patients should be stratified by coeliac andor SMA involvementThe most common predictive factors for eligibility for surgical exploration and resection were CA “ decline and PET SUV max response which are indicators of tumour response to treatment The combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success Both the ability of CA “ response and the inability of radiographic response RECIST and NCCN criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials In the MPACT study both CA “ and PET response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 Recent surgical series of patients with borderline resectable and LAPC have also corroborated their impact in the localised setting25 Correlation of clinical response with plasma levels of endogenous CTGF and pamrevlumab exposure as shown in the prior study by Picozzi et al16 may provide added prognostic and predictive insightWith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel In addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone Pamrevlumab is well tolerated and considered safe compared with the SOC drugs for patients with PDAC These observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in LAPC In addition there were no signals of increased surgical morbidity or wound healing problems with CTGF blockade by pamrevlumab In fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres Similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationFinally while survival data are not yet mature both patients who were eligible for surgery and those that Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cwere ultimately resected had longer PFS and OS highlighting the importance of surgical resection of the tumour Therefore more investigation into newer agents targeting LAPC and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseaseIn conclusion this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in LAPC with prespecified criteria for surgical exploration The use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates Further evaluation of this drug combination in the neoadjuvant treatment setting for LAPC is warranted and a larger phase III trial with resection and survival endpoints is ongoingContributors FibroGen Inc was the study sponsor that designed the study in consultation with the Principal Investigator VP and surgical co investigator FGR All authors except those of the sponsor contributed patients to the study FibroGen was responsible for data collection and analysis All authors reviewed the manuscript and signed off on its accuracyFunding The study was funded by FibroGen Inc San Francisco CAdisclaimer The corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article if accepted to be published in ESMO editions and any other BMJPGL products to exploit all subsidiary rights as set out in our licenceCompeting interests MC MZ SP EK and EC are employees of FibroGen and hold stock andor stock options

Thyroid_Cancer

" COVID19 pathways for brain andheart injury in comorbidity patients A role of medical imaging and artificial intelligencebased COVIDseverity classification A review Computers in Biology and Medicine 101016jcompbiomed2020103960 0cThis is a PDF file of an that has undergone enhancements after acceptance such as the additionof a cover page and metadata and formatting for readability but it is not yet the definitive version ofrecord This version will undergo additional copyediting typesetting and review before it is publishedin its final form but we are providing this version to give early visibility of the Please note thatduring the production process errors may be discovered which could affect the content and all legaldisclaimers that apply to the journal pertain Published by Elsevier Ltd 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS1 Anudeep Puvvula MBBS FCD12 Mainak Biswas PhD3 Misha Majhail14 Luca Saba MD5 Gavino Faa MD6 Inder M Singh MD7 Ronald Oberleitner BS MBA8Monika Turk MD PhD9 Paramjit S Chadha BE1 Amer M Johri MD FASE10 J Miguel Sanches PhD11 Narendra N Khanna MD DM FACC12 Klaudija Viskovic MD PhD13 Sophie Mavrogeni MD PhD FESC14 John R Laird MD FACC FACP15 Gyan Pareek MD16 Martin Miner MD17 David W Sobel MD16 Antonella Balestrieri MD5 Petros P Sfikakis MD18 Gee Tsoulfas MD19 Athanasios Protogerou MD PhD20 Durga Prasanna Misra MD FRCP21 Vikas Agarwal MD FRCP21 Gee D Kitas MD PhD FRCP22 Puneet Ahluwalia MS MCh24 Raghu Kolluri MD25 Jagjit Teji MD26 Mustafa AlMaini MD27 Ann Agbakoba MD28 Surinder K Dhanjil MSc FSVU29 Meyypan Sockalingam MBBS FRCR30 Ajit Saxena MD12 Andrew Nicolaides MS PhD FRCS31 Aditya Sharma MD32 Vijay Rathore MD33 Janet N A Ajuluchukwu MD34 Mostafa Fatemi PhD FAIMBE FIEEE FASA FAIUM35 Azra Alizad MD FAIMBE FAIUM36 Vijay Viswanathan MD PhD37 P K Krishnan MD38 Subbaram Naidu PhD Life FIEEE39 1Stroke Monitoring and Diagnostic Division AtheroPoint„¢ Roseville CA USA 2Annu™s Hospitals for Skin and Diabetes Nellore AP INDIA 3JIS University Kolkata West Bengal INDIA 4Oakmont High School and AtheroPoint„¢ Roseville CA USA 5Department of Radiology Azienda Ospedaliero Universitaria Cagliari ITALY 6Department of Pathology AOU of Cagliari Italy 7Stroke Monitoring and Diagnostic Division AtheroPoint„¢ Roseville CA USA 8Behavior Imaging USA 9The HanseWissenschaftskolleg Institute for Advanced Study Delmenhorst GERMANY 10Department of Medicine Division of CardiologyQueen™s University Kingston Ontario CANADA 11Institute of Systems and Robotics Instituto Superior Tecnico Lisboa PORTUGAL 12Department of Cardiology Indraprastha APOLLO Hospitals New Delhi INDIA 13University Hospital for Infectious Diseases Zagreb CROTIA 14Cardiology Clinic Onassis Cardiac Surgery Center Athens GREECE 15Heart and Vascular Institute Adventist Health St Helena St Helena CA USA 16Minimally Invasive Urology Institute Brown University Providence Rhode Island USA 17Men™s Health Center Miriam Hospital Providence Rhode Island USA 18Rheumatology Unit National Kapodistrian University of Athens GREECE 19Aristoteleion University of Thessaloniki Thessaloniki GREECE 20National Kapodistrian University of Athens GREECE 21Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow UP INDIA 22Academic Affairs Dudley Group NHS Foundation Trust Dudley UK 23Arthritis Research UK Epidemiology Unit Manchester University Manchester UK 24Max Institute of Cancer Care Max Superspeciality Hospital New Delhi INDIA 25OhioHealth Heart and Vascular Ohio USA 26Ann and Robert H Lurie Children™s Hospital of Chicago Chicago USA 27Allergy Clinical Immunology and Rheumatology Institute Toronto CANADA 28University of Lagos NIGERIA 29AtheroPoint LLC CA USA 30MV Center of Diabetes Chennai INDIA 31Vascular Screening and Diagnostic Centre and University of Nicosia Medical School CYPRUS Journal Preproof 0c32Division of Cardiovascular Medicine University of Virginia Charlottesville VA USA 33Nephrology Department Kaiser Permanente Sacramento CA USA 34Department of Medicine Lagos University Teaching Hospital Lagos NIGERIA 35Dept of Physiology Biomedical Engg Mayo Clinic College of Medicine and Science MN USA 36Dept of Radiology Mayo Clinic College of Medicine and Science MN USA 37MV Hospital for Diabetes and Professor M Viswanathan Diabetes Research Centre Chennai INDIA 38Neurology Department Fortis Hospital Bangalore INDIA 39Electrical Engineering Department University of Minnesota Duluth MN USA Corresponding Author Dr Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS Fellow American Institute of Medical and Biological Engineering Fellow American Institute of Ultrasound in Medicine Fellow Asia Pacific Vascular Society Stroke Monitoring and Diagnosis Division AtheroPoint„¢ Roseville Roseville CA USA Phone Email jasjitsuriatheropointcom Journal Preproof 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Abstract Artificial intelligence AI has penetrated the field of medicine particularly the field of radiology Since its emergence the highly virulent coronavirus disease COVID19 has infected over million people leading to over deaths as of July 1st Since the outbreak began almost s about COVID19 have been published pubmedncbinlmnihgov however few have explored the role of imaging and artificial intelligence in COVID19 patients”specifically those with comorbidities This paper begins by presenting the four pathways that can lead to heart and brain injuries following a COVID19 infection Our survey also offers insights into the role that imaging can play in the treatment of comorbid patients based on probabilities derived from COVID19 symptom statistics Such symptoms include myocardial injury hypoxia plaque rupture arrhythmias venous thromboembolism coronary thrombosis encephalitis ischemia inflammation and lung injury At its core this study considers the role of imagebased AI which can be used to characterize the tissues of a COVID19 patient and classify the severity of their infection Imagebased AI is more important than ever as the pandemic surges and countries worldwide grapple with limited medical resources for detection and diagnosis We conclude that imaging and AIbased tissue characterization when considered alongside COVID19 symptoms and their pretest probabilities offer a compelling solution for assessing the risk of comorbid patients These methods show the potential to become an integral part of tracking and improving the healthcare system both during the pandemic and beyond Keywords COVID19 comorbidity pathophysiology heart brain lung imaging artificial intelligence risk assessment Journal Preproof 0c Introduction In December a novel coronavirus referred to as œsevere acute respiratory distress syndrome coronavirus  SARSCoV2 [] appeared in Wuhan the capital of Hubei Province in PR China The disease caused by the virus was initially named œnovel coronavirus pneumonia NCP by the Chinese government but was subsequently renamed œcoronavirus disease  COVID19 by the World Health anization WHO On January 30th it was declared a public health emergency of international concern PHEIC [] It is believed that SARSCoV2 is primarily transmitted through saliva droplets or nasal discharge [] The first evidence of humantohuman transmission was found by Jasper FukWoo Chan et al in their study at The University of Hong KongShenzhen Hospital [] Due to its contagiousness Ro27 the virus has reached epidemic levels affecting countries and causing over million infections and more than deaths as of July 1st [] shown in Figure Recent literature suggests that patients with preexisting diseases are likely to experience severe complications from COVID19 [] In one study on admitted diabetic and nondiabetic COVID19 patients the mortality rate vs and the rate of admission to the intensive care unit ICU vs were significantly higher for diabetic patients Diabetic patients also experienced severe inflammatory responses and cardiac hepatic and renal coagulopathy [] The prevalence of heart and brain injuries was also higher in COVID19 patients with concomitant chronic conditions like diabetes kidney disease dyslipidemia hypertension [] chronic obstructive pulmonary disease COPD and cardiovascular diseases [] Recent studies have shown that SARSCoV2 invades the thin lining of the epithelial cells of the arteries leading to atherosclerosis [] and arterial inflammatory disease”one of the major causes of cardiovascular diseases CVDs which also causes heart and brain injuries [ ] This could be due to a reduced expression of angiotensinconverting enzyme ACE2 causing endothelial dysfunction which in turn aggravates existing Journal Preproof 0catherosclerosis [ ] It has also been observed that comorbid patients when subjected to imagescreening show mild to severe pretest probability PTP for COVID19 [] The conventional cardiovascular risk factors CCVRF in these comorbid patients appear strongly correlated either to their heart imaging or to surrogate biomarkers of coronary artery disease such as carotid artery disease Both imaging and biomarkers could be helpful in severity predictions for COVID19 [] Figure illustrates the associations between SARSCoV2 and other comorbidities such as diabetes as well as the comparative survival rates for COVID19 patients with and without diabetes Figure World map showing COVID19 spread over countries courtesy John Hopkins University ACE2 expression causes scars in the vessels and can even rupture the walls of the arteries [] For this reason CCVRF should be considered alongside imaging in patients who present with COVID19 and many comorbidities [] The second stage is the one at which a patient is most severely affected by Journal Preproof 0cCOVID19 and has the highest probability of cardiac injury or release of troponin T TnT Imaging has been shown to offer benefits in monitoring the tissue scars caused by COVID19 [] Figure a Association of SARSCoV2 with other comorbidities and b comparison of the mortality rate of diabetic and nondiabetic COVID19 patients reproduced with permission [] Multiple modalities can be utilized to determine whether a patient has the sequelae of COVID19 including magnetic resonance imaging [] computed tomography [] and ultrasound [] The advantage of these imaging modalities is the visual access they provide to the scar tissue caused by the disease A disadvantage however is their inability to provide a œrisk assessment The application of artificial intelligence AI can enhance the information provided by these imaging modalities resulting in a more accurate characterization of the tissue and the disease process [] In fact the combination of AI and medical imaging has been shown to improve diagnosis and risk stratification speed up patient evaluation enhance disease monitoring and accelerate early intervention [ ] Thus this review will focus on the use of AIbased tissue characterization of images of comorbid patients affected by COVID19 The layout of this paper is as follows Section presents the pathophysiology of the four pathways leading to brain and heart injury Section summarizes the evidence related to the use of Journal Preproof 0cimaging during the COVID19 pandemic Section elaborates on the use of AIbased tissue characterization for risk assessment Finally the paper concludes with a critical discussion The Pathophysiology of SARCoV2 Leading to Brain and Heart Injury Several studies suggest that SARSCoV2 uses the ACE2 receptor to gain access to cells by binding to the SPIKE protein ˜S™ protein on their surface [] see Figure ACE2 and angiotensinconverting enzyme ACE1 are homolog carboxypeptidase enzymes that have different vital functions in the reninangiotensinaldosterone system RAAS pathway [] ACE2 is widely expressed in myocardial cells [] type pneumocytes enterocytes and astrocytes in the brain [ ] Thus it is recognized as a cause of extrapulmonary complications Figure shows the overall picture of how SARSCoV2 causes brain and heart injuries via four different pathways These include i the neuronal pathway ii the hypoxia pathway iii the RAAS pathway and iv the immune pathway We will discuss these pathways and the injuries they lead to which may manifest as viral encephalitis infectious toxic encephalopathy or acute cerebrovascular disease i The Neuronal Pathway Figure the pathway I Recent epidemiological studies have demonstrated similarities at the genomic level between SARSCoV1 MERS and SARSCoV2 [ ] Meanwhile previous experimental studies have shown that beta coronaviruses in general”such as SARSCoV1 and MERS”can spread into and directly infect the brain when inhaled as droplets via the nasal epithelium [ ] Figure depicts the olfactory nerve and the olfactory bulb []”labeled as œa and œb respectively”on the image of the sagittal brain in the neuronal pathway Based on recent reports we are aware that patients infected by SARSCoV2 show symptoms of dysgeusia loss of taste and anosmia loss of smell [ ] Bohmwald et al further validate that coronaviruses that infect through the olfactory nerve and bulb can reach the brain and cerebrovascular fluid CSF within seven days Additionally these viruses have been observed to cause inflammation and demyelination [] The Journal Preproof 0cauthors demonstrated in an experimental study of mice that removing the olfactory bulb from the pathway can lead to the restriction of CoV in the central nervous system CNS [] Based on this evidence we believe that the neuronal pathway is one possible track for SARSCoV2 ii The Hypoxia Pathway Figure pathway II In this pathway decreased levels of ACE2 proliferate in the lung parenchyma cells after the coronavirus has passed through causing exaggerated neutrophils accumulation enhanced vascular permeability and the formation of diffuse alveolar and interstitial exudates This ultimately leads to pulmonary edema and acute respiratory distress syndrome ARDS [] ARDS is characterized by severe abnormalities in blood gas composition resulting from an oxygen and carbon dioxide mismatch which leads to low blood oxygen levels [ ] This ongoing hypoxia can lead to myocardial ischemia and heart injury [ ] see Figure pathway IIA Hypoxia in the brain increases anaerobic metabolism in the mitochondria of the brain cells [] leading to cerebral vasodilatation edema and impaired flow This can result in cerebral ischemia and acute cerebrovascular diseases such as acute ischemic stroke [ ] see Figure pathway IIB iii The RAAS Pathway after SARSCoV2 Figure pathway III The RAAS pathway is crucial in regulating blood pressure as well as the balance of fluid and electrolytes Any disturbance in this pathway can trigger the pathogenesis of cardiovascular diseases [] Before a SARSCoV2 infection triggers the RAAS Angiotensin I Ang I cleaves to Angiotensin II Ang II via ACE1 Ang II causes vasospasm It is also a proinflammatory agent with prothrombotic and proliferative effects that are detrimental to vascular tone and hemostasis [ ] Thus as a counterregulatory mechanism ACE2 degrades Ang II and generates Ang which counteracts the negative impacts of Ang II [ ] Both ACE2 and Ang have cardiocerebral vascular protective effects [] After the triggering of SARSCoV2 infection it results are in the deregulation of RAAS causing heart and brain injury in two different pathways The main culprit is an increase in Ang II which is caused by the decrease in ACE2 levels Figure pathway IIIA First an increase in Ang II levels stimulates the adrenal cortex of the kidney resulting in an increased production of aldosterone Aldosterone is a steroid hormone that causes sodium and water Journal Preproof 0creabsorption to increase at the distal tubule and collecting duct of the nephron [] This reabsorption increases blood volume and causes an elevation in blood pressure which results in the endothelial dysfunction that causes brain and heart injury [] The second effect of an increase in Ang II levels ie as a consequence of decreased ACE2 levels is endothelial dysfunction leading to intimal damage in the arterial walls [] which can be seen during the imaging of the arterial wall see Figure pathway IIIB This pathway can also trigger a cytokine storm as high levels of Ang II can cause an increase in proinflammatory cytokines see the bridge line between the RAAS and immune pathways iv The Immune Pathway Figure pathway IV Several recent studies have reported SARSCov2 viral pneumonia [ ] having an exaggerated inflammatory response known as a œcytokine storm This response appears to present at advanced stages of severe COVID19 with increased levels of inflammatory cytokines leading to multiplean failure [] The rise in inflammatory markers”including IL6 IL7 IL12 IL15 IL22 TNFα and CXCL10”results in the destabilization of plaque This in turn can cause plaque rupture resulting in heart and brain injury [ ] The Role of Imaging in Comorbid Patients with COVID19 As the previous section discussed COVID19 uses four pathways ie neuronal hypoxia RAAS and immune to cause critical heart and brain injuries in patients with comorbidities The prevalence of myocardial injury and brain injury caused by COVID19 [ ] points to a need for increased use of medical imaging to expedite assessments differential diagnoses and patient management [ ] with proper safety measures [] The seriousness of a patient™s COVID19 symptoms helps to determine which imaging modality is appropriate portable or nonportable and invasive or noninvasive BMode ultrasound imaging is portable and can be used for lowrisk patients Meanwhile Xray magnetic resonance imaging [] and computed tomography [] are nonportable and can be used for mediumrisk patients Intravascular ultrasound IVUS [] and ventriculography are invasive imaging modalities used in highly critical cases [ ] Amongst all the imaging modalities ultrasound is noteworthy because it is radiationfree portable quick repeatable inexpensive Journal Preproof 0cand can be performed in isolation thus lowering the chance of spreading the COVID19 infection [ ] There are several examples of medical imaging that have led to proper treatment and healthcare management during the pandemic ultimately reducing the mortality rate Xray imaging of the chest has demonstrated irregular patchy hazy reticular and widespread groundglass opacities indicating the progression of COVID19 at various stages this information can support the healthcare team in developing the most appropriate treatment plan [] Chest CT scans of COVID19 patients revealed in almost of the patients that the disease was affecting at least one of the five lobes of their lungs [] Chest MRI scans of COVID19 patients showed pulmonary tissue consolidation in six diffusionrestricted regions in six and lung damage in seven [] Meanwhile heart MRI studies of recovered patients showed that of the patients had myocardial edema At the same time late gadolinium enhancement was found in implying that COVID19“related cardiac injury is longstanding and requires frequent monitoring even after recovery [] In a different study MR scans of a COVID19 patient revealed myocardial inflammation signifying myocardial injury due to a cytokine storm related to the SARSCoV2 infection as discussed in Section Pathway IV [] Several studies have also evaluated the effects of COVID19 on the brain In one MRI scans revealed hemorrhagic rim enhancing lesions within the bilateral thalami medial temporal lobes and subinsular regions [] shown in Figure In another brain MRI scans were completed for patients of which produced abnormal findings [] Additionally evidence of liver injury and gall bladder abnormality was found in a joint CT and ultrasound study of the abdomen [] Recent MRI scans of COVID19 patients™ olfactory bulbs have revealed the cause of olfactory function loss to be the interaction between SARSCoV2 and the ACE2 protein expressed by the olfactory epithelium which leads to inflammatory obstruction [] Journal Preproof 0cFigure We have shown in four pathways how COVID19 can cause Brain and heart injury Brain image in pathway I httpdebugliescom20200123olfactorydisturbanceshaveimplicationsinmentalandemotionalwellbeinghealth Courtesy of Debug Lies Invasive imaging is another option for diagnosing COVID19 patients who have critical comorbidities In one such study IVUS along with stenting was performed with precautions on a COVID19 patient with myocardial infarction [] shown in Figure A detailed discussion of precautions is included in section In another study takotsubo syndrome a form of myocardial injury triggered by COVID19 was detected using ventriculography [] In various studies the medical imaging of COVID19 patients had been crucial to ascertaining the extent of tissue damage and critical infection although there were no visible symptoms [ ] Therefore medical imaging is the preferred way to ascertain the extent of cardiac and brain tissue damage throughout the lifetime of COVID19 patients COVID19 Journal Preproof 0cpatients with comorbidities are especially vulnerable and so they need to be screened through medical imaging from the first day of their diagnosis Medical imaging can help patients with deep vein thrombosis DVT as they are highly susceptible to severe tissue damage from COVID19 A study showed that COVID19 patients suffering from DVT had a worse prognosis than patients without DVT Patients with DVT were admitted to the ICU more frequently discharged less frequently and suffered more deaths than those without DVT [] Figure MRI scan of COVID19 patient showing hemorrhage MRI images demonstrate T2 FLAIR hyperintensity within the bilateral medial temporal lobes and thalami A B E F with evidence of hemorrhage indicated by hypointense signal intensity on susceptibilityweighted images C G and rim enhancement on postcontrast images D H reproduced with permission [] Journal Preproof 0c Figure Application of chest CT and IVUS for a COVID19 patient suffering from myocardial infarction a Chest CT scan with viral pneumonia showing fibrinous focal exudative changes b when the patient complained of chest pain the ECG report showed the STsegments elevations in V1V5 lead c d CAG radiology that the proximal segment of LAD was occluded e f The blood flow of LAD restoration after DESs was implanted g the dissection distal shown by IVUS to the stent in LAD from “ o'clock h the low echogenic shadow with scattered higher echogenic flicker indicating a thrombus i after a DES was implanted and the stent was well expanded the dissection could not be seen j The thrombus disappearance after the intervention reproduced with permission [] Journal Preproof 0c Although medical imaging can be very useful to patients and doctors alike the exponential pandemic curve inadequate medical facilities [] and a limited number of radiologists make the assessment diagnosis and management processes challenging tedious and errorprone Therefore although medical imaging can make diagnoses faster as stated above it will be of limited use Given this fact new age techniques such as artificial intelligence AI [] applications in medical imaging for tissue characterization can make computeraided assessments and diagnoses faster The main reason for this is that the AI can be scaled up to match the pandemic curve thereby meeting the immediate demands of medical image diagnoses during the COVID19 pandemic AIbased tests can categorize the nature of a patient™s risk in one of the categories namely norisk low lowmedium LM highmedium HM lowhigh LH or highhigh HH risk depending on the patient™s symptoms and their severity [ ] as shown in Figure The imaging modality also varies with the degree of risk as follows no imaging for norisk portable imaging for low and LM risk nonportable imaging for HM and LH and invasive imaging for HH A probability PTP is performed to accurately interpret diagnostic results to categorize the patient into one of the four groups [] After that for norisk patients nonimaging biomarkers can be collected for risk assessment using AIbased data protocols For lowrisk patients portable 2D3D imaging such as ultrasound is used whereas nonportable and invasive 2D3D imaging such as MRICTXRayechocardiography can be used for LM patients For HH patients invasive imaging techniques such as IVUS and ventriculography can be used Based on the data provided by various 2D3D scans AIbased medical imaging is applied for risk assessment Further treatment is then planned based on this imaging process In the next section deep learning DLbased medical imaging is proposed for medical imaging scans particularly ultrasounds for COVID19 patients Machine Learning and Deep Learning for Tissue Characterization Using AI and associated technologies in healthcare can significantly slow down diagnosis times Journal Preproof 0cespecially during the COVID19 pandemic as patient numbers are continually growing and there are few specialists available [] Figure Role of AIbased risk assessment on COVID19 patients having comorbidity Journal Preproof 0cAlthough some caution must be exercised regarding its fullscale deployment [] its overall usefulness in healthcare management during times of crisis cannot be ignored [ ] In general AI in healthcare refers to all artificial intelligencebased technologies that make educated decisions regarding a patient™s diagnosis monitoring treatment and management The importance of AI has specifically increased many folds when imaging comes into play mainly because of large volumetric data sizes and the extensive need to characterize and quantify the disease via lesion images [] Tissue imaging and its characterization is of prime importance since it has a direct influence on decisions related to COVID19 severity for a patient [] The main benefit of AI methods is that the machines can be used to train by mimicking the physician™s cognitive actions and such trained models can be used to predict the disease™s severity in asymptomatic patients Within a short period several machine learning MLbased techniques used the power of AI to manage COVID19 [ ] ML and DL Architectures ML Architecture ML is a twostage process In stage I different features are extracted from the lesion COVID images the extractions are then operated on by an ML statistical model called a training system to generate offline coefficients These coefficients are then transformed by the test lesion images which yield an intelligent classification or inference A typical ML system for predicting risk class is shown in Figure CUSIP is an imagebased phenotype that uses the event equivalent gold standard EEGS [ ] model DL Architecture DL refers to a visual cortex that imitates multiple layers of a neural network applied directly to tissue images to extract features and for characterization purposes [] The convolution neural network CNN [] shown in Figure is one such DL network architecture that is widely used to characterize medical images It performs a series of convolution maxpooling operations to extract features and perform characterizations ML and DL both follow the supervised learning Journal Preproof 0capproach by which models are trained using offline data Figure Typical lowcost machine learning architecture utilizing EEGS model As discussed in previous sections there are four pathways through which a COVID19 infection leads to heart and brain injuries AI can be used via medical imaging to detect the extent of tissue damage in these pathways and help medical professionals to develop an effective treatment plan for patients There are several instances in which the AI paradigm has been used for tissue characterization based on Journal Preproof 0cmedical images during the pandemic as well as during standard times Some uses of AI are described anwise following a proposed model for characterizing DLbased tissues Figure A convolution neural network courtesy of AtheroPoint„¢ CA USA ML Architecture used for Tissue Characterization for Stroke Risk Stratification There are two types of tissue characterization that can be carried out using AI i MLbased [ ] and ii DLbased [] Various MLbased technologies have been developed to classify symptomatic and asymptomatic plaque from ultrasound images For example an MLbased technique based on support vector machines SVM was developed to characterize the symptomatic and asymptomatic plaques of carotid scans that indicated the presence of plaque [ ] SVM classifiers work by determining the maximum margin between two data clusters First a texture analysis [] is used to extract the features ie standard deviation entropy symmetry and run percentage in

Thyroid_Cancer

"Type and diabetes confer an increased risk of pancreatic cancer PaC of similar magnitudesuggesting a common mechanism The recent finding that PaC incidence increases linearly with increasing fastingglucose levels supports a central role for hyperglycaemia which is known to cause carbonyl stress and advancedglycation endproduct AGE accumulation through increased glycolytic activity and nonenzymatic reactions Thisstudy investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanismsinvolvedMethods Pdx1CreLSLKrasG12D mice were interbred with mitosis luciferase reporter mice rendered diabetic withstreptozotocin and treated or not with carnosinol FL92616 a selective scavenger of reactive carbonyl speciesRCS and as such an inhibitor of AGE formation Mice were monitored for tumour development by in vivobioluminescence imaging At the end of the study pancreatic tissue was collected for histologyimmunohistochemistry and molecular analyses Mechanistic studies were performed in pancreatic ductaladenocarcinoma cell lines challenged with high glucose glycolysis and glycoxidationderived RCS their proteinadducts AGEs and sera from diabetic patientsResults Cumulative incidence of invasive PaC at weeks of age was in untreated diabetic vs in FL92616gtreated diabetic and in nondiabetic mice FL92616 treatment suppressed systemic and pancreaticcarbonyl stress extracellular signalregulated kinases ERK activation and nuclear translocation of Yesassociatedprotein YAP in pancreas In vitro RCS scavenging and AGE elimination completely inhibited cell proliferationstimulated by high glucose and YAP proved essential in mediating the effects of both glucosederived RCS andtheir protein adducts AGEs However RCS and AGEs induced YAP activity through distinct pathways causingreduction of Large Tumour Suppressor Kinase and activation of the Epidermal Growth Factor ReceptorERKsignalling pathway respectivelyContinued on next page Correspondence giuseppepuglieseuniroma1it Stefano Menini and Carla Iacobini contributed equally to this work1Department of Clinical and Molecular Medicine œLa Sapienza University Viadi Grottarossa Rome ItalyFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMenini Journal of Experimental Clinical Cancer Research Page of Continued from previous pageConclusions An RCS scavenger and AGE inhibitor prevented the accelerating effect of diabetes on PainINsprogression to invasive PaC showing that hyperglycaemia promotes PaC mainly through increased carbonyl stressIn vitro experiments demonstrated that both circulating RCSAGEs and tumour cellderived carbonyl stressgenerated by excess glucose metabolism induce proliferation by YAP activation hence providing a molecularmechanism underlying the link between diabetes and PaC and cancer in generalKeywords Pancreatic ductal adenocarcinoma Hyperglycaemia Reactive carbonyl species Methylglyoxal Advancedglycation endproducts Carnosine derivatives Yesassociated protein Large tumour suppressor kinase Epidermalgrowth factor receptor Extracellular signalregulated kinases BackgroundPancreatic cancer PaC is the tenth most common incident cancer but the seventh leading cause of cancerrelated death worldwide [] because of the poor 5yearsurvival outcomes [] Due to the rising prevalence ofrisk factors such as obesity and type diabetes PaC isexpected to become the second leading cause of cancerrelated death in the US by [] Type diabetes wasfound to be associated with a “7fold higher risk ofPaC in the first year after diabetes diagnosis and nearlytwofold thereafter [ ] Though type diabetes is themain contributor to this problem the entity and temporal trajectory of PaC risk were recently reported to besimilar in type diabetes [] suggesting a commonmechanism related to hyperglycaemia This concept issupported by the recent finding that PaC incidence increases linearly with increasing fasting glucose levelseven within the normal range []thus hindering the understanding ofPrevious studies have shown that type diabetes induced by a highfat diet promotes PaC [ ] Howeverthis experimental model of the metabolic syndrome doesnot allow assessing the role of hyperglycaemia independent of confounding factors such as obesity and hyperinsulinemiathemechanisms underlying the risk conferred by hyperglycaemia We have recently demonstrated that advancedglycation endproducts AGEs promote proliferation ofhuman pancreatic ductal adenocarcinoma PDA cell linesand that exogenous AGE administration markedly accelerates invasive tumour development in a mouse model ofKrasdriven PaC [] Accumulation of AGEs in diabetesis mainly due to increased formation of reactive carbonylspecies RCS derived from glucose autooxidation egglyoxal GO but also from cell metabolism of excess glucose through glycolysis eg methylglyoxal MGO []In turn RCS react with amino groups of proteins causingstructural and functional modifications The resulting irreversible adducts ie AGEs accumulate in tissues wherethey can exert further biological effects through interaction with specific receptors [ ]Carnosine betaalanylLhistidine is an endogenousinhibits AGEhistidinecontainingdipeptidethat[]carnosine derivativesformation by scavenging RCS [] Though LCarnosinewas proven to be effective in several carbonyl stressrelated disease conditionsincluding metabolicdisorders [“]its therapeutic use in humans ishampered by the presence of high levels of serum carnothus prompting the search for carnosinasesinase[“] The novelresistantbioavailable compound carnosinol ie 2S23aminopropanoylamino31Himidazol5ylFL [“] was shown to be highly effective in attenuating diabetesassociated vascular complications [] and obesityrelated metabolic dysfunctions [ ]Moreover it was recently shown that Lcarnosine is effective in counteracting glycolysisdependenttumourgrowth by quenching RCS []propanollesionsthe progression of preneoplasticThis study aimed at investigating whether hyperglycaemia associated with experimental type diabetes favourstomalignancy in a wellvalidated mouse model of PaC byincreasing carbonyl stress To this end mice weretreated with the RCS scavenger and inhibitor of AGEformation FL92616 An additional objective was toanalyse the effect of the diabetic milieu and of FL926 on the activity of Yesassociated protein YAP a keydownstream target of KRAS signalling required for progression of pancreatic intraepithelial neoplasias PanINsto invasive PaC [ ] and for MGOinduced tumourgrowth []MethodsIn vivo studyThe experimental protocols comply with the principles ofwwwnc3rsukarriveguidelines and were approved by the National Ethics Committee for Animal Experimentation ofof HealthAuthorization no 14702015PR The mice were housedin single cages with woodderived bedding material in aspecific pathogenfree facility with a 12h lightdark cycleunder controlled temperatures “ °C Mice werecared for in accordance with the Principles of LaboratoryAnimal Care National Institutes of Health publ no “ revised and with national laws and receivedItalian Ministrythe 0cMenini Journal of Experimental Clinical Cancer Research Page of water and food ad libitum The primary and secondaryendpoint were the development of invasive PaC and thedevelopmentprogression of PanINs respectivelyDesignThe effect of diabetes on PaC progression was investigatedin Pdx1CreLSLKrasG12D KC mice which develop autochthonous PaC in a pattern recapitulating human pathology with high fidelity by developing the full spectrum ofPaC progression from preneoplastic lesions PanINs toadenocarcinoma and metastasis [ ] KC mice wereinterbred with mitosis luciferase MITO“Luc reporter miceto obtain KCMito KCM mice [ ] The LSLKrasG12D lineage was maintained in the heterozygousstate Mice were screened by polymerase chain reactionPCR using tail DNA amplified by specific primers to theLoxP cassette flanking mutated KrasG12D wild type KrasCre recombinase and MITO genes as previously reported[ ] In the MITOLuc mouse an artificial minimal promoter derived from the cyclin B2 gene and induced by NFY drives the expression of the luciferase reporter specificallyin replicating cells Therefore both normal eg bone marrow and tumour actively proliferating cells may be localized by a bioluminescence imaging BLIbased screen [ ] We have previously shown that KCM mice developpreinvasive PanINs and invasive ductal PaC with thesame penetrance latency and histological features as thosedescribed for KC mice [] According to the Ethics Committee recommendations to limit the number of animalsthe experiments were stopped when it was sufficient toconfirm or reject the working hypothesis in a statisticallyand clinically meaningful mannerFigure shows the flowchart and timeline of study design Thirtythree KCM mice were rendered diabeticFig Flowchart and timeline of study design Please refer to the text for detailed description In dashed boxes groups of nondiabetic KCMmice Ctr that served as control for the effect of STZ STZnonDiab or FL92616 FL treatment Ctr FL on PaC development and progressionTo avoid unnecessary suffering three diabetic mice Diab and one Diab mouse treated with FL DiabFL were killed and weeks respectivelybefore the end of the study STZ streptozotrocin BLI bioluminescence imaging 0cMenini Journal of Experimental Clinical Cancer Research Page of with streptozotocin STZ and followed for weeksie up to weeks of age After an overnight fast weekold mice were intraperitoneally injected with mg·kgˆ’ STZ SigmaAldrich St Louis MO USA Success rate defined as the percentage of STZinjected micewith glucose levels mgdL for the entire studyperiod was Three days after injection diabetic mice were randomized to receive no treatmentDiab n or FL92616 gift of Flamma SpAChignolo d™Isola Italy [] at a dose of mg·kgˆ’ ˆ™dayˆ’ in the drinking water DiabFL n andinjected weekly with IU of insulin glargine to preventexcessive weight loss and ketoacidosis FL92616 wasshown to have a suitable absorption distribution metabolism excretion and toxicity ADMET profile and thegreatest potency and selectivity toward RCS among allother carnosine derivatives [] The FL92616 dosewas chosen based on previous results from our group[]showing high efficacy in preventing diabetesinduced renal injury and from other investigators indicating a good safety profile at the dose of “ g·kgˆ’ ·dayˆ’ [ ] Neither histological abnormalities of theliver kidney lung and heart nor functional abnormalities attributable to toxicity on these tissues wereobserved in this study or in a previous one [] STZtreated mice not fulfilling the criteria for diabetes diagnosis STZnonDiab n served as control for STZeffect on PaC seven of these mice failed to develophyperglycaemia whereas two had spontaneous recoveryfrom diabetes within weeks Vehicle salineinjectedKCM mice were used as nondiabetic controls and either left untreated Ctr n or treated with FL926 Ctr FL n to check for any drug effectMice were subjected to in vivo BLI every otherweek [ ] and daily manual palpation of the abdomen to check for tumour growth and avoid the lossof animals along with the need to cope with the related ethicalissues ie compliance with the 3Rsprinciples Briefly min after administration of Dluciferin mgkg body weightintraperitoneal Perkin Elmer Hopkinton MA USA photon emissionfrom the different body areas was acquired for minand analysed with a CCD camera Xenogen IVIS Lumina System Perkin Elmer A specific region ofinterest ROI corresponding to the abdominal areaoccupied by the pancreas was manually selected andthe total photon flux ps from this ROI was evaluated with Living Image software Caliper Life Sciences Perkin Elmer [ ]At the end of the study mice were anaesthetizedwith ketamine mg·kgˆ’ Imalgene ip and xylazine mg·kgˆ’ Rompum ip and killed by cervical dislocation According to the Ethics Committeerecommendations to avoid suffering three Diab andone DiabFL mice presenting with both positive BLIand a palpable abdominal mass or poor general condition were killed and weeks respectively beforethe end of the study The lungs and the middle partof the gastrointestinal tractincluding the pancreasand the liver were dissected and exposed to theCCD camera for min for photon emission assessment The pancreas was dissected photographed andthen one part was stored at ˆ’ °C forweightedmolecular analysis whereas the other part was processed for histologicalimmunohistochemical analysis[] At time of collection a technician CC seeAcknowledgementstoallow blinded analysisrecoded biologicalsamplesMetabolic parametersBody weight and blood glucose were monitored weeklyAt the end of the study the levels of haemoglobin HbA1c an indicator of longterm glycaemic control wereassessed by using the Mouse HbA1c Assay Kit Crystal Chem Zaandam Netherlands and serum AGEsand total protein carbonyls PCOs two carbonyl stressmarkers were measured by ELISA OxiSelect„¢ Advanced Glycation EndProduct Competitive ELISA Kitno STA817 and OxiSelect„¢ Protein Carbonyl ELISAKit no STA310 respectively Cell Biolabs Inc SanDiego CA USAPancreas histologySix 4μmthick nonserial pancreatic sections stainedwith haematoxylin and eosin were examined to confirm the presence of invasive PaC Pancreas withoutinvasive PaC were analysed to grade dysplastic ductsie PanINs according to previously established criteria [] The numbers oflowgrade PanIN1ABand highgrade PanIN23 dysplastic ducts werecounted and expressed as a percentage of total ductsin the specimen []Pancreatic AGEsERK phosphrylation status nuclear YAP and itstarget gene connective tissue growth factor CTGFLevels of AGEs pERK and CTGF protein in homogenates and of active nonphosphorylated YAP1 innuclear extracts of pancreas of mice were assessed byWestern blot Human PDA tissues n were obtained from the Pathology Unit of Sant™Andrea HospitalRome Italyin agreement with the ethical guidelinesestablished by the locally appointed Ethics CommitteePancreatic tissue distribution of AGEs and activatedYAP1 in mouse and human specimens were evaluatedby dual label immunofluorescence and immunoperoxidase respectively [ ] For immunofluorescence agoat polyclonalrabbitantiAGE antibodyand a 0cMenini Journal of Experimental Clinical Cancer Research Page of sections weremonoclonal antibody to active nonphosphorylatedYAP1 were used as primary antibodies followed by appropriate secondary fluorescent antibodies see Supplementary Table S1 for antibodies in Additional file Sections were analysed at a fluorescence microscopeZeiss AXIO A1 equipped with an Axiocam colorcamera Carl Zeiss Italy Milan Italy For immunoperoxidase formalinfixed paraffin embedded sections μm thick were rehydrated and treated with H2O2in PBS for min to block endogenous peroxidase activity Heat mediated antigen retrieval was performed withœAntigen Unmasking Solution Citric Acid Based H Vector Laboratories Burlingame CA USA forAGE staining or TrisEDTA buffer pH for YAPstaining both for min Nonspecific binding wasblocked by incubation in Protein block serum free AgilentDako Santa Clara CA USA for min at roomtemperature Thenincubated withAvidinBiotin blocking Kit SP2002 Vector Laboratories for min an antiAGE antibody Abcam Cambridge UK ab23722 or an antibody directed to theactive nonphosphorylated YAP1 Abcam ab205270at °C overnight and the appropriate biotinylated secondary antibody at room temperature for min seeSupplementary Table S1 for antibodies in Additional file Finally sections were stained with UltraTek Horseradish Peroxidase ABL015 ScyTek Laboratories UTUSAfor min followed by 33diaminobenzidineDABH2O2 ChromogenSubstrate Kit High ContrastACV500 ScyTek Laboratories until the reaction product was visualized min and counterstained withhematoxylin AGE positive staining and nuclear expression of YAP were measured in random fields of eachsection at a final magnification of 250X and 400X respectively by means of the interactive image analyzerImagePro Premier ImmaginiComputer MilanItaly AGE positivity was expressed as the mean percentage of field™s area occupied by the specific stain Expression status of active YAP in tumor specimens wasassessed by counting the number of nuclei positive forYAP and expressed as the mean ratio of YAPpositive nuclei to total nucleiand AGEstheIn vitro studyThe in vitro study investigated the putative role ofRCStumourpromoting effect of high glucose HG and the protective effect of the carbonylsequestering agent andAGE inhibitor FL92616as mediators ofDesignHuman MIA PaCa2 Catalogue No Lot No14A02 and Panc1 Catalogue No Lot No10G011 cells SigmaAldrich were used for assessing theeffects of HG and FL92616 on cell proliferation Experiments aimed at investigating the molecular mechanismsunderlying the glucosemediated effects and the protection by FL92616 were conducted on MIA PaCa2 cellsMycoplasma contamination in cell cultures was regularlytested by PCR MycoSPY Kit Biontex Laboratories GmbHMunchen Germany Human PDA cells were maintainedin DMEM supplemented with FBS and incubated indifferent conditions for three daysie normoglycaemia normal glucose mM hyperglycaemia HG mM treated with MGO or GO μM SigmaAldrich two RCS and AGE precursors or the preformed AGE Nεcarboxymethyllysine CML μgmLprepared as previously reported [ ] with or withoutFL92616 mM and exposed to DMEM lowglucose medium containing of pooled sera from nondiabetic or diabetic individuals before and after AGE removalfrom diabetic serum by an immunoadsorptionmethod see below with or without FL92616 mMInformed consent was obtained from nondiabetic anddiabetic individuals Moreover both YAP and EpidermalGrowth Factor Receptor EGFR were silenced to assessthe role of YAP and EGFR pathway in RCS and AGEinduced cell proliferation see belowRemoval of AGEs from diabetic serumAGEs were removed from diabetic serum using animmunoadsorption method To immunoprecipitateAGEmodified proteins μl of diabetic serum wasincubated for h with μl of Pierce NHSactivatedmagnetic beads Thermofisher Scientific covalentlyconjugated with μg of antiAGE antibody Abcamsee Supplementary Table S1 for antibodies in Additional file according to the manufacturer instruction To confirm the efficiency of AGE depletionAGE concentration in both treated unbound serumfraction and untreated diabetic serum was evaluatedin triplicate by ELISA OxiSelect„¢ Advanced GlycationEndProduct Competitive ELISA Kit no STA817Cell Biolabs Inc San Diego CA USA Followingthis procedure the concentration of AGEs in diabeticserum was reduced by about reaching a concentration similar to that of the nondiabetic serum seethe œResults sectionYAP and EGFR silencingYAP and EGFR were silenced using smallinterferingRNAs siRNAs and irrelevant scrambled siRNAs as control Thermo Fisher Scientific Waltham MA USAValidated predesigned siRNA oligonucleotides and related TaqMan assays are detailed in SupplementaryTable S2 see Additional file Lipofectamine RNAiMAX Thermo Fisher Scientific transfections were performed using nM of each siRNA 0cMenini Journal of Experimental Clinical Cancer Research Page of Cell survival and proliferationCell viability and proliferation were evaluated by Cytoselect WST1 Cell Proliferation Assay Cell Biolabs following the manufacturer instructionsYAP1 its upstream regulators large tumour suppressor Kinase 1LATS1 and EGFRERK pathway and itsmolecular targets CTGF WTN5A and EMP2 in inhuman PDA cells Cells were extracted in SDS buffer containing protease and phosphatase inhibitorsSigma Aldrich Nuclear protein extracts were obtainedfrom cell monolayers with the Nuclear Extract Kit Active Motif Corp Carlsbad CA USA Protein concentrations were determined using the Bradford Assay KitBioRad Hercules CA USA Nuclear protein levels ofYAP1 and cellular protein levels of total and EGFRphosphorylated at Tyr1068 pEGFR total and pERK and LATS1 a key kinase of the Hippo pathway []were assessed by Western blotting see SupplementaryTable S1 for antibodies in Additional file KRAS activity was evaluated by the KRAS activation Assay Kit noSTA400K Cell Biolabs Inc according to the manufacturer™s protocol Briefly mg of lysate was subjected topulldown and μg of lysate was used to measure totalKRAS Pulldown and totallysates were subjected toWestern blotting procedure using the primary antibodyagainst KRAS provided by the kit The mRNA levels ofCTGFCCN2 WTN5A and EMP2 three recognized molecular targets of YAP [ ] were assessed by realtime PCR RTPCR using a StepOne RealTime PCRSystem and TaqMan Gene Expression assays ThermoFisher Scientific [] listed in Supplementary Table S3see Additional file Statistical analysisResults are expressed as mean ± SD mean ± SEM orpercentage Differences between cell typestreatmentsor animal groups were assessed by oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisons or twoway ANOVA followed bythe Bonferroni posttest as appropriate Betweengroup differencesin PaC incidence were assessedusing the Chisquared test and Fisher™s exact test tocompute a Pvalue from a contingency table A Pvalue of was considered to be significant Allstatisticalincluding linear regression analysiswere performed on raw data using GraphPad Prismversion for Windows GraphPad Software SanDiego CA USAtestsResultsIn vivo studyMetabolic parametersSTZtreated KCM mice developed hyperglycaemiastartingandabout h postinjection Fig 2ashowed a slight decline in the growth curve vs Ctrmice which reached statistical significance only at and weeks of age Fig 2b Despite no differencein body weight Fig 2c blood glucose Fig 2d andHbA1c levels Fig 2e FL92616 treatment preventedthe diabetesassociated increase in circulating AGEsFig 2f and total PCOs Fig 2g as assessed at theend of the studyInvasive PaC development Representative BLI imagesat the end of the study period and total photon fluxinduction from pancreas at and weeks of ageare shown in Fig 3a At sacrifice pancreas weightwas significantly P increased in Diab ± g vs Ctr ± g and vs DiabFL ± g KCM mice Pancreasbody weight percent ratiowas almost tripled in Diab vs Ctr mice whereas nostatistical difference was observed between DiabFLand Ctr mice Fig 3b and Table As assessed byhistology Fig 3c cumulative incidence ofinvasivePaC at weeks of age was in Diab mice vs in DiabFL and in Ctr mice Fig 3d and Table Representative BLI images and pancreas histologyfrom Ctr Diab and DiabFL are shown in Fig 3cdNeither the Ctr FL nor the STZnonDiab groupshowed significant differences in the incidence invasive PaC and pancreasbody weight percent ratio vsthe Ctr group Table Furthermore no betweengroup differences were observed in tumour invasiveness except for an apparent reduction in DiabFL vsDiab group Table However the few cases of PaCin DiabFL n and Ctr n mice prevent toperform statistical comparisons among groupsformetastatic disease Representative ex vivo BLI andhistology images of liver and lung metastases are presented in Supplementary Fig S1 in Additional file Grading of dysplastic ducts in mice free of invasivePaC Table showed significant differences betweenDiabFL and Diab mice for the percentage of normalducts which was higher and of highgrade PanINswhich was lowerin the FL92616 treated arm Inaddition Ctr FL mice presented with higher normalducts and lowerlowgrade PanINs vs Ctr micewhereas no difference was observed between STZnonDiab and Ctr micePancreatic AGEs ERK phosphrylation status nuclear YAP and connectivegrowth factorCTGF Pancreatic accumulation of AGEs Fig 4aand levels pERK Fig 4b CTGF Fig 4c awellestablished transcriptional target of YAP [ ] and nuclear YAP1 Fig 4d were increased inDiab vs Ctr mice and increments were prevented bytissue 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig Glucose and HbA1c levels body weight and hyperglycaemiaassociated carbonyl stress Blood glucose levels and body weight during thestudy period a and b and at the end of the study period weeks of age1 c and d and HbA1c levels e and serum levels of AGEs f andtotal PCOs g at the end of the study period weeks of age1 in control Ctr Ctr treated with FL92616 Ctr FL diabetic Diab and Diabtreated with FL92616 DiabFL KCM mice Statistical significance between groups for time course of blood glucose a and body weight c wascalculated using twoway ANOVA followed by the Bonferroni posttest Each time point represents mean ± SD of animals until the 17th weekof age and “ animals from the 18th to the 22nd week of age Statistical significance for blood glucose c body weight d serum levels ofAGEs e and PCOs f at weeks of age1 was assessed using oneway ANOVA followed by the StudentNewmanKeuls test for multiplecomparisons Each dot represents one case and bars represent mean ± SEM P or P vs Ctr    P vs Diab 1Except for threeDiab and one Diab FL mice which were killed and weeks respectively before the end of the study see œResults section for further detailslabelFL92616 treatment Dualimmunofluorescenceanalysis confirmed the association between AGEs andnuclear YAP1 in PaC lesions from Diab mice Fig4e A significant positive relationship between AGEaccumulation and nuclear YAP1 levels was also observed in human PDA Fig 4fgIn vitro studyProliferation of human PDA cellsHG concentration mimicking diabetic hyperglycaemiapromoted PDA cell growth and this effect was prevented by FL92616 Fig 5ab The AGE precursors RCS MGO and GO and the preformed AGE 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig In vivo BLI and gross and microscopic examination of pancreas Representative BLI at the end of the study period and total photon fluxps induction from pancreas at and weeks of age1 a pancreasbody weight percent ratio b representative pancreas histology coriginal magnification 100X scale bar μm and cumulative incidence of PaC d in control Ctr diabetic Diab and Diab treated with FL DiabFL KCM mice at the time of sacrifice Statistical significance between groups for pancreasbody weight percent ratio a wascalculated using oneway ANOVA followed by the StudentNewmanKeuls test for multiple comparisons Each dot represents one case and barsrepresent mean ± SEM Statistical significance for PaC incidence b was assessed using the Chisquared test and Fisher™s exact test P vsCtr  P vs Diab Is islet invasive PaC arrows PanINs 1Except for three Diab and one Diab FL mice which were killed and weeksrespectively before the end of the study see œResults section for further detailsCML also stimulated PDA cell proliferation FL926 was able to inhibit cell proliferation induced byMGO and GO but not CML Fig 5c Treatmentwith CML but not with MGO induced ERK activation and FL92616 was ineffective in counteractingphosphorylation status Fig 5d However the proliferating effect of both the RCS MGO and the AGECML was associated with YAP1 nuclear persistenceand activity Again FL92616 efficiently preventedeffectCMLtheofonERKthe nuclear translocation of YAP1 induced by MGObut failed to counteract the effect of CML Fig 5eConsistently FL92616 treatment reversed the MGOinduced upregulation of gene expression of CTGFWnt Family Member 5A WNT5A and EpithelialMembrane Protein EMP2three wellrecognizedYAP target genes [ ] Conversely FL92616was ineffective in preventing the modulatory effect ofCML on the mRNA level of these genes Supplementary Fig S2 in Additional file 0cMenini Journal of Experimental Clinical Cancer Research Page of CtrDiab1Diab FL1Ctr FL ± ±     ± Table Pancreatic cancer PaC incidence PancreasBodyweight Wt percent ratio and metastasisPaC NtotPancreasBody Wt Metastasis Ntot PaC ± STZnonDiab ± Cumulative incidence of PaC and PancreasBody weight Wt percent ratio incontrol Ctr diabetic Diab Diab treated with FL92616 DiabFL Ctr treatedwith FL92616 Ctr FL and streptozotocintreated nondiabetic STZnonDiab KCM mice at the end of the study weeks of diabetes weeks ofage1 The number of KCM mice with metastasis liver and or lung on thetotal number of PaC cases is also shown KCM LSLKrasG12D Pdx1Cre MITONtot number of casestotal number of mice Ntot PaC number of casestotal number of PaC PaC ductal adenocarcinoma and hepatic andor lungmetastasis were confirmed by histology P or P vs Ctr   P or  P vs Diab Statistical significance between groups forPancreasBody Weight percent ratio was calculated using oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisonsStatistical significance for PaC rate was assessed using the Chisquared testand Fisher™s exact test Except for three Diab and one Diab FL mice which were killed and weeks respectively before the end of the studyMechanisms underlying RCS and AGEinduced YAPactivationSilencing of YAP1 using two independent siRNAssiYAP1 and Fig 6a significantly inhibitedthe transcription activity of YAP target genes induced by both MGO and CML in PDA cells Fig6b In MGOtreated cells YAP induction was associated with a decrease in protein levels of LATS1 awellestablished negative regulator of YAP activity[] whereas CML treatmentfailed to modulateLATS1 Fig 6c Instead treatment with CML butnot with MGO was found to induce EGFR phosphorylation pEGFR Fig 6d EGFR silencing Fig6e almost completely reversed YAP1 nuclear translocation Fig 6f KRAS activation and ERK phosphorylation Supplementary Fig S3ABinduced by CMLEffects of serum from diabetic patients on proliferation ofhuman PDA cellsThe levels of AGEs were ± μgmL in thepooled sera from diabetic patients and ± μgmLin pooled sera from nondiabetic individuals The diabetic serum induced a 3fold increase in PDA cell proliferation compared to the nondiabetic serum This effectwas greatly reduced by prior selective AGE removalfrom the diabetic serum AGE levels ± μgmLand almost completely reversed by combining AGE removal from serum and FL92616 treatment of PDAcells Fig DiscussionDespite the epidemiological evidence of increased PaCrisk in both type [] and [ ] diabetestheunderlying mechanisms still remains to be elucidatedHere we showed that STZinduced type diabeteswhich is characterized by marked hyperglycaemia andinsulin ia without weight gain [] significantlyaccelerated tumour progression in a mouse model ofKras“driven PaC The absence of obesity and insulinresistance argues in favour of the hypothesis that thePaCpromoting effect of diabetes is directly related tothe adverse effects of hyperglycaemiaIn additionRCS trapping and AGE inhibition by FL92616 efficiently prevented the acceleration of PanIN progression to invasive PaC induced by diabetes Thedifference in the incidence of PaC between the twodiabetic groups ie untreated and treated with FL occurred despite similar increases of bloodglucose levels supporting the conceptthat glucosemetabolites but not glucose per se were responsiblefor PaC promotion STZtreated mice that failed todevelop or reversed hyperglycaemia showed the samePaC incidence as the Ctr group thus ruling out aneffect of STZ on invasive PaC development in DiabmiceOur finding of an association between AGE accumulation and YAP induction in PaC in

Thyroid_Cancer

"Cellular recognition of microbial DNA is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens Cyclic GMPAMP synthase cGAS and its downstream effector stimulator of interferongenes STING are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype I interferons IFNs and other inflammatory cytokines Accumulating evidence suggests that the activation ofthe cGASSTING axis is critical for antitumor immunity The downstream cytokines regulated by cGASSTINGespecially type I IFNs serve as bridges connecting innate immunity with adaptive immunity Accordingly a growingnumber of studies have focused on the synthesis and screening of STING pathway agonists However chronicSTING activation may lead to a protumor phenotype in certain malignancies Hence the cGASSTING signalingpathway must be orchestrated properly when STING agonists are used alone or in combination In this review wediscuss the dichotomous roles of the cGASSTING pathway in tumor development and the latest advances in theuse of STING agonistsKeywords cGASSTING Innate immunity Type I interferon STING agonists Antitumor response CancerdevelopmentIntroductionThe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses[]Protection againston patternrecognition receptors PRRs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens Correspondence zqliucsueducn Juyan Zheng and Junluan Mo contributed equally to this work1Department of Clinical Pharmacology Hunan Key Laboratory ofPharmacogenetics and National Clinical Research Center for GeriatricDisorders Xiangya Hospital Central South University Changsha People™s Republic of China2Institute of Clinical Pharmacology Engineering Research Center for appliedTechnology of Pharmacogenomics of Ministry of Education Central SouthUniversity Changsha People™s Republic of ChinaFull list of author information is available at the end of the adaptive immunity [] Abnormal RNA or DNA RNADNA hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns PAMPs [ ] Cells associated with innate immunity recognize different microbial PAMPsthrough specific PRRs thereby playing key roles in hostresistance to microbial infection [] The pathways governing RNA recognition such as retinoid acid induciblegene I RIGIlike receptors have been reviewed elsewhere and will not be covered herein In the case of DNArecognition one of the best known PRRs is Tolllike receptor TLR9 which senses extracellular CpG hypomethylated DNA that has entered the cytosol through thephagosomelysosome system [] In addition the AIM2like receptor AIM2 inflammasome can be triggered afterthe entry of doublestranded DNA dsDNA into the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZheng Molecular Cancer Page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as IL1and IL18 and the activation of gasdermin D leading topyroptosis [“] Nevertheless the most notable PRR iscGAS a direct cytosolic dsDNA sensor which was identified by Dr Chen™s group in [] Once cGAS bindsto dsDNA the cGASSTING pathway is activated to further induce the expression of type I IFNs and other inflammatory cytokinesthus triggering innate immuneresponses [] Mounting evidence suggests that cGASSTING signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis Hence in this review we summarize themechanism of cGASSTING activation and elaboratefindings regarding its dual effects on tumor developmentCurrent advances in the use of STING agonists as a novelstrategy for antitumor therapy are also reviewedInsights into the cGASSTING signal transductioncascadecGAS is an innate immune sensor that identifies variouscytosolic dsDNAincluding DNA with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderivedDNA and selfDNA Table In the cytoplasm cGAS isactivated by interacting with dsDNA in a sequence[“]independent butStructural and biochemical analyses have revealed thatthe Cterminal lobe of cGAS contains a conserved zinclengthdependent mannerionbinding module that mediates DNA binding andcGAS dimerization [ ] DNA ligands promotecGAS activation primarily by inducing conformationalchanges around the catalytic site and in the DNAbinding structures of cGASthe GScontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cGAS activation byDNA [] In addition to the primary DNAbinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops [] The proximity ofthe two DNAbinding sites in cGAS leads to a cGASDNA complex assembly in which two cGAS moleculesembrace two molecules of dsDNA [ ] The cGASdimers are anized in œheadtohead alignment nextto the DNA [] and thus form stable œladderlike networks between one long curved dsDNA helix or two independent dsDNA strands [ ] In this way eachindividual cGASdsDNA complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsDNA as more likely to activate cGAS [] In additionlong DNA is more efficient than short DNA in drivingthe liquidliquid phase separation of cGAS and the formation ofcriticallydependent on the concentration of cGAS and DNA inthe cytoplasm [] cGAS and dsDNA are spatially concentratedcGASdimerization and activation [“] Once cGAS andcGAS liquidlike dropletsin liquiddropletsistofacilitateTable Classification of the cytosolic dsDNA that activates the cGASSTING signaling axisClassificationSelfDNASource of dsDNAMicronucleiPossible mechanismsRupture of the micronuclei membrane leads to exposureof chromatin DNA that is recognized by cGAS whichactivates the cGASSTING pathwayReferences[]MitochondrionNuclear RNAPathogenderived DNADNA virusHSV1 HSV2 KSHV adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus RetrovirusHIV SIV murine leukemia virusRNA virusWest Nile virus dengue virus VSVSARSCOV2BacteriaListeria monocytogenes Mycobacteriumtuberculosis Listeria Shigella FrancisellaChlamydia and NeisseriaMitochondrial stress induces mtDNA leakage into thecytosol thus activating the STING pathway and inducingproduction of cytokinesFacilitated by endogenous retroelements nuclear RNAcan be reversely transcribed into DNA that activatescGASSTING signaling[][]DNA viruses invade host cells and release pathogenderivedDNA to induce STING activation[“]DNA intermediates generated from reverse transcription maybe recognized by cGAS to stimulate downstream STINGsignaling[]Infection with RNA viruses might cause cellular damage andcell death which results in the release of cellular DNA andfurther activation of the cGASSTING axis SARSCoV2 bindingto ACE2 can lead to excessive angiotensin II signaling thatactivates the STING pathway in mice[“]Bacteria produce CDNs such as cyclic diGMP and cyclicdiAMP which can directly bind to and activate STING[ “]HSV1 herpes simplex virus HSV2 herpes simplex virus KSHV Kaposi sarcoma“associated herpesvirus HIV human immunodeficiency virus SIV simianimmunodeficiency virus VSV vesicular stomatitis virus CDNs cyclic dinucleotides and SARSCOV2 severe acute respiratory syndrome coronavirus 0cZheng Molecular Cancer Page of dsDNA interacts structural switches rearrange the catalytic pocket to enable cGAS to catalyze the synthesis of²²cyclic GMPAMP ²²cGAMP with ATP andGTP as substrates The first step in this process is theformation of a linear dinucleotide ²pppG ²²pAwith ATP serving as the donor and ²OH on GTP serving as the acceptor Then the intermediate product flipsover in the catalytic pocket placing GTP at the donorposition and AMP at the acceptor position to form asecond ²² phosphodiester bond [ ] Notablyalthough dsRNA or singlestrand DNA ssDNA is ableto bind to cGAS neither can rearrange the catalyticpocket which may explain the exclusive activation ofcGAS by dsDNA Ultimately cGAMP acts as a secondmessenger to bind to and activate STING a small endoplasmic reticulum ERlocated protein KD withfour putative transmembrane domains [ ] Normally in a resting state STING is retained in the ER byinteracting with the Ca2 sensor stromalinteractionmolecule STIM1 [] The cytosolic ligandbindingdomain LBD of STING exists as the most functionalunit capable of integrating with ²² cGAMP or CDNscyclic dinucleotides such as cdiAMP cdiGMP or ²²cGAMP from bacteria Upon interaction the obviousclosure of the ligand binding pocket in the LBD is observed which is related to the activation of STING []Next STING transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe ER to the perinuclear area facilitated by cytoplasmiccoat protein complex II COPII and ADPribosylationfactor ARF GTPases [ ] In the Golgi STING ispalmitoylated atCys88 andCys91 a posttranslational modification necessary forSTING activation [] Modified STING recruits thekinase TANKbinding kinase TBK1 in turn the Cterminal domains of STING are phosphorylated byTBK1 and then phosphorylated STING recruits interferon regulatory factor IRF3 which is also phosphorylated by TBK1 and dimerizes ultimately dimerizedIRF3 enters the nucleus and exerts its function in thetranscription of type I IFNs and interferonstimulatedgenes ISGs [] In parallel STING can also bind toand stimulate IκB kinase IKK to mediate the production of nuclear factorκB NFκBdriven inflammatorygenes Upon signal transduction termination STING istransferred to endolysosomes for degradation [] Considering that cGAMP can be transferred through gapjunctions or delivered in viralexosome packages cGASSTING signaling may be activated in the cytoplasmwithout dsDNA [ ] Moreover newly produced typeI IFNs activate heterodimer interferon receptors IFNAR1 and IFNAR2 through paracrine signaling and thusinduce the transcription of ISGs [ ] In summaryonce virusderived DNA and selfDNA are located intwo cysteine residuesthe cytoplasm they can be sensed by cGAS and a cGASdsDNA complex is formed to catalyze the synthesis of ²²cGAMP with ATP and GTP Then ²²cGAMP and bacteriaderived CDNs induce STING activation and mediate the release of downstream type IIFNs TNFα and IL6 which are prerequisites for antimicrobial defense and antitumor effects The wholeprocess shows that the dsDNAcGASSTING axis canlead to the activation of both innate and adaptive immunity Fig The antitumor functions of the cGASSTINGsignaling pathwayRecent evidence has revealed the close association of thecGASSTING pathway with cancer development Thissignaling pathway is generally regarded as a potent regulator of cancer immunity A STINGmediated immunesupportive microenvironment can hamper malignancyoccurrence []stressbyTumor cell cytosolic dsDNA induces STING activationUnder normal circumstances DNA is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity [] However DNA leaks aberrantly in tumorcells [ ] Cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism [] Under these intense states nuclear andmitochondrial DNA is fragile and easily damaged whichleads to eventual DNA leakage in the forms of micronuclei chromatin fragments andor free telomeric DNA[ ] Chromosomal instability CIN is the primary source of cytoplasmic DNA in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance [] Excessive proliferation of cancer cells results in unstable genomes [] usuallychromosomal missegregation during mitosis Due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner [] The vulnerable membraneof micronuclei easily exposes the inner DNA to the cytoplasm and activates the cGASSTING signaling axis [] Exogenous stimuli such as chemotherapy and irradiation can also cause DNA damage In addition to leakednuclear DNA oxidative stressinduced mitochondrialDNA leakage is another crucial initiator of STING pathway activation Several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death Therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial DNA escape [ ] Other sourcessuch as apoptotic cellderived DNA exosomal DNAExoDNA and transposable elements have also beencharacterized 0cZheng Molecular Cancer Page of Fig The cGASSTING DNA sensing signaling pathway Various DNA derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic DNA sensor cGAS cGAS catalyzes the synthesis of ²²cGAMP in the presence of ATP and GTP then ²²cGAMP bindsto the ER adaptor STING which also can be activated by CDNs derived from bacteria Upon activation STING translocates from ER to Golgicompartments where it activates TBK1 and IKK which phosphorylate IRF3 and IκBα respectively Then IRF3 and IκBα dimerize and enter nucleusinitiating the transcription of Type I IFN TNF and IL6 The primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cGAS“STING activation intumor cells [ ]Type I IFNs mediators of STING and adaptive antitumoreffectscGASSTING signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner On the one hand DNA damage can provokeacute STING signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype SASP which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] In contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein BCL2associated X BAX and downregulating the BCL2 apoptosis[] On the other hand STINGsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type I IFNs to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination []STING activation in nonmalignant cells causes tumorsuppressive effects as well STING signaling protectsagainst colitisassociated carcinomas CACs induced byazoxymethane AOM and dextran sulfate sodiumDSS which induce DNA damage in intestinal epithelialcells and further trigger STING activation Downstreamcytokines of STING signaling such as IL1 and IL18prevent neoplastic transformation by facilitating woundrepair More importantly STING signaling can also provoke cytotoxic T cell responses to control tumorigenesis[] Necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor ATFlike BATF3drivenlineage of dendritic cells DCs [] BATF3 DCs take intumorassociated antigens and migrate towardsthe 0cZheng Molecular Cancer Page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific CD8 T cellsThen CD8 T cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells In turn damaged cancercells release more antigens that are further captured byDCs the whole process forms a positive feedback loopcalled the cancerimmunity cycle [] Tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and T cell priming and activation withtumor antigenspecific T cell priming and activationrelying on DCs and type I IFN release [] The involvement of type I IFNs in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate PRR pathways as potential immunomodulators Mice lacking TLR9 myeloid differentiationprimary response gene MyD88 cytosolic RNA sensor MAVS or the purinergic receptor P2X7R maintainintact antitumor immunity responses whereas mice deficient in STING or IRF3 present with impaired CD8 Tcell priming and activation [ ] In fact dying tumorcells can release multiple damageassociated molecularpatterns DAMPs to trigger innate immune responsesin DCs among these released stimuli tumor cellderivedDNA is a pivotal inducer In general the phagocytosis ofapoptotic cells causesimmune silence because ofDNasebased degradation [] Nevertheless tumor cellreleased DNA can be preserved in the DC endolysosomal compartment through an unknown mechanism [] cGAS recognizes DNA invading the cytoplasm andinduces the activation of STING cascades excretion oftype I IFNs and expression of ISGs Additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial DNAmight be packaged in exosomes Exosomal DNAExoDNA animates STING signaling once it is absorbedby tumorinfiltrating DCs [] Finallytumor cellderived cGAMP can also be transferred to host DCs bythe folate transporter SLC19A1 and then directly bindsto STING activating it in DCs [] A recent study moredirectly demonstrated that cellautonomous STING promoted the maintenance of stem celllike CD8 T cellsand augmented antitumor T cell responses and mechanistically cGASSTINGmediated type I interferon signaling reinforced the stem cell“like CD8 T celldifferentiation program mainly by restraining Akt activity []Immune cellderived type I IFNs have crucial functions in antitumor immunity control On the one handtype I IFNs boost cross presentation by various mechanisms first they stimulate the maturation of DCs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of MHC I molecules on the cellsurface [ ] finally they accelerate DC migrationtowardslymph nodes where they can crossprimetumorspecific CD8 T cells [] On the other handtype I IFNs drive the expression of multiple chemokinessuch as CXCL9 and CXCL10 both of which are necessary for cytotoxic T lymphocyte CTL transfer and infiltration [] Similarly type I IFNs restrain the defaultimmune suppressive action of regulatory T Treg cellsby downregulating phosphodiesterase PDE4 and upregulating cyclic AMP cAMP [] Consequently typeI IFNs serve as bridges linking the cGASSTING pathway with CD8 T cellmediated antitumor immunityThe antitumor mechanisms of the cGASSTING signaling axis are illustrated in Fig Indeed previous studies revealed that STING activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [“] Additionally STING expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent STING expression [] Lower STING expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] Consistentlycompared with that in the MCFG10A mammary epithelial cell line lower STING expression was detected inmalignant breast cancer cellincluding MCF7HBL100 and T47D cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] Collectivelythat cGASSTING signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinesSTING pathway agonists as cancer therapeuticsThe immunostimulatory potential of the cGASSTINGpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmentTME can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectorT cells Recent drug research has focused on the development of STING agonists because of their potential inanticancer therapy [ ] To date various kinds ofSTING agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates DMXAA and its analogsand small molecular agonists In addition some conventional antitumor therapeutics can also indirectly activateSTING such as chemotherapy radiotherapy RT andtargeted therapy [] In addition STING agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination STING 0cZheng Molecular Cancer Page of Fig The antitumor immunity effect of the cGASSTING pathway DNA damage leads to the formation of dsDNA in tumor cells upon itsstimulation STING signaling is activated and promotes the release of Type I IFN which is crucial for DC maturation STING signaling activation inDCs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cGAMP gap junctions Then DCs migrate towards the tumordraining lymph node and crossprime tumor specific CD8 T cells withthe help of Type I IFNs Finally T cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowCyclic dinucleotides CDNsCDNs constitute a main type of STING agonist whichmainly originate from bacteria The known naturalCDNs consist of exogenous cyclic diGMP cdiGMPcdiAMP ²²cGAMP and endogenous ²²cGAMPAmong these cdiGMP cdiAMP and ²²cGAMPare synthesized by bacteria and identified as secondarymessengers that mediate STING signal transduction inprokaryotic cells while ²²cGAMP functions as theinitiator of STING in mammalian cells [] The antitumor potential of these natural dinucleotides was firstproven by the finding that cdiGMP could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomaH508 cells was inhibited with μM cdiGMP []Intraperitoneal ip injection of highdose cdiGMPdirectly activated caspase3 and triggered T1 tumoripcell apoptosis in vitro nmol of cdiGMP reduced thegrowth of T1 tumor cells in vitro by and nmreduced it by while lowdose cdiGMP nmol accelerated the adaptive T cell response by converting a subgroup of myeloidderived suppressor cellsMDSCs into immune stimulatory cells producing IL12injection of ²²cGAMP [] Consistentlymgkg expedited dramatic leukemic elimination in ElTCL1 transgenic mice bearing chronic lymphocyticleukemia CLL and promoted tumor shrinkage of multiple myeloma in vivo [] From the perspective of endogenous CDNs ²²cGAMP mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing CT26 colon adenocarcinomain a dosagedependent manner relying on DC activationand T cell crosspriming [] More recently OhkuriT further demonstrated that intratumoral it injection of ²²cGAMP μg25 μLdose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0cZheng Molecular Cancer Page of cancer T1luc squamous cell carcinoma mSCC1colon cancer CT26 and melanoma B16F10 mousemodels [] Notably the it injection of ²²cGAMPinhibited not only tumor growth but also lung metastases in mice bearing B16F10 cellderived tumors suggesting that cGAMPinduced CD8 Tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth []termedvaccineSTINGVAXConsidering the superior properties of STING signaling in activating adaptive immunityit is rational toutilize STING agonists such as CDNs as cancer vaccineadjuvants to increase tumor immunogenicity [] Fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor GMCSF and bacteriaderived or synthetic CDNs Theyobserved that after it injection of STINGVAX with μg of CDNs per vaccine dose the volume of B16melanoma tumors was dramatically reduced in a dosedependent manner Compared to mice receiving GMCSF cancer vaccine alone STINGVAXtreated mice hadmore infiltrating CD8 IFNγ T cells in the tumormicroenvironment The in vivo antitumor effect of STINGVAX was also verified in models of colon carcinomaCT26 pancreatic carcinoma Panc02 and upper aerodigestive squamous cell carcinoma SCCFVII []Although natural CDNs are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting First native CDNs are easily degraded by enzymes inside the cellor in the bloodstream Second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede CDNs from penetrating cell membranes to activate cytosolic STING leading to low bioavailability andpoor retention of the CDNs in specific cells and tissuesThird unintentional toxicities and narrow therapeuticwindows are also unavoidable Thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening [] Regarding agonistdelivery Smith reported that biopolymer implantscodelivering cdiGMP μg and chimeric antigen receptor T CART cells resulted in significant tumor regression in mice bearing pancreatic tumors[]Moreoveriv administration of cdiGMPYSK05Lip equivalent to μg of cdiGMP aYSK05liposome delivery system encapsulating cdiGMP led to a tremendous decrease in metastatic lesionsin a B16F10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced [] Chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cGAMP cGAMPNP could activate the STING axis more effectively than solublecGAMP and converted the immunosuppressive TME toa tumoricidal state in a transplanted B16F10 cell melanoma model and in a genetically engineered triplenegative breast cancer model [] Moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of STING agonistsIntroduction of a dinucleotide cyclasecoding gene intothe Escherichia coli Nissle strain was an attempt at realizing this effect however advancements to the systemare needed []tobysnakeApartdigestionresistancecompoundatoms The modifiedfrom improving delivery methods CDNswith superior properties are currently being synthesized and tested For instance to prevent enzymatichydrolysis of cGAMP the nonbridging oxygen atomsin cGAMP phosphodiester linkages were replaced by²²sulfurcGsAsMP showed resistance against degradation byENPP1 a major ²²cGAMP hydrolasetherebyleading to a longer halflife and sustained high affinity for human STING hSTING[] Syntheticdithio mixedlinkage CDNs with both Rp Rp R Rand Rp Sp R S dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforSTING A novel superior modified product ML RRS2 CDA also termed ADUS100 had the potencyto activate all hSTING variants and mouse STINGmSTING ADUS100 had higher efficiency in activating STING signaling than endogenous or exogenous CDNs mainly because of its enhanced stabilityand lipophilicity Its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding B16 melanoma T1 breast cancer and CT26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of ADUS100 three mg doseswhen tumor volumes reached mm3 [] TheremarkableforhSTING laid the foundation for its clinical use Related clinicaltrials of ADUS100 are outlined inTable In addition to ADUS100 some other novelSTING agonists have been well designed IACS8779and IACS8803 are two highly potent ²²thiophosphate CDN analogs that induced striking systemicantitumorin a B16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with ADUS100or cGAMP [] The characteristics and preclinicalapplications of all these mentioned CNDs are summarized in Table Because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of CDNs have beenand high affinityresponsescurativeeffect 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonistsClassificationCharacteristicsApplicationmodelsNatural CDNagonistscdiGMPPoor membrane permeabilitysuitable for various codeliverytechnologiesColon cancer H508cells T1 metastaticbreast cancerTreatmentinformation μM nmol ip nmol ip nmol ip²²cGAMP²²cGAMPHigher binding affinity formSTING than for hSTINGHigher affinity for hSTING thanits lineage isomers binds tovarious STING nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembraneChronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipCT26 colonadenocarcinoma mgkgbreast cancer T1lucsquamous cellcarcinomasmSCC1 μg25 μLdose it μg25 μLdose itcolon cancer CT26 μg25 μLdose itmelanoma B16F10 μg25 μLdose itTherapeutic effects References[ ][][ ]Inhibitsproliferation tumorregression tumorregressionAccelerates TcellresponseLeukemiaeliminationSuppressesgrowthRestrainstumorigenesisImproves survivalrateDelays tumrowthDelays tumrowthDelays tumrowthDelays tumrowthSTINGVAXSyntheticCDNagonistsPotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercGAMPNPsBiopolymer scaffolds cdiGMP and CAR T cellscdiGMPYSK05Lip²²cGsAsMPADUS100IACS8779IACS8803NonCDNagonistsFAALiposomal nanops NPsdeliver cGAMP intracellularlymore effectively than realizedwith soluble cGAMPEradicates tumors moreeffectively than systemicdeliveryYSK05 is a lipid that can efficientlydeliver cdiGMP to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapeMore resistant to degradation byENPP1 tenfold more potent atinducing IFN secretion potentialuse as a cancer vaccine adjuvantImproves stability and lipophilicityhigher affinity for hSTING thannatural CDN agonists capable toactivate all hSTING variants andmSTINGStimulates a superior systemicantitumor response thanADUS100 and cGAMPCauses hemorrhagic necrosisfailed in a phase I clinical trialdue to species specificity μg CDNs itReduces tumorvolume[]B16 melanomacolon carcinomaCT26pancreaticcarcinoma Panc02B16F10 melanomaivTNBCCreates atumoricidal state[]Pancreatic cancer μg cdiGMPTumor regression[]B16F10 mousemelanoma μg cdiGMP ivDecreasesmetastasisTHP1 monocytesB16 melanomathree mg doses it T1 breast cancerthree mg doses itMC26 colon cancerthree mg doses itDurable tumorregressionDurable tumorregressionDurable tumorregression[][][]B16 melanoma μg on day and posttumor implantationAntitumorresponse[]Murine colontumorsExtensive tumorrejection[ ]DMXAAFirst discovered as a vascularRat mammary mgkg ipHigh anticancer[ 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonists ContinuedClassificationCharacteristicsApplicationmodelsTreatmentinformationInduces proinflammatory cytokinesin a STINGdependent mannerHuman fibroblastsAntiviral activity[]Selectively induces STINGdependentsynthesis and secretion of bioactiveIFNs no evidence of binding directlyto STINGActivates STING

Thyroid_Cancer

Drug repurposing identifying novel indications for drugs bypasses common drug development pitfalls to ultimately deliver therapies to patients faster However most repurposingdiscoveries have been led by anecdotal observations eg Viagra or experimentalbasedrepurposing screens which are costly timeconsuming and imprecise Recently more systematic computational approaches have been proposed however these rely on utilizing theinformation from the diseases a drug is already approved to treat This inherently limits thealgorithms making them unusable for investigational molecules Here we present a computational approach to drug repurposing CATNIP that requires only biological and chemicalinformation of a molecule CATNIP is trained with diverse small molecules and uses different drug similarity features such as structural target or pathway based similarityThis model obtains significant predictive power AUC Using our model we createda repurposing network to identify broad scale repurposing opportunities between drugtypes By exploiting this network we identified literaturesupported repurposing candidatessuch as the use of systemic hormonal preparations for the treatment of respiratory illnessesFurthermore we demonstrated that we can use our approach to identify novel uses fordefined drug classes We found that adrenergic uptake inhibitors specifically amitriptylineand trimipramine could be potential therapies for Parkinson™s disease Additionally usingCATNIP we predicted the kinase inhibitor vandetanib as a possible treatment for Type Diabetes Overall this systematic approach to drug repurposing lays the groundwork tostreamline future drug development effortsa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Gilvary C Elkhader J Madhukar NHenchcliffe C Goncalves MD Elemento O Amachine learning and network framework todiscover new indications for small moleculesPLoS Comput Biol e1008098 101371journalpcbi1008098Editor Avner Schlessinger Icahn School ofMedicine at Mount Sinai UNITED STATESReceived September Accepted June Published August Copyright Gilvary This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data is available atthe following URL wwwgithubcomcoryandarCATNIPFunding JE is supported by NLM of the NationalInstitutes of Health under award numberF31LM013058 The content is solely theresponsibility of the authors and does notnecessarily represent the official views of theNational Institutes of Health OE and his laboratoryare supported by NIH grants 1R01CA1945471U24CA210989 P50CA211024 UL1TR002384PLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingThe funders had no role in study design datacollection and analysis decision to publish orpreparation of the manuscriptCompeting interests OE is cofounder and equityholder in OneThree Biotech and Volastra twocompanies that use data science and machinelearning to develop novel therapies In addition OEis an advisor and equity holder in Freenome andOwkin NM is cofounder and equity holder inOneThree Biotech a company that use datascience and machine learning to develop noveltherapies CG is cofounder and equity holder inOneThree Biotech a company that use datascience and machine learning to develop noveltherapies This does not alter our adherence to allPLOS Computational Biology policies on sharingdata and materialsAuthor summaryCurrently clinical approval of a drug is an arduous process that results in an overwhelming number of compounds failing due to safety or efficacy concerns which leaves patientswithout novel lifesaving treatments The idea of drug repurposing is to take approveddrugs or compounds that were shelved due to reasons other than safety and identify newdiseases for them to treat This would allow drugs if they are sufficiently effective toquickly go through the FDA approval process and be available to patients quicker whichalso cuts the ever growing cost of novel compound research and development Here weintroduce CATNIP a computational model that can predict novel indications for specificdrugs or entire drug classes This approach analyzes drug similarity across a wide range ofbiological chemical and clinical features giving a complete picture of each drug™s mechanism and possible indications Interestingly CATNIP can be used for drugs that not onlyare previously approved but also shelved compounds which are often overlooked in previous repurposing analyses Most importantly CATNIP successfully identified noveltreatments for both Parkinson™s disease and Type Diabetes which are currently undergoing preclinical validationIntroductionWith over million spent bringing a single drug to market over the course of yearsdrug development has remained a costly and timeconsuming affair[] In response there hasbeen an increase in interest in drug repurposing the identification of novel indications forknown safe drugs Successes in this area have been seen in the past most notably in sildenafileg Viagra which was originally intended to treat hypertension and angina pectoris but waslater repurposed to treat erectile dysfunction Other examples of compounds repurposed fornew therapeutic applications include minoxidil[] and raloxifene[] which are now used totreat androgenic alopecia and osteoporosis respectively However most of these repurposingopportunities were discovered through inefficient approaches including anecdotal observations or hypothesisdriven investigations and a more efficient approach could lead to manymore repurposing opportunitiesComputational approaches for repurposing drugs are appealing in that they can be systematically and quickly applied to many drugs at a low cost compared to their experimental counterparts One computational approach that has proven to be invaluable in other areas of thedrug development pipeline is machine learning Machine learning is the use of computationalalgorithms to learn from available data to make novel predictions and gain new insight Usingthis technique one can create unbiased algorithms to match seemingly disparate drugs bycomparing their common features[] such as clinical indication toxicity profile[] or therapeutic target[ ] Previously our lab used a ˜similarity™ approach leveraging the principlethat similar drugs tend to have similar characteristics to predict a drug™s target by investigatingthe known targets of other drugs that were predicted to be œsimilar to the investigated drugbased on shared features[] We found that DRD2 a dopamine receptor was the predicted target for the compound ONC201 After identifying and experimentally validating this targetclinical trials were shifted to focus on gliomas which are now successfully completing phasetwo trials at the time of this publication[] The approach of leveraging drug similarity couldimmensely aid drug repurposing efforts with the appropriate dataPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingOthers have successfully used this ˜similarity™ approach to repurpose drugs and demonstrated high predictive power when tested against FDA approved drugdiseases[] However these methods have primarily linked drugs together using a diseasecentric approachinstead of using features related to the drug itself ie drugcentric These repurposingopportunities are identified by predicting diseases similar to the diseases a drug is alreadyknown to treat Disease similarities can be based on semantic pathophysiological or clinicalsimilarities related to the drug™s clinical indication For example PREDICT a repurposingmethod developed by Gottlieb et al[] exploits the semantic similarity of disease terms asa form of diseasedisease similarity Such approaches while reliable limit the scope of therepositioning effort in several ways First the vast majority of small molecules never reachclinical approval and would be overlooked in this type of analysis Second the use of a diseasecentric approach biases repurposing predictions toward exclusively similar clinical diseases ie cancer drugs to other cancer types [] We postulated that using solely druginformation such as chemical and biological features would be a more effective andbroader approach to drug repurposingHere we propose a novel approach to drug repurposing which operates by a platform wecall Creating A Translational Network for Indication Prediction CATNIP CATNIP is amachinelearning algorithm that learns to predict whether two molecules share an indicationbased solely on the drug™s chemical and biological features using unique drugs The systematic application of CATNIP to molecule pairs creates a network with million nodesthat can then be used to identify potential drug repurposing opportunities By identifying feature importance through the use of chemical structure and target information to make broadscale predictions CATNIP is able to effectively bridge between different therapeutic indications to advance methods of drug repurposing In this report we have identified various candidate drug classes that are predicted to have therapeutic activity outside of their intendedindication in diseases such as Parkinson™s disease and Type DiabetesResultsVariance in drug indication nomenclature can be standardizedWe collected a wide variety of drugs N including both approved and investigationalmolecules with a diverse set of indications to ensure that our drug network covered a largeportion of the known chemical space A subset of these drugs FDA approved drugs and indications taken from DrugBank [] were used as a goldstandard of drugindicationassociations in the training set for the model Disease names are often not standardized whichcan lead to many diverse names for the same disease This problem leads to many drug pairsappearing to not have shared indications when they are associated with two different namesfor the same disease To address inconsistencies in nomenclature for drug indications such asœprostate carcinoma and œcarcinoma of the prostate the MetaMap tool [] was applied tomap disease names to UMLS concepts Methods This standardization of medical terminologies allowed us to reconcile various variations in the database allowing us to confirm thatdrugs did in fact treat the same disease Examples of these variations and their mappingsmay be seen in Table Using MetaMap we clustered the DrugBank indications into standardized indications A multitude of indication types were included in this standardization including but not limited to oncological mental health and neurological diseasesS1A Fig Our rigorous standardization of drug indications ensured an accurate training setallowing for the discovery and modeling of drugindication relationshipsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cTable Indication nomenclatures and their mappingsMetamap MappedIndicationIndicationDrugBankIndication IDDrugBankNumber of unique drugsassociated with IndicationUnique drugs associated with Indication IDMachine learning approach to drug repurposingProstateCarcinomaAcne VulgarisAdvanced ProstateCarcinomaAdvanced carcinomaof the prostateSevere AcneAcneDBCOND0070333DBCOND0020265DBCOND0077433DBCOND0019842DementiaVascularIdiopathicPulmonaryFibrosisPaget DiseaseModerate AcnevulgarisMild VascularDementiaDementia VascularDementiasIdiopathic PulmonaryFibrosis IPFMild IdiopathicPulmonary FibrosisPaget™s DiseasePaget™s Disease ofBoneDBCOND0022329DBCOND0022662DBCOND0029264DBCOND0060453DBCOND0031843DBCOND0093824DBCOND0038793DBCOND0030189101371journalpcbi1008098t001IDCyproterone acetate Esterified estrogensGoserelinCyproterone acetate Doxycycline TetracyclineAloe Vera Leaf Benzoyl peroxide Chloramphenicol ClioquinolGlycolic acid Linoleic acid Octasulfur Salicylic acid SilverSpironolactoneEthinylestradiol Minocycline Nestimate TazaroteneMemantineDonepezilGalantamine Trazodone TrifluoperazineNintedanib PrednisolonePirfenidoneAlendronic acid Pamidronic acid Risedronic acid ZoledronicacidEtidronic acidDrug pairs sharing indications have other similar characteristicsWe hypothesized that pairs of drugs that shared at least one indication would have other similar drug characteristics S1 Table To test this hypothesis we integrated the similarity of twodrugs across chemical and biological drug properties and created a computational model topredict if two drugs will share an indication Fig All of the drug similarity features S1Table collected could significantly distinguish between drug pairs known to share an indication and those not known to share an indication S2“S5 Figs For example we found thatdrug pairs with a shared clinical indication according to their listed DrugBank indicationstended to have significant overlap in targets Dstatistic pvalue S2A FigThe feature which best discriminated between drug pairs that shared a clinical indication versus drug pairs that do not was the similarity between the KEGG pathways that each drug™s targets are involved in Dstatistic p S4C Fig Pathway similarity was calculatedas the Jaccard Index between the KEGG pathways that contain each drug™s gene targets Methods The difference in effect size between the target similarity and the pathway similarity Dstatistic vs respectively indicates that the drugs do not necessarily have to targetthe same exact genes but rather the same biological pathway in order to share a clinical indication Additionally we found that drug pairs that share an indication had a more similarchemical structure than drug pairs that did not share an indication Dstatistic pvalue S5A Fig A biological network containing both physical and nonphysicalinteractions was curated containing proteincoding genes drugs and TFsThis curated network provided another feature for our model allowing us to utilize previouslyestablished interactions between proteins to aid with distinguishing drug pairs that share anindication Overall these features seem to indicate sufficient power in differentiating drugsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingFig Schematic of CATNIP repurposing approach A The use of drug similarity properties to predict if two drugs will share an indication using agradient boosting model the model is referred to as CATNIP B Schematic showing the use of CATNIP output scores to create a network with the scoresused as edge weights The colors of each drug represent the known disease and this demonstrates how one could identify novel indications for drugsthrough the network101371journalpcbi1008098g001PLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingFig CATNIP model accurately predicts drugs that share an indication and can be used for repurposing A Receiveroperating characteristic curve for CATNIP the performance for drug pairs with high and low structural similarity is alsoshown B A network of all drug pairs with a CATNIP score higher than Nodes drugs are colored based on ATCclassification and a specific example of repurposing between ATC classifications is highlighted C A graph of all ATCclassification and the median CATNIP score between the drugs belonging to each of them only including drug pairswith CATNIP score The edges between ATC Classifications with the highest median CATNIP scores are colored red101371journalpcbi1008098g002that share and do not share indications which we hypothesized can then be leveraged to createa predictive modelDrug pairs that share indications can be predicted by modelUsing these diverse drug properties as features we trained a Gradient Boosting model to predict if two drugs share a clinical indication A Gradient Boosting model showed superiorresults when compared with other algorithms Methods S2 Table The model output is adrug similarity score hereby referred to as a œCATNIP score which allows us to classifydrug pairs that share clinical indications We performed a 5fold crossvalidation analysis andachieved significant predictive performance with an areaunderthereceiveroperator curveAUC of Fig 2A We confirmed the statistical significance of our model with aPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingprecisionrecall curve PRC because of the class imbalance in our dataset between drug pairsthat share indications against those that do not Shared Not Shared Whencompared to random predictions our model showed significant improvement vs areaunder PRC S6 Fig We retained a low percent of false positive predictions at various cutoffs false positives and false positives at a model prediction probability oftwo drugs sharing an indication of and respectively providing extra confidencethat our predictions can lead to strong repurposing candidatesWe found that the predictive model greatly benefited from the addition of diverse datatypes While structure similarity showed the highest feature importance of any single featureS11 Fig when used as a single feature within a gradient boosting model it only achieved anAUC of S12 Fig Interestingly when only supplying the model with ontology featuresa Jaccard index for the GO terms of the known targets of each drug within a drug pairachieved an AUC of However even at the highest AUC of any single feature typeit is significantly below the performance when combining all feature typesIn certain cases a high predictive performance is expected such as when two drugs arestructurally similar or share targets It has been shown before that structurally similar drugshave a high probability of treating the same indication[] However there continue to bedrug pairs known to treat the same indication that are not structurally similar For exampletamoxifen[] and anastrozole[] are structurally dissimilar compounds Dice similarity that treat the same indication Metathesaurus term Cancer Breast We recalculated our performance metrics to evaluate how our model performed in classifying drug pairsthat shared indications when only exposed to drug pairs with low structure similarityDice High performance was retained under with an AUC Fig 2A Additionally we found that our model performed similarly well when only exposed to drug pairs thatdid not have any known shared targets AUC Fig 2A These performance metricsconfirm that our model is robust enough to predict if a drug pair will share an indication evenfor more difficult prediction tasksNetwork clusters identify drugs with similar clinical characteristicsWe constructed a repurposing network by calculating a CATNIP score for all possible drugpairs found within DrugBank and assigning the drugs as nodes and the CATNIP score as theedge weight We pruned the network using a cutoff value of for the CATNIP scoresFig 2B which included different drug pairs This cutoff is equivalent to a predictedprobability of to share an indication and allowed for a balance between confidencewithin our predictions and drug diversity and availabilityWe hypothesized that drugs sharing at least one indication would cluster together in ournetwork To confirm this theory we classified each drug per its 1st order Anatomical Therapeutic Chemical ATC classification This identification is a method of distinguishing theclinical use of a drug that is widely used in European and North American chemoinformaticsdatabases[] Using ATC we observed clearly defined clusters within the repurposing network Fig 2B Many clusters featured multiple ATC classifications suggesting potential repurposing opportunities For example one cluster included the thiazolidinediones rosiglitazoneand pioglitazone ATC classification ˜Alimentary Tract and Metabolism™ and the fibratesfenofibrate and bezafibrate ATC classification ˜Cardiovascular system™ These two clusteredATC classifications were connected by a high CATNIP score between bezafibrate andpioglitazone an antidiabetic drug a relationship driven by the shared targeting of PPARa andPPARg resulting in the improvement of lipid and glucose metabolism Bezafibrate has shownefficacy in the treatment of Type Diabetes in numerous retrospective and preclinical studiesPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingATC Code ReferenceRespiratory SystemRespiratory SystemRespiratory System[“][“][“][ ]Table Literature Support for ATC Repurposing PredictionsATC Code DermatologicalsAlimentary Tract and MetabolismSensory ansSystemic Hormonal Preparations Excluding Sex Hormones AndRespiratory SystemInsulinsSensory ans101371journalpcbi1008098t002Alimentary Tract and[“]Metabolismincluding Phase trials[“] however is still not an approved antidiabetic The identification of bezafibrate as a potential diabetes treatment is a key example of how CATNIP can beused to identify repurposing opportunitiesWe reasoned that the connections between ATC classifications across all the drug clusterscould provide additional aid for drug repurposing purposes Using the pruned network CATNIP Score we collected all the scores between drugs of differing ATC classificationsFrom this collection we were able to determine the median score associated between each pairof ATC classifications The ATC classifications with the highest median CATNIP scores hadliterature support for numerous repurposing efforts between them Table For exampledrugs with the ATC classifications of œRespiratory System and œSystemic Hormonal Preparations excluding sex hormones and insulins were strongly connected to each other median CATNIP score This connection was driven by highly scored pairs of drugs includingrimexolone to mometasone CATNIP score and prednisone to triamcinolone CATNIP score These connections are supported by the fact that hormonal agents like glucocorticoids and beta adrenergic agonists have been used for decades to relax the airway musculature in patients with reactive airways disease and chronic obstructive pulmonary disease[]Interestingly our analysis identified glucagon a peptide hormone that increases blood glucoselevels as a candidate for œRespiratory System repurposing and this use already has clinicalsupport[][] Additionally drugs classified as œRespiratory System and œDermatologicalwere also observed to be highly associated because of interactions such as the one betweenciclesonide and hydrocortisone CATNIP score Ciclesonide and hydrocortisone do infact share a clinical indication œAsthma Bronchial giving added confidence to our findingsThese types of network observations are important in laying the groundwork for suggestingnovel clinical repurposing strategies for FDAapproved drugsCATNIP identifies novel disease areas for drug classesWe investigated the ability to leverage CATNIP scores to identify repurposing opportunitiesby evaluating specific drug classes Drug classes are predefined in DrugBank In order to identify actionable repurposing possibilities we narrowed this list down to classes containinginhibitors antagonists or agonists of specific gene or protein families We focused our attention on specific disease areas that are attractive for drug repurposing opportunities due to alack of current treatments or high rates of acquired resistance The specific disease areas wereœmental disorders œneurological diseases œdiabetes and œcancer cancer was furtherdivided into specific cancer types due to the large variance in disease pathology between typesMethodsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingWe hypothesized that CATNIP scores could be used to identify specific drug classes thatwould be efficacious for a new disease area For each drug class and disease area we foundthe statistical difference in the CATNIP score distribution between two sets of drug pairsThe first set included pairs that had one drug within the drug class and the other drugapproved for the disease in question while the other set included drug pairs that had onedrug within the drug class and the other drug not approved for the disease in questionMethods We compared the effect size estimated by the Wilcoxon location shift for alldrug classdisease pairs that had a significant difference in distribution compared to drugclassnondisease pairs FDR Supplementary Data By using CATNIP scores wefound that many wellknown drug classdiseases associations could be recovered For example œmuscarinic antagonists were highly ranked for œneurological diseases and many suchagents are FDAapproved for this indication[] In addition we found that œkinase inhibitors were closely associated with the treatment of cancer and œdopamine antagonists forthe treatment of œmental disorders[ ] Wilcoxon Location Shift “ forœkinase inhibitors and select cancer types Location Shift for œdopamine antagonists and œmental disorders pvalue S7 Fig In fact almost all drug classdiseaseassociations contained at least one FDAapproved drug for the respective disease giving usadded confidence in our model Of note each drug was allowed to be categorized intonumerous drug classes leading to unexpected yet easily explained results for exampleœdopamine antagonists appearing as a top drug class for œneurological diseases This isdue to risperidone a drug traditionally used for schizophrenia and mood disorders alsohaving a secondary indication of Alzheimer™s type severe dementiaOur method reached significant levels of predictive power for predicting both drug class”disease associations and individual drugdisease association When predicting drug classdisease associations under our most lenient conditions calling cases where at least one drugwithin the class was known to treat the disease a true positives our method achieved a sensitivity of greater than However this improved to a sensitivity of when we implementedstricter cutoffs ie only calling drug classdisease associations true positives if of drugswithin the class treated that disease S10 Fig We additionally compared our method™s abilityto predict individual predictions to that of a previously highlighted method Gottlieb et al™sPREDICT[] We found our method had a slightly higher AUPRC vs andhigher sensitivity vs S4 Table S1 Methods While these results indicate modest improvements over PREDICT it is important to note that unlike in PREDICT diseaseinformation is not a required feature in CATNIP™s machine learning approach This meansthat CATNIP can be applied towards investigational molecules with no previously knownindications Additionally by not using disease information as a feature repositioning of drugswith known indications using CATNIP is not directly biased by the associated disease indication and instead uses mechanistic features chemical structure and properties targets etc aspart of the repositioning strategyNext we further interrogated the drug classes associated with œneurological diseases andœdiabetes specifically CATNIP scores could correctly identify drug classes known to treatthese diseases Table To identify possible repurposing candidates we focused our attentionon drug classes shown to have a large positive effect size with this CATNIP analysis but are notcurrently approved for treatment For œneurological diseases the use of adrenergic uptakeinhibitors traditionally used as antidepressants was the top repurposing candidate for œdiabetes alpha antagonists and kinase inhibitors were identified as possible novel treatmentsTable We believe further investigation into these drug classes and diseases could lead tosuccessful clinical applicationsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingTable Top Predictions of Drug Class Repurposing OpportunitiesClassDiabetesDiseasePrediction RankAlpha1 AntagonistsKinase InhibitorProtein Kinase InhibitorsProtein Synthesis InhibitorsCytochrome P450 CYP2E1 InhibitorsMonoamine Oxidase InhibitorsNeurologicalAdrenergic Uptake InhibitorsAdrenergic alpha AgonistsProtease Inhibitors101371journalpcbi1008098t003CATNIP interpretability reveals reasoning for repurposing candidatesFrom our list of repurposing candidates we chose two novel drug classdisease associations tofurther investigateAdrenergic uptake inhibitors applied to Parkinson™s disease First we evaluated therelationship between œneurological diseases and œadrenergic uptake inhibitors We focusedon the drug pairs with the highest CATNIP scores ie those predicted with the highest confidence to share at least one indication Fig 3A Of all the adrenergic uptake inhibitors wefound that amitriptyline and trimipramine two antidepressants had the highest CATNIPscores with the œneurological diseases drugs The drugs that shared the strongest connectionswith amitriptyline and trimipramine were drugs approved for Parkinson™s disease PD Specifically metixene atropine pergolide and benzatropine were associated with amitriptylineaccording to CATNIP and trimipramine was associated to benzatropine and rotigotine Trimipramine was also strongly connected with orphenadrine which is sometimes used off labelin PD but will not be included in the following analysesUsing the CATNIP model we evaluated which features contributed towards the predictionof amitriptyline and trimipramine to share an indication with PD drugs We found that targetgene ontology and pathway similarity all strongly contributed to the predictions for both amitriptyline and trimipramine Fig 3B S8 Fig Since target similarity and distance between targets in a proteinprotein interaction network were among the top contributing features weinvestigated which gene targets were shared amongst these drug pairs We found that amitriptyline targets three specific gene classes that are also targeted by at least one of the PD drugsmuscarinic acetylcholine receptors Gcoupled protein receptors GPCRs and alpha adrenergic receptor Trimipramine also targets muscarinic acetylcholine receptors alphaadrenergicreceptors and dopamine transporters which is similar to benzatropine a PD drug All thesereceptors have welldefined relationships with PD and other neurological diseases[ ]which adds support for repurposing amitriptyline andor trimipramineAmitriptyline may be an ideal candidate for use in PD patients We evaluated the sharedmolecular function gene ontology terms shared between amitriptyline and all four PD drugsGPCR activity was once again identified S1“S4 Files We then interrogated the biologicalpathways these drug targets are involved in and found many broad GPCR pathways overlapping between amitriptyline and the PD drugs S9 Fig including the Reactome pathway œGASTRIN_CREB_SIGNALLING PATHWAY VIA PKC AND MAPK Several recent studiessupport the link between gastrinreleasing peptide signaling to brain function[] ThroughCATNIP we have identified œadrenergic uptake inhibitor

Thyroid_Cancer

"metastasis is a major cause of death in cancer patients new antimetastatic strategies are needed to improvecancer therapy outcomes Numerous pathways have been shown to contribute to migration and invasion ofmalignant tumors Aspartate hydroxylase ASPH is a key player in the malignant transformation of solid tumorsby enhancing cell proliferation migration and invasion ASPH also promotes tumor growth by stimulation ofangiogenesis and immunosuppression These effects are mainly achieved via the activation of Notch and SRCsignaling pathways ASPH expression is upregulated by growth factors and hypoxia in different human tumors andits inactivation may have broad clinical impact Therefore small molecule inhibitors of ASPH enzymatic activity havebeen developed and their antimetastatic effect confirmed in preclinical mouse models ASPH can also be targetedby monoclonal antibodies and has also been used as a tumorassociated antigen to induce both cluster ofdifferentiation CD and CD4 T cells in mice The PAN3011 vaccine against ASPH has already been tested in aphase clinical trial in patients with prostate cancer In summary ASPH is a promising target for antitumor andantimetastatic therapy based on inactivation of catalytic activity andor immunotherapyKeywords ASPH Small molecule inhibitor Metastasis ImmunotherapyBackgroundCancer is a multifactorial disease with an approximate million fatalities in Worldwide it is the secondleading cause of death [] The complex modifications inthe genome affected by the interactions between hostand environment lead to cancer development and progression Despite advancements in characterizing themolecular mechanisms of oncogenesis tumor progression and metastasis [] delayed cancer detection limitedsurgical options therapeutic resistance and tumor recurrence are serious obstacles in decreasing the prevalence and fatality rate of cancer Since metastasis is theprimary cause of deaths from cancer the design oftherapeutictarget mechanisms oftumorcell migration and invasiveness is essential In thisapproachesthat Correspondence smahelmnaturcunicz1Department of Genetics and Microbiology Faculty of Science CharlesUniversity BIOCEV Vestec Czech RepublicFull list of author information is available at the end of the regard a growing number of investigations of signalingpathways involving products of oncogenes and tumorsuppressor genes in human carcinomas has helped toelucidate the mechanisms underlying malignant transformation of cells and facilitated the development ofnew and more efficient therapeutic methodsAspartate hydroxylase ASPH has been identified asone of the cell surface proteins associated with malignant transformation of tumor cells [ ] ASPH belongsamong the most important biological targets to controlmigration and invasion of tumor cells as its overexpression has been observed in “ of human solid tumors [“] The overexpressed ASPH is transportedfrom the endoplasmic reticulum to the plasma membrane which results in exposure of the Cterminal regionto the extracellular environment where it is accessible toantibody binding Recently molecular targeted therapyhas been developed against this target using small molecule inhibitors SMI that can inhibit the catalytic site The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKanwal Journal of Experimental Clinical Cancer Research Page of in the Cterminal region Moreover as antigenic epitopes that reside on the ASPH protein can efficientlystimulate cluster of differentiation CD and CD8 Tcell responses unique to tumor cells harboring ASPHthis enzyme can be used as a tumor associated antigenTAA in immunotherapy [ ]ThedioxygenasesStructure of the ASPH gene and isoformsASPH is a type II transmembrane protein of approxito the family of αmately kDa that belongsketoglutaratedependenthydroxylated products of ASPH hydroxylation were firstdetected in blood coagulation proteins [“] ASPHwas initially identified in the bovine liver as an enzymeresponsible for catalyzing the hydroxylation of aspartyland asparaginyl residues in calcium binding epidermalgrowth factor cbEGFlike domains of various proteins[] Fig Thereafter the human ASPH gene wascloned and characterized [] This gene spanning base pairs long region of genomic DNA and containing exons is located at the position q123 of thehuman chromosome The ASPH sequence is highlyconserved in mammalian evolution The sequence of thehuman protein is from about identical to the sequences of rat and mouse analogs and the catalytic siteis quite conserved among proteins of these three species[] The whole ASPH protein consists of five domainsan Nterminal cytoplasmic a universal transmembranea positively charged luminal a calcium binding and a Cterminal catalytic domain [] Tissue specific transcription is directed from two putative promoters P1 and P2which differ in their regulation sequences [ ] Whilethe transcription from the P1 promoter was observed inmost human tissues the P2 promoter is activated by thecalciumdependent transcription factor myocyte enhancer factor MEF2 particularly in muscle tissues []The ASPH gene undergoes extensive alternative splicingresulting in four protein isoforms ie ASPH humbugjunctate and junctin [ ] These proteins vary in theCterminal region which affects their function [ ]The two longest ASPH transcript variantsthat aretranscribed from the P1 and P2 promoters and differ inthe length of the ²untranslated region encode the fulllength ASPH protein This protein contains the catalyticCterminal domain that catalyzes the posttranslationalhydroxylation in the cbEGFlike domains of numerousproteins Supplementary Fig including receptors receptor ligands and extracellular adhesion moleculesthat influence cell motility and invasiveness [ ] Thetruncated isoforms humbug junctate and junctin sharethe Nterminal part with the ASPH protein but lackcatalytic function They are involved in calcium homeostasis [] Humbug has a potential role in cell adhesionand calcium flux and similar to ASPH its overexpressionhas been correlated with aggressive tumorcell behavior[]reticulummembranebound protein that is known for its functionin the regulation of the intracellular Ca2 concentrationJunctin is a structural membrane protein and as an integral part of the complex consisting of the ryanodine receptor calsequestrin and triadin influences calciumrelease from the sarcoplasmic reticulum [ ]sarcoendoplasmicJunctateisaLocalization in cells tissue distribution andexpression regulationASPH is predominantly a cellsurface protein [] that isalso localized in the endoplasmic and sarcoplasmicreticulum [] Furthermore a recent study identifiedmitochondriallocalization of ASPH in hepatocellularcarcinoma HCC In that study ASPH overexpressioncorrelated with an instability of mitochondrial DNA andmitochondrial dysfunction that may lead to more aggressive pathological outcomes in HCC []ASPH is abundantly expressed in proliferating placental trophoblastic cells [ ] and in decidua and endometrial glands [] and has a potential role in placentalimplantation and fetal growth [] On the contrary theASPH expression in normal adult tissues is relativelylow or negligible However ASPH expression is inappropriately activated during oncogenesis when ASPH is required for generation of malignant and metastaticphenotypes The elevated expression of ASPH at bothFig ASPH catalytic reaction Aspartyl and asparaginyl residues in cbEGFlike domains are hydroxylated 0cKanwal Journal of Experimental Clinical Cancer Research Page of transcription and translation levels has been shown in awide range of transformed cell lines as well as humancarcinoma tissues including hepatocellular pancreaticcolon prostate lung breast ovarian and cervical carcinoma cholangiocarcinoma neuroblastoma and gastriccancer Table The first study that demonstrated thesignificantly higher expression of both ASPH mRNAand protein in HCC and cholangiocarcinoma relative totheir normal adjacent tissue counterparts was by Lavaissiere [] Subsequently they verified the role of upregulated ASPH protein production and its enzymaticfunction in the malignant transformation on biliary epithelium the NIH3 T3 cell line and animal models []The level of ASPH also correlated with cell motility andinvasiveness in in vitro experiments [ ] In thestudy by Maeda [] the overexpression of theASPH protein was in accordance with worse clinical andhistopathological characteristics of the intrahepatic cholangiocarcinomas and prognosis of patients Similar findings were obtained in other studies for hepatocellular[ ] nonsmall cell lung [] and colon carcinomas[] and glioblastoma multiforme [] Recentlytheprognostic significance of 2oxoglutaratedependent oxygenase expression was demonstrated by analysis of expression profile datasets of tumor samples and nontumor samples ASPH has been identified asone of the genes which upregulated expression couldserve for risk stratification of patients with cancertypes [] In glioblastoma the prognostic significance ofASPH was suggested by profiling of alternative mRNAsplicing []ASPH gene expression is upregulated via Wntcatenin [] and insulininsulinlike growth factor IGF1insulin receptor substrate IRS1 signaling [ ]through extracellular signalregulated kinaseERKmitogenactivated protein kinase MAPK andphosphatidylinositol3kinaseprotein kinase B PI3KAkt pathways Fig for review see ref [] InsulinIGF1IRS1 signaling affects cell growth and survival andcan be involved in oncogenesis in various human tumorsTable Summary of the studies which have identified the elevated ASPH expression in human tumor tissuesStudyPositive cases of studied samples nnTumor tissuesDetectionmethodIHCAntibody recognized region ofASPH proteinFB50 Ab NterminusFB50 Ab NterminusFB50 Ab NterminusFB50 Ab NterminusFB50 Ab Nterminus or 15C7 Abcatalytic domainFB50 Ab NterminusmAb G3 hybridomaPolyclonalFB50 mAb NterminusIHCIHCIHCIHCRTqPCRIHCRTqPCRIHCIHCIHCRTqPCRIHCFB50 Ab NterminusLavaissiere []HepatocellularCholangiocarcinomaBreastColonPalumbo []Pancreatic adenocarcinomaSepe et al[]Primitive neuroectodermalmedulloblastoma neuroblastomaMaeda et al[]Cantarini []CholangiocarcinomaHepatocellularMonte et al[]HepatocellularYang et al[]Wang et al[]Dong et al[]Tang et al[]Lin et al[] types of tumor tissuesaHepatocellularPancreatic cancerHepatocellularBreast 75fold higher level of mRNAcompared to normal tissue 7fold higher level of mRNAcompared to normal tissuePancreatic ductal adenocarcinomaOgawa []aLiver kidney breast cervical ovarian Fallopian tube laryngeal lung thyroid pancreatic thymic prostate bladder esophagus gastric gall bladder colon andrectum cancer and cholangiocarcinomaFB50 Ab NterminusIHC 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig Regulation of ASPH expression and ASPH involvement in signaling pathways The expression of the ASPH protein can be regulated atseveral levels The ASPH gene can be amplified in tumor cells and its transcription activated by INIGF1 and Wnt catenin pathways or inducedby hypoxia At the posttranscriptional level miR200a and miR135a can downregulate ASPH expression Stability of the ASPH protein can bereduced by phosphorylation with GSK3 Conversely ASPH can enhance GSK3 activity by inhibition of its phosphorylation with AKT and p38kinases ASPH also supports cell proliferation epithelialmesenchymal transition migration invasion and angiogenesis and consequently tumrowth and metastasis by hydroxylation of the Notch receptor and ligands ex JAG and interaction with pRb vimentin and ADAMs Finallyinactivation of NK cells by ASPH has been demonstrated Green arrow activation signal red bar inhibitory signal[] The catenindependent Wnt pathway regulatescell proliferation motility and differentiation and is oneof the most frequently modified pathways in humanmalignancies Upon aberrant activation of Wnt signalingcatenin is accumulated in the cytoplasm and subsequently translocated to the nucleus [] where an interaction between catenin and Tcellfactorlymphoidenhancerbinding factor TCFLEF proteins forms atranscriptional regulatory complex which enhances theexpression of Wnt target genes including IRS1 []ASPH was proposed as a common link between Wntcatenin and insulinIGF1IRS1 pathways and downstream signaling []The regulation of ASPH gene expression in tumorsmight also be affected by a copy number variation Inthe study by Kadota [] the ASPH gene locus hasbeen identified as one of the DNA regions with focalamplification in primary breast cancer In colorectal cancer ASPH gain or amplification was found in ofsamples [] Next a suppressant role of the microRNAmiR200a in posttranscription regulation of the ASPHexpression in hepatoma cells has been found [] MiR200a belongs to miR200 family which plays significantrole in preventing cancer initiation and metastasis forreview see ref[] Similarly miR135a has beenshown to suppress ASPH in endometrial cancer []Moreover consistent with the protein sequence analysis that recognized numerous prospective phosphorylation sites of glycogen synthase kinase3 GSK3casein kinase CK2 protein kinase A PKA and protein kinase C PKC on ASPH [] several studies demonstrated that phosphorylation can regulate the ASPHprotein expression [ “] Inhibition of the GSK3activity did not modify mRNA expression but increased 0cKanwal Journal of Experimental Clinical Cancer Research Page of the ASPH protein level [] Direct phosphorylation ofASPH by GSK3 probably decreases ASPH stability andthus reduces cell mobility [] ASPH protein expressionwas also increased by inhibitors of PKA PKC and CK2[] Mutational analysis of potential sites of phosphorylation demonstrated complex and nonuniform effects ofASPH phosphorylation on protein expression enzymaticactivity and subcellular localization [ ] ThereforeASPH phosphorylation probably regulates the functionof this protein by various mechanismsASPH expression can also be regulated by hypoxia andoxidative stress In human neuronal cells this effect wasmediated by hypoxia inducible factor alpha HIF1αthat is stabilized under hypoxiaoxidative stress whenthe prolyl hydroxylase domain PHD proteins and factorinhibiting HIF FIH are inactivated Consequently theHIF1 heterodimer made up of subunits HIF1α andHIF1 functions as a transcription factor likely enhancing ASPH expression by binding to hypoxiaresponsiveelements [] In hypoxic regions of glioblastoma bothHIF1α and ASPH were highly expressed particularly inmore aggressive mesenchymal subtype of glioblastomasuggesting a possible involvement of ASPH in mesenchymal transition [] Brewitz showed reducedASPH hydroxylation activity at low oxygen concentrations and suggested an ASPH role in oxygen hypoxiasensing ASPH upregulation induced by hypoxia couldcompensate for reduced enzymatic activity [] Moreover a recent study reported an oxidative stress state ofthe castrationresistant prostate cancer cells upon ASPHoverexpression which was reversed by silencing ASPHexpression or generating hypoxic conditions resulting inimpaired cell proliferation and invasion []ASPH protein interactions and signaling pathwaysThe ASPH hydroxylation consensus sequence is confined within cbEGFlike domains that are found in proteins of diverse function including Notch receptors andligands clotting factors structural proteins of the extracellular matrix and ligands of the tyro3Axl family ofreceptor tyrosine kinases []The Notch signaling cascade is a remarkably conservedpathway Notch proteins Notch1 Notch4 are singlepasscell surface receptors that mediate communication betweencells and their expression is crucial for proper embryonicdevelopment [] Notch signaling mainly results in cell differentiation but also plays a significant role in proliferationapoptosis and the maintenance and selfrenewal of stemcells Dysregulation of the Notch pathway is directly linkedto cancer vascular disorders and congenital defects [] In mammals Notch signaling activated by binding ofone of two families of canonical Notch ligandsjaggedJAG1 and JAG2 and delta like DLL1 DLL3 and DLL4leads to the generation of the cleaved Notch intracellulardomain NICD fragment and its nuclear translocation Inthe nucleus the NICD fragment interacts with the DNAbinding complex CSL CBF1RBPjκ SuH Lag1 Thiscomplex is then converted from a repressor into an activator leading to increased transcription of target genes suchas hes family bHLH transcription factor HES1 HESwith YRPW motif HEY1 CD44 epithelial cell adhesionmolecule EPCAM cmyc protooncogene matrix metallopeptidase MMP29 cyclin D1 cyclooxygenase vascular endothelial growth factor VEGF and proliferatingcell nuclear antigen PCNA [ ]Upregulation of ASPH results in enzymatic modification of the cbEGFlike repeats in the Notch receptorextracellular domain and its ligands which promotes thereceptor interaction with the ligands and the activationof Notch signaling [ ] Furthermore the interactionof ASPH with a disintegrin and metallopeptidase domainADAM stabilizes this complex and enhances theS2 cleavage ofthe Notch receptors and subsequentNICD fragment release [] The activation of the targetgenes in malignant cells increases cell proliferation migration and invasion []through the epithelialtomesenchymal transition EMT that is probably upregulated by the interaction of ASPH with vimentin []Consequentlythis activation supports tumor growthand metastasis The ASPHNotch axis also stimulatesthe release of exosomes that transfer proteins involvedin invasion metastasis metabolism and immunosuppression [ ]The SRC kinase pathway is another important pathwayin malignant cell transformation that regulates a complex signaling network promoting angiogenesis invadopodia formation and maturation and metastasis []ASPH has been identified as an SRC pathway activatorOverexpressed ASPH directly interacts with ADAM12 and strengthens the SRC activation by these proteinswhich promotes MMPmediated extracellular matrixdegradation and tumor invasiveness []ASPH can also contribute to malignant phenotype ofcells by interaction with other proteins Iwagami et alrevealed the interaction of ASPH with GSK3 that prevents GSK3 inactivation by phosphorylation with upstream kinases [] This mechanism was confirmed ina castrationresistant prostate cancer model [] GSK3 is a multifunctional kinase that is involved in variousprocesses including glycogen metabolism cell divisionand cell fate determination Some types of tumors aresensitive to GSK3 inhibitors [] Recently Huang elucidated a direct binding of ASPH with retinoblastoma protein pRb leading to pRb phosphorylation[] They also showed that this effect was mediated byincreased binding of cyclindependent kinase CDK CDK4 and cyclins D1 and E with pRb and wasdependentAsASPH enzymaticonactivity 0cKanwal Journal of Experimental Clinical Cancer Research Page of phosphorylation of pRb inactivates its tumorsuppressorfunction ASPH can contribute to the progression of cellcycle via interaction with pRbEffect of ASPH on an immune systemTumor generation and progression are influenced bycancer immunoediting that involves immunosurveillanceand escape from a host immune system [] In theseprocesses various mechanisms of both innate and adaptive immunity are included [] Immune cells that infiltrate developing tumors are initially antitumorigenicbut in tumor microenvironment they can be modifiedinto cells with protumorigenic properties []As potential targets of ASPH hydroxylation are alsoexpressed on immune cells this enzyme could affect thefunction of immune system particularly in tumor microenvironment when ASPH is overexpressed on cancercells Indeed such effect was demonstrated for humannatural killer NK cells by using recombinant ASPHwhich reduced viability and cytotoxicity of these cells viaenhancing caspase signaling and decreasing the surfaceexpression of activating receptorsrespectively []Antibodies against ASPH inhibited these effectsInteraction of ASPH with other immune cells has notbeen studied However we suppose possible influence ofASPH on different tumorinfiltrating cells This assumption comes from the involvement of Notch signaling indifferentiation and function of various immune cells fibroblasts mesenchymal cells and endothelial cells Forinstance Notch activation contributed to stimulation ofproinflammatoryantitumorigenic M1 polarization inboth bone marrowderived primary macrophages [] and tumorassociated macrophages[] WhenNotch signaling was abrogated protumorigenic M2polarization was induced even by stimulators of M1polarization [] miR125a has been identified as adownstream mediator of Notch signaling in macrophages [] Similarly the Notch pathway plays an important role in differentiation of other types of myeloidcells and probably all subsets of CD4 and CD8 T cells[] Different Notch receptors and their interactionwith different ligands contribute to these processes []Moreover noncanonical Notch signaling is implicatedin regulation of immune cells [] While activation ofNotch signaling in some cells eg T helper cells cytotoxic CD8 T cells and M1 macrophages supports induction ofincluding antitumorimmunity in other cells particularly regulatory T cellsit leads to immunosuppression [] Thus immunostimulatory effect of Notch signaling is often inhibited intumor microenvironment to enable the tumor cells toescape from the host immunity [] Therapeutics affecting Notch signaling in malignant diseases are being developed and tested in clinical trials but their effects onimmune reactionsimmune reactions and possible combination with immunotherapy have not been properly studiedASPH as a therapeutic targetOncogenic abilities of ASPH have been experimentallydemonstrated using tumor cell lines and mouse and ratmodels of different types of human tumors with ASPHoverexpression including cholangiocarcinoma [ ] hepatocellular carcinoma [ ]neuroblastoma [] pancreatic cancer [ ] glioma [] breast carcinoma [] castrationresistant prostatecancer [] and colorectal cancer [] In studies analyzingASPH function various approaches were utilized to revealsignaling pathways affected by ASPH Particularly ASPHexpression was diminished by using small interfering RNAs[ ] short hairpin RNAs [ ] or theCRISPRCas9 system [ ] The importance of ASPHenzymatic activity in these processes was shown by the sitedirected mutagenesis [ ] or treatment by SMIs [ ] In vitro assays showed ASPH involvement in cell proliferation migration and invasion Cellularalterations included EMTinhibition of apoptosis andstemness acquisition Tumor growth and invasivenesscould further be supported by ASPHinduced extracellularmatrix degradation angiogenesis and transendothelial migration Notch and SRC signaling are probably major pathways influenced by ASPH Fig and contributing toincreased aggressiveness of tumor cells that was verified inin vivo models Thus these studies also demonstrated thatASPH is a suitable target for cancer treatment especially bySMIs or immunotherapySmall molecule inhibitorsSMIs of ASPH Fig have been developed and used totest the role of ASPH in a wide range of cancer modelsincluding subcutaneous orthotopic and patient derivedxenograft in vivo models [ ] A small orallybioavailable inhibitor has severalintrinsic advantagesover immunotherapy approaches Not only can these inhibitors inhibit the catalytic activity of ASPH unlike conventional antibodies that simply bind to the protein butthey can also penetrate into the cell and inhibit ASPHcatalytic activity in the endoplasmic reticulum Differentcancers have different ASPH expression patterns andwhile surface expression is quite common in pancreaticcancer and hepatocellular carcinoma intracellular overexpression patterns have also been observed []The first ASPH SMIs published were the tetronimidesMOI500 and MOI1100 Tetronimides were originallysynthesized in by Dahn [] and are redoxactivemimics of ascorbic acid and 2oxoglutarate MOI500 isa mixed inhibitor that inhibits both ASPH and the FatMass and Obesity protein FTO [] and is not onlyorally bioavailable but also can penetrate the blood 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig Small molecule inhibitors of ASPHthereand kinasesbrain barrier MOI1100 is a more potent inhibitor ofASPH and is also more selective [] Despite investigation against a wide range ofirondependent dioxygenasesare no other knownenzymatic targets for MOI1100 Enhanced activity wasobserved by replacing the chlorine with a trifluoromethylgroup [] as in MOI1151 and even a greater improvement in in vivo activity was found by replacing the trifluoromethyl group with a carboxymethyl group as inMOI1182 although it is not yet clear if the nature ofthis enhancement is due to increased inhibitory activityorenhanced solubility parameters MOI1182 isreported to have the ability to suppress invasive activityat a concentration of nM [] SMIs of ASPH have acharacteristic in vitro concentration dependent profilewhere the activity of the SMI plateaus at value around viability [] emphasizing the noncytotoxic properties of this class of inhibitorsNatural products and inhibitors of other enzymes thathave been repurposed as ASPH inhibitors have alsorecently been reported in the patent literature includingbosutinibcepharanthineCN2019101414327 and guaianolides related to nortrilobolideCN2019104575886CN2019101414219CN2019101414187 0cKanwal Journal of Experimental Clinical Cancer Research Page of sesquiterpene with complex anticancerBosutinib is a wellknown inhibitor of BCRABL and SRCtyrosine kinases approved for the treatment of chronic myelogenous leukemia [] Cepharanthine is a natural productactivityincluding AMPactivated protein kinase AMPK activationand nuclear factor kappa B NFκB inhibition [] Nortrilobolide and related compounds are reported to be potentcytotoxic agents with subnanomolar sarcoendoplasmicreticulum calcium ATPase SERCA inhibition [] Recently a family of potent pyridine dicarboxylates have alsobeen published [] utilizing a mass spectrometrybasedinhibition assay [] These compounds are related toknown irondependent dioxygenase inhibitors 23pyridinedicarboxylate 24pyridine dicarboxylate and 26pyridinedicarboxylate The synthesized pyridine dicarboxylateswere assayed for activity against a range of other enzymesto include PHD2 FIH and lysinespecific demethylase 4EKDM4E in addition to ASPH with varying degrees of selectivity However while cellbased activities have not beenevaluated the dicarboxylate nature of the compounds maybe useful for cell surface ASPH inhibitors that may nothave cell penetrating activity []immunity As a target of humoralImmunotherapyASPH can be used not only as a target of the inhibitors inactivating its enzymatic activity but also as a target of immune reactions leading to destruction of tumor cells andtumor growth suppression Since ASPH is cell surface displayed on tumor cells it represents a tumorassociatedantigen that can be targeted by both cellmediated andhumoralimmunityASPH on the surface of cancer cells can be bound by antibodies that mediate antibodydependent cellular cytotoxicity ADCC complement dependent cytotoxicity CDCor antibodydependent cellular phagocytosis ADCP []When the ASPH antigen is processed in tumor cells orantigen presenting cells antigenic peptides are presentedon these cells by human leukocyte antigen HLA class Ior class II molecules and recognized by CD8 or CD4 Tlymphocytes respectively [] that can be stimulated byimmunization breaking tolerance to selfantigens []Induction of ASPHspecific CD4 and CD8 T cells wasexamined in blood samples of HCC patients Using synthetic peptides derived from ASPH after prediction of HLAclass I and HLA class IIrestricted epitopes it has beenfound that ASPH is a highly immunogenic protein that activates both types of analyzed T cells [] Thus efficientantitumor reactions could be stimulated by immunizationThe first vaccine against ASPH was based on matureddendritic cells DC loaded with the ASPH protein andtested in an orthotopic rat model of intrahepatic cholangiocarcinoma [] This study showed that vaccinationstimulated cytotoxicity against cancer cells in an in vitroassay and decreased tumor growth and metastasis BothCD8 and CD4 cells contributed to an antitumor effectinduced in a mouse model of HCC by immunizationwith ASPHloaded DCs []The next antiASPH vaccine was based on a bacteriophage lambda display system The viral capsid proteingpD was fused with the N or Cterminus of ASPH andimmunogenicity ofthese nanopforming constructs was verified in two mouse tumor models []The vaccine PAN3011 containing these constructs hasalready been examined in a phase clinical trial in patients with biochemically relapsed prostate cancer[] This study demonstrated safety and immunogenicity of PAN3011 and indicated an antitumor effect interms of the reduction of prostate specific antigen PSAor PSA doubling time ASPHspecific immune responseswere mediated by both antibodies and T lymphocytesAs ASPH is a type II transmembrane protein its Cterminus carrying the enzymatic domain is exposed outside cells and can be bound by antibodies that can be usedfor diagnostic and therapeutic purposes Development ofASPHspecific antibodies has been described in several s [“] The human IgG1 PAN622 recognizesthe catalytic domain of ASPH This antibody is not directly cytotoxic for tumor cells but is internalized and candeliver cytotoxic moieties into cells [] In the subsequent study with a mouse model of metastatic breast cancerandradioimmunotherapy with promising results [] MouseIgG1 monoclonal antibody binding to the CterminalASPH domain mediated ADCC by human NK cells []Recently a secondgeneration antibody approach hasbeen disclosed The prepared antibody binds to the extreme Cterminus of ASPH US that is involved in specific substrate recognition [] Thereforethis antibody has direct ASPH inhibitory activity anddoes not require any radioisotope or cytotoxic payloadfor potential therapeutic activityPAN622 wasbioimagingusedforConclusionsASPH is an important enzyme in malignant transformationof cells It stimulates tumor cell proliferation migration andinvasion but it can also affect other cells in tumor microenvironment Two main pathways Notch and SRCthrough which ASPH promotes the tumor growth havebeen identified It has also been shown that ASPH expression is induced by some growth factors and hypoxia and isregulated at various levels The overexpression of ASPHand its downstream targets has been detected in numeroushuman malignancies Since ASPH is not expressed in appreciable level in normal adult tissues and the catalytic domain is localized on the cell surface it has been proposedas one of the most exciting potential therapeutic targetsFig Small inhibitory molecules orally bioavailable havebeen developed and successfully tested in several cancer 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig ASPH as a therapeutic target ASPH expression is upregulated by growth factors and hypoxia Its enzymatic activity can be inhibited bySMIs or monoclonal antibodies which results in reduction of cell proliferation angiogenesis immunosuppression and cell migration and invasionConsequently tumor growth and metastasis are also reduce

Thyroid_Cancer

"dramatic spread of Coronavirus Disease COVID19 has profound impacts on every continent and life Due to humantohuman transmission of COVID19 nuclear medicine staffs also cannot escape the risk of infection from workplaces Everystaff in the nuclear medicine department must prepare for and respond to COVID19 pandemic which tailored to the characteristics of our profession This provided the guidance prepared by the Korean Society of Nuclear Medicine KSNM incooperation with the Korean Society of Infectious Disease KSID and Korean Society for HealthcareAssociated InfectionControl and Prevention KOSHIC in managing the COVID19 pandemic for the nuclear medicine department We hope that thisguidance will support every practice in nuclear medicine during this chaotic periodKeywords Coronavirus COVID19 Nuclear medicine Prevention and control Practice guideline HoYoung Leedebobkrgmailcom Department of Nuclear Medicine CHA Bundang Medical CenterCHA University of Medicine Professor Pocheon Republic ofKorea Department of Nuclear Medicine Seoul National UniversityBundang Hospital Professor Seongnam Gyeonggido Republic ofKorea Department of Nuclear Medicine Samsung Medical CenterSeoul Republic of Korea Department of Nuclear Medicine Seoul National UniversityHospital Seoul Republic of Korea Department of Nuclear Medicine Chosun University HospitalGwangju Republic of Korea Department of Nuclear Medicine Korea University Guro HospitalSeoul Republic of Korea Department of Nuclear Medicine Hanyang University Guri HospitalSeoul Republic of Korea Department of Nuclear Medicine National Cancer CenterGoynag Republic of Korea Department of Nuclear Medicine Seoul Medical CenterSeoul Republic of Korea Division of Nuclear Medicine Department of RadiologyEunpyeong St Mary™s Hospital College of Medicine The CatholicUniversity of Korea Seoul Republic of Korea Department of Nuclear Medicine Soonchunhyang University SeoulHospital Bucheon Republic of Korea Department of Nuclear Medicine Inje University Haeundae PaikHospital Busan Republic of Korea Department of Nuclear Medicine Keimyung University DongsanMedical Center Daegu Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityCheonan Hospital Cheonan Republic of Korea Department of Nursing Soonchunhyang University BucheonHospital Bucheon Republic of Korea Division of Infectious Disease Department of Internal MedicineKangdong Sacred Heart Hospital Hallym UniversityChuncheon Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityBucheon Hospital Bucheon Republic of Korea Department of Nuclear Medicine Korea University Anam Hospital Korean Society of Nuclear Medicine Quality Control CommitteeSeoul Republic of KoreaBucheon Republic of Korea 0cIntroductionSince the first reports of Coronavirus Disease COVID in Wuhan China the infection had spread worldwiderapidly and COVID19 has reached pandemic levels InSouth Korea since its outbreak in February COVID has affected profoundly every aspect of communities Thehumantohuman transmission of COVID19 provides challenges for all healthcare facilities and healthcare providersIn the face of the COVID19 pandemic the Korean Societyof Nuclear Medicine KSNM Korean Society of InfectiousDisease KSID and Korean Society for HealthcareAssociated Infection Control and Prevention KOSHIC haveprepared the guidance for the nuclear medicine department tominimize confusion and ensure that nuclear medicine physicians and technicians continue to provide their services whileprotecting the patients and workers and preventing the transmission of the virus The Quality Control Committee ofKSNM reviewed several reports and recommendations previously published by the European Association of NuclearMedicine EANM [] Society of Nuclear Medicine andMolecular Imaging SNMMI American Society of NuclearCardiology ASNC [] International Atomic Energy AgencyIAEA and others [“] This guidance is basically in compliance with the COVID19 guidelines of the Korea Centersfor Disease Control and Prevention KCDC [“] Finallythis document was prepared in cooperation with KSID andKOSHIC KSNM emphasize that this guidance must be considered in the context of following the state and hospital infection control policies and flexibly applied according tochanges in circumstances and evidenceGeneral Principles During COVID19PandemicIn a pandemic situation such as COVID19 if necessarythe condition of the scheduled patient can be checked inadvance to adjust the examination schedule Nonurgent elective studies or therapy should be postponed in COVID19confirmed or COVID19suspectedpatients Rescheduling the studiestherapy must be donein a discussion with the referring clinicians Only urgent studies or therapy could be performed inCOVID19confirmed or COVID19suspected patientswhenever clinically appropriate The priority of studytherapy should be based on a casebycase indepth discussion between nuclear medicine physicians and referring clinicians In case of performing the urgent studiestherapy consult with the infection control offices of eachinstitution to comply with the infection control rules ofownNucl Med Mol Imaging “ COVID19suspected patients should undergo COVID testing before performing the studiestherapy Lung ventilation scan should not be performed in anyCOVID19confirmed or COVID19suspected patients Lowdose radioiodine therapy may be considered in caseof acute hyperthyroidism patients who are unable to tolerate antithyroid medications As lowdose radioiodinetherapy lower than GBq of I131 can be performedin an outpatient setting in South Korea COVID19infected patient can be administrated lowdoseradioiodine in the isolation room or negative pressureroom without any additional monitoring related toradioiodine therapyConsideration During the StudyTherapy Patient transportationScheduling COVID19confirmed or COVID19suspected patient as last study of the day to preventcrossinfection in the nuclear medicine department Ensure that other patients or caregivers should notaccess the nuclear medicine department to minimizethe exposure to COVID19 patient during the studytherapy Transfer the COVID19infected patient to the nuclear medicine department using negative pressuretransport bag to minimize exposure and contact todroplet COVID19 patients should wear masks at all timesof procedures If necessary add gowns gloves etc Devices and scanner management Mainly use disposable instruments or items Do notreuse disposable items such as oxygen masks nasalprongs suction tubes or suction lines The protocolfor reusable devices is as follows Cleaning After use the equipment contaminated with blood bodyfluids secretions and feces should be delivered to awashing room with care not to contaminate the surrounding environment The washing place should be separated from the spaceused for cleaning other items or other patients After immersing the contaminated equipment in a washing spacewash the product carefully to avoid splashing Wash enough to remove blood body fluids secretionsand feces from remaining 0cNucl Med Mol Imaging “ Staff undertaking cleaning should wear KF94 or N95masks longsleeved waterproof gowns goggles or faceshields hats shoe covers or rubber boots and doublegloves outer gloves are rubber gloves Disinfection and sterilization Depending on the risk level of the device according tothe Spaulding Classification of medical equipmentdevices noncritical devices require lowlevel disinfectionsemicritical devices require highlevel disinfectionsterilization and critical devices must be sterilized Disinfectants and sterilization methods by device classification should be followed in accordance with the notificationof the Ministry of Health and Welfare Be sure to check the disinfectant manufacturer™s recommendations The recommended disinfection process suchas dilution and application time of disinfectant and theeffective period and concentration of disinfectant arestrictly followed Laboratory and scan room management Only the minimum number of staffs should be placedin the nuclear medicine department All participatingstaffs should wear appropriate personal protectiveequipment PPE eye protection with goggles or faceshield medical protective masks N95KF94 or equivalent respirator disposable latex gloves disposablegown disposable shoe covers etc Cover the scanner couch or other equipment with aplastic cover to prevent contamination Every effort should be made to minimize theCOVID19 exposure to medical staff during injection of radiopharmaceuticalsSelect the protocol with the shortest duration of uptake time and scan time to minimize the time spentby the COVID19 patient in the departmentIn case of studies requiring an uptake phaseCOVID19 patients should be waiting in separatespace If possible COVID19 patients wait in negative pressure transport bag If negative pressuretransport bag is not available use bed or stretcherin waiting room with disposable cover Considerusing standard radiopharmaceutical dose to shortenthe procedure time After the completion of image acquisition the scanroom and patient™s space area should be disinfectedaccording to the standard protocol After image acquisition remove the plastic cover ofthe scanner and disinfect the scanner surface Remove and discard PPE adequately when leavingthe camera room or care area and immediately perform hand hygieneIn case of performing the radiolabeling of theCOVID19 patients™ blood products every processwith infectious materials openingstirringmixingdispensing COVID19 patient™s blood sampleradiolabeling etc should be done in class II biosafety cabinet according to the Biosafety Level Regulation Disinfection of laboratory with properdisinfectants ethanol hydrogen peroxide or ppm sodium hypochlorite should bedone Used PPE and disposable covers are removed withcaution not to contaminate the clean area and disposed in a container for biosafety waste Employee management All employees should be trained in the preventionand management of COVID19 infection and adhereto the rules of infection prevention Considering the skill level fatigue etc of the working staff sufficient personnel are allocated to securethemPriority from exemption is given to employees withhighrisk underlying diseases such as diabetesmellitus chronic obstructive pulmonary diseaseCOPD endstage renal disease ESRD chroniccardiac disease etc or pregnant women Cleaning and environmental management General principle Personnel responsible for cleaning or disinfectionshould complete the infection preventioneducation Employees should wear PPE KF94 or N95 respirators fullbody protective clothing or aprons goggles or face shields shoe covers or rubber bootsdouble gloves outer gloves are rubber gloveswhen cleaning or disinfectingIf there are organic substances on the surface of theenvironment it cannot be properly disinfectedTherefore wipe the surface before disinfecting theenvironmentIn order to prevent the possibility of microbialspraying cleaning should be performed using acleaning solution or a mop moistened with a disinfectant rather than a cleaning method using abroom or a vacuum cleanerInstead of spraying disinfectants thoroughly cleanthe surface of the environment using a clean towelmoistened with the disinfectant or a commerciallyavailable disinfecting tissue towel 0cNucl Med Mol Imaging “ Use cleaning tools as disposable as possible or exclusively However when the cleaning tool isreused the used cleaning tool is sterilized usingan appropriate disinfectant and then dried andstored Disinfection of a patient™s space areaIn the case of the space area used by the patientmark the place where contamination was confirmedbefore cleaning and disinfecting the surface andseal the contaminated object to prevent others frombeing exposed Ventilation before during and after cleaningdisinfection disinfection after ventilation for hbased on air cycles per hour Wear PPE Wipe with a cloth cloth etc wet withthe diluted disinfectant Wipe the touched wall surface and all frequently used areas and keep it for atleast min After then wipe the surface with acloth dampened with clean water cloth etcResumption of use Consider the characteristics ofeach type of disinfectant used and the purpose of thefacility After disinfection the virus is killed but thedecision at the time of resumption of use cannot beapplied in batch due to different characteristics ofdisinfectants so it is necessary to consider the precautions for each productFor details on disinfecting methods such as surfacedisinfection and washing refer to œDisinfectionGuidelines to Prevent the Spread of COVID19 atPublic and Multipurpose Facilities 3rd editionRefer to the method of disinfecting the patient spaceareaSelect an environmental disinfectant Select an approved or declared disinfectant by the Ministry ofEnvironment and follow the usage usage and precautions for each product Disinfectant list of the Ministry of Environmenthttpecolifemegokr Precautions when using environmentaldisinfectants Select the disinfectant after confirming informationsuch as approval from the Ministry of Environmentand Environment When using environmental sterilizers make sure tofollow the manufacturer™s recommendations suchas checking the expiration date safe usage for eachproduct and precautions and preparing the diluentaccording to the manufacturer™s instructions The disinfecting method of sprayinginjecting disinfectant is not applied to surface disinfectionbecause it causes aerosol infection increased riskof inhalation and the range of contact between thedisinfectant and the surface is insufficient so thedisinfecting effect is insufficient Disinfectant hazard information must be checkedand used carefully Do not mix different disinfectants Do not placenear flammable materials Disinfectant should beused in a wellventilated area As the disinfection effect may decrease over timedilute as much as necessary and use it immediatelyDo not store the remaining amount and discard itimmediately Laundry managementStore clean laundry in a separate space Employees handling laundry should be trained toprevent infection Employees handling contaminated laundry shouldwear PPE N95 masks or equivalent respiratory protection gowns gloves overshoes etc and performhand hygiene after removing PPE The laundry used for the patient is disposed of according to the relevant regulations see WasteManagement Act Medical Institution LaundryManagement Rules etc Thoroughly ensure that pathogens are not exposed topersonnel handling the laundry or surrounding environment during the entire process of collectingtransporting and washing laundry Waste management Waste related to COVID19 patients is managed bythe rules of hospital infectious control policySharp tools such as needles or blades are collectedin containers for impervious and nonpermanentwaste and containers should be stored in the placewhere the items are usedSimple infectious waste contaminated or possiblycontaminated with COVID19 patients™ sample isautoclaved and discarded Radioactive waste shouldbe discarded in compliance with national regulationwith caution not to contaminate the staff or areaConclusionConsidering that outbreaks of novel viruses have been periodically appearing these days nuclear medicine staffs should getused to guidance and policies for infectious disease in working 0cNucl Med Mol Imaging “place to protect patients worker themselves and furthermorevaluable medical resources Basically this guidance can beapplied in case of any other humantohuman transmissiondisease for operating the nuclear medicine department Alsoalways bear in mind the rapid change in the situation thisguidance should be used in conjunction with the currentgovernment and local hospital policiesCompliance with Ethical StandardsConflict of InterestJiIn Bang HoYoung Lee Young Seok ChoHongyoon Choi Ari Chong Jae Sun Eo Ji Young Kim Tae SungKim HyunWoo Kwon Eun Jeong Lee Eun Seong Lee Hye LimPark Soo Bin Park Hyekyung Shim BongIl Song Ik Dong YooKyung Jae Lee Hong Jae Lee Su Ha Han Jin Seo Lee Jung Mi Parkand Sung Hoon Kim declare that they have no conflict of interestEthical Approval This work does not contain any studies with humanparticipants or animals performed by any of the authorsInformed Consent Not applicableReferences Paez D Gnanasegaran G Fanti S Bomanji J Hacker M SathekgeM et al COVID19 pandemic guidance for nuclear medicine departments Eur J Nucl Med Mol “ Skali H Murthy VL AlMallah MH Bateman TM Beanlands RBetter N et al Guidance and best practices for nuclear cardiologylaboratories during the coronavirus disease COVID19 pandemic an information statement from ASNC and SNMMI J NuclCardiol “ httpsdoiorg101007s12350020021232 Huang HL Allie R Gnanasegaran G Bomanji J COVID19“nuclear medicine departments be prepared NuclMedCommun MossaBasha M Medverd J Linnau K Lynch JB Wener MHKicska G et al Policies and guidelines for COVID19 preparedness experiences from the University of Washington Radiology httpsdoiorg101148radiol2020201326 Zhang X Shao F Lan X Suggestions for safety and protectioncontrol in Department of Nuclear Medicine during the outbreak ofCOVID19 Eur J Nucl Med Mol “ Buscombe JR Notghi A Croasdale J Pandit M O'Brien J GrahamR et al COVID19 guidance for infection prevention and controlin nuclear medicine Nucl Med Commun “ Standard guideline for healthcareassociated infection control andprevention Korean Center for Disease Control and Prevention andKorean Society for HealthcareAssociated Infection Control andPrevention httpcdcgokrCDCcmscontentmobile2675626_viewhtml Accessed 2nd Jun Korean Society for HealthcareAssociated Infection Control andPrevention Korean Center for Disease Control and Preventionhttpwwwcdcgokrboardesmida20507020000bid0019actviewlist_no366579 Accessed 2nd Jun Guidelines in response to coronavirus disease for local governmentKorea Centers of Disease Control and Prevention2020 httpswwwcdcgokrboardboardesmida20507020000bid0019actviewlist_no367279tagnPage1 Accessed 2ndJun Disinfection guidelines to prevent the spread of COVID19 at public and multipurpose facilities Korea Centers of Disease Controland Prevention httpswwwcdcgokrboardboardesmida20507020000bid0019 Acessed 15th Jun Publisher™s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c"

Thyroid_Cancer

"production process errors may bediscovered which could affect the content and all legal disclaimers that apply to the journalpertain Published by Elsevier 0cSARSCoV2 another kind of liver aggressor how does it do that Sonia A LozanoSepulveda1 Kame GalanHuerta Natalia Mart­nezAcu±a Daniel ArellanosSoto and Ana Mar­a RivasEstilla1 1Department of Biochemistry and Molecular Medicine School of Medicine and Hospital Universitario œDr Jose E Gonzalez Autonomous University of Nuevo Len Monterrey Ana Mar­a RivasEstilla Department of Biochemistry and Molecular Medicine School of Medicine and Hospital Universitario œDr Jose E Gonzalez Autonomous University of NL Mxico Corresponding author Nuevo Leon Ave Francisco I Madero y Ave Gonzalitos sn Col Mitras Centro Journal Preproofbut complications such as pneumonia respiratory distress syndrome and multian Monterrey Nuevo Len Mxico Telfax Email amrivas1yahooca Abstract Clinical manifestations of SARSCoV2 infection include more frequently fever and cough failure can occur in persons with additional comorbidities Liver dysfunction is one of the most striking affections among patients suggesting that SARSCoV2 may represent a new king of liver aggressor However the molecular process underlying this phenomenon is 0cstill unclear In this work we overview the most recent findings between the molecular biology of the virus pathogenic mechanisms and its relationship to liver disease observed in patients Abbreviations AaDO2 ACE2 AIH ALT AST COVID19 GGT GI GTEx Alveolararterial Oxygen Gradient Angiotensinconverting enzyme Autoimmune Hepatitis Alanine transaminase Aspartate aminotransferase Coronavirus infectious disease Gamma glutamyl transpeptidase Gastrointestinal GenotypeTissue Expression Metabolicassociated fatty liver disease Nonstructural proteins Reading Frame Journal Preproofcomplex disease in many severely ill patients In other infected subjects an infection is Keywords SARSCoV2 liver liver impairment COVID19 ACE2 MAFLD nsp ORF Introduction SARSCoV2 is the etiological agent of the disease known as COVID19 which causes a disease characterized by pneumonia cough fever occasional diarrhea headache cardiac injury and in some patients™ liver alterations COVID19 has been found to be an extremely reported to be so severe that it can lead to a disproportionate and mortal reaction of the immune system known as a cytokine storm All of these factors make COVID19 highly unpredictable it is what specialists call a multisystem disease 0cAround the world cases of liver dysfunction denoted by elevated hepatic enzymes in serum such as AST aspartate aminotransferase and ALT alanine transaminase have been documented among patients infected with SARSCoV2 There is still no certainty whether the COVID19related liver damagedysfunction is due mainly to the viral replication per se drugs toxicity or other coexisting comorbidities whether sexrelated difference could help to explain why infected men are more healthy or harmful relationship between the liver and its viral aggressor In this paper we describe a brief overview of the implications for researchers in the field of It is important to understand how liver function can be altered by direct infection with this predisposed to develop COVID19associated liver dysfunction than infected women to analyze if there is any genetic predisposition related to impaired liver function during the œrespiratory virus which mechanisms of viral pathogenesis are involved to evaluate disease and of course the crosstalk between viral and cellular proteins that mediate this Journal Preproofliver disease of the most recent findings between the molecular biology of the virus This emerging viral illness is typically characterized by fever dry cough myalgia headache sore throat diarrhea and may be aggravated with shortness of breath and respiratory failure [] The angiotensinconverting enzyme ACE2 the functional receptor of the spike glycoprotein of SARSCoV2 is widely distributed in the anism Historically Hamming and colleagues reported ACE2 expression in the surface of lung alveolar epithelial cells enterocytes of the small intestine arterial and venous endothelial pathogenic mechanisms and its relationship to liver disease observed in patients Clinical characteristics and liver injury in patients with COVID19 0ccells and arterial smooth muscle cells [] Posterior transcriptomic and proteomic analyses confirmed their findings and added high ACE2 expression in adipose tissue bone marrow duodenum endometrium heart kidney small intestine smooth muscle testis and thyroid [] ACE2 is also expressed in liver but in lesser extent One of the most worrisome severe cases of COVID19 [] Regarding the gastrointestinal GI tract and liver over COVID19associated liver injury is defined as any liver damage that occurs during disease progression andor COVID19 treatment in patients with or without a history of previous complications is the unusual formation of blood clots in many patients with COVID19 even those who were receiving anticoagulants Researchers at Mount Sinai in New York published studies suggesting that clots in the lungs play an important role in the most of COVID19 patients develop GI symptoms such as anorexia diarrhea nausea and vomiting and a significant proportion present with altered liver function tests [] Journal PreproofDecreased albumin levels are associated with severe infection and poor prognosis Still AST elevation is the most common abnormality in patients presenting with COVID19 observed more frequently in men and is mainly documented in more severe cases [] liver disease In general the incidence of increased liver biochemical markers in hospitalized patients with COVID19 mainly AST and ALT and slightly elevated bilirubin varies between to of cases [] The increase in liver enzymes is there are no reports of acute or subacute liver failure in patients with COVID19 The largest cohort study that included cases of COVID19 from China showed that had preexisting chronic liver disease Lei and colleagues reported that impaired liver function was related to mortality in COVID19 patients [] Elevated AST was more frequent and significant than the increase of ALT in severe 0chospitalized patients Moreover elevated AST was shown to be associated with highest mortality risk [] In the study reported by Yijin Wang [] they found that of COVID patients had elevated AST activity The median levels of ALT were UL vs UL respectively AST were UL vs UL respectively in abnormal and normal aminotransferase groups Liver enzymes abnormality were associated with disease severity protein levels In addition they found by ultrastructural examination of coronavirus ps in hepatocytes in COVID19 cases SARSCoV2 infected hepatocytes displayed as well as a series of laboratory tests including higher Alveolararterial Oxygen Gradient AaDO2 higher gamma glutamyl transpeptidase GGT lower albumin and lower total conspicuous mitochondrial swelling endoplasmic reticulum dilatation and glycogen granule decreased Histological findings showed apoptosis and binuclear hepatocytes Journal Preproofshowed a disease course similar to that reported in non immunosuppressed population coronavirus the interaction of its proteins with cellular proteins and consequently the immunosuppressive therapy for autoimmune hepatitis AIH developing COVID19 Taken together both ultrastructural and histological evidence indicated a typical lesion of viral infection [] All these findings by different reports demonstrates that SARSCoV2 infection in liver is a crucial cause of hepatic impairment in COVID19 patients However alteration of cellular metabolism that give rise to systematic alterations and metabolic Report from Alessio Gerussi et al[] demonstrated that patients under today the cellular and molecular mechanisms that are altered by infection with this changes are still unknown 0cMolecular biology of SARSCov2 Coronaviruses are enveloped viruses that contain a positively polarized unsegmented RNA genome belonging to the Coronaviridae family and the order of Nidovirales they are distributed in humans and other mammals[] The size of the SARSCoV2 virions is approximately to nm in diameter [“] SARSCoV2 has a genome that consists polymerase RdRp which is nsp12 and is responsible of the replication and transcription of the virus[] which are encoded by the various genetic loci on the genome [] At the center of the virion lies a nucleocapsid composed of the genomic RNA and the nucleocapsid protein[] The virus glycoprotein S consists of two subunits S1 which is at the amino terminus and that encodes for structural proteins and a larger region that encodes two reading frames ORF 1a and 1b which together encode for the nonstructural virus proteins from nucleocapsid protein which is within the phospholipid bilayer and nonstructural proteins nsp1 to nsp16 []The virions have a structural Sspike protein outer spiky glycoprotein Mmembrane protein a type III transmembrane glycoprotein Nof nucleotides [] encoding amino acids and it is composed of a region Journal Preproofvirus [] as well as protein M which is a type III transmembrane glycoprotein[] and participates in the cellular membrane rearrangements for the replication and transcription complexes[] Among the encoded proteins is an RNAdependent RNA provides the receptor binding site and S2 which is at the carboxyl terminus responsible for membrane fusion [] The envelope protein E has a role in the assembly and release of the Nonstructural proteins have several functions during de viral cycle For example nsp 0cThe virus enters the cell by endocytosis through the interaction between envelope glycoprotein S with the cell receptor ACE2 and with the participation of the type II transmembrane serine protease TMPRSS2 [] Once it enters the cell the N protein with viral genome are released within the cytoplasm then cellular proteases degrade the capsid and the virus genome is left free Next the polyprotein containing the viral proteins that are How does the virus select which cells to infect Viruses can infect only certain species of hosts and only permissive cells within that host Permissive cells make all the necessary proteins and viral factors to allow virus to replicate Once a virus gets inside a cell it hijacks the cellular processes to produce virally encoded proteins that will replicate the virus™s genetic material []Viral replication may cause exocytosis[] will translate into viral proteins this entire process will occur in the cell cytoplasm The processed to form the replication complex is translated and then the complementary strand of negative sense pregenomic RNA is synthesized to be used as a template to replicate the structural proteins that will be synthesized in the endoplasmic reticulum membrane to assembly the viral p and finish the cycle through the release of the viruses by positive sense viral genome[] Furthermore the replication and transcription complex will lead to a series of smaller positive sense subgenomic RNAs these are the ones that Journal PreproofBoth SARSCoV and the new SARSCoV2 are very similar in structure and pathogenicity but the major structural protein S protein is slightly different between them[] Compared to other beta coronaviruses the presence of a furinlike cleavage site in SARSCoV2 enables the S protein priming and facilitates an increase on the efficiency of the spread of SARSCoV2 as is reported wide world [] 0cbiochemical changes producing cell damage called cytopathic effects Like other coronaviruses SARSCoV2 requires cellular receptors to initiate its internalization to the cells that carry these factors []Li SARSCoV2 uses the angiotensinconverting enzyme ACE2 as a host cell receptor SARSCoV2 spike S protein binds ACE2 with significantly high affinity[] In addition the main host protease that suggested to promote the pathogenesis of this coronavirus [] program httpsportalgdccancergov They compared ACE2 expression levels across human tissues between males and females and between younger and older persons in these individual tissues Furthermore other reports have analyzed the correlations between ACE2 In order to provide insights into the mechanism of SARSCoV2 infection Li [] analyzed the expression of ACE2 in various normal human tissues using the datasets from the GenotypeTissue Expression GTEx project and The Cancer Genome Atlas TCGA transmembrane serine protease[] Other host proteases such as furin have also been mediates Sprotein activation on primary target cells and initial viral entry is the type II Journal Preproofreported by Li ACE2 expression levels showed no significant difference between have no significant association with sex age or race Is the liver a direct target for SARSCoV2 males and females between younger and older persons or between Asian and nonAsian races This finding suggests that the infection risk of SARSCoV2 and SARSCoV may expression levels and immune signature enrichment levels in individual tissues As As we expected because the systemic manifestations of COVID19 it has been reported that SARSCoV2 has an anotropism beyond the respiratory tract including the kidneys 0cliver heart and brain and possibly that anotropism influences the course of Covid19 disease and aggravates preexisting conditions The ACE2 protein is found at high levels in the GI tract as the colon biliary system and liver [] On the other hand it is well documented a SARSCoV2 RNA shedding in the GI tract [] supporting its tropism for architecture express ACE TMPRSS1 receptors [] The presence of these two host factors in the liver suggests that a direct viral cytopathic effect occurs Also in SARS infection the presence of viral RNA in liver tissue was documented but not as extensively as the new coronavirus Data published by Gordon [] suggest that mitochondrial proteins interact directly with the virus which helps to understand the potential mechanism by which elevated AST the GI tract and liver cells and these may be sites of active viral replication and either direct or indirect tissue injury Indeed a large part of the cells distributed in the liver Journal Preproofeffect the exacerbated inflammatory response in COVID19 may play a central role in profiles are detected in these patients [] Furthermore in addition to this intracellular More recently [] identified the clinical and laboratory characteristics of COVID19 patients with abnormal liver transaminases and they reported that SARSCoV2 is able to which high levels of IL6 have been reported [] which are involved in both infect liver cells and cause liver impairment by direct cytopathic effect inflammatory and repair responses in liver disease Mechanisms of liver pathogenicity 0cIf SARSCoV2 replication has direct adverse effects on liver function it is still unknown Findings in liver biopsy of patients killed by COVID19 showed moderate micro vesicular steatosis and mild portal and necroinflammatory activity[] This seems to indicate that a direct injury occurred while the infection that could have been directly caused by SARSCoV2 Another possibility is that a druginduced liver injury occurred To date there are the following possible mechanisms Figure infection The massive release of cytokines by the immune system in response to the viral infection can result in a cytokine storm and symptoms of sepsis that are the cause of death in of fatal COVID19 cases [] In these cases uncontrolled inflammation induces multian damage leading including liver failure Biomarkers of inflammation such as Creactive protein PCR serum ferritin LDH Ddimer IL6 IL2 are have been found to be significantly elevated in Immune damage from exacerbated inflammation in response to SARSCoV2 Journal Preproofpathogenesis of SARS CoV “related liver disease more studies should be liver is a potential target for direct infection with this virus To understand the performed for evidence of viral mechanisms of replication in different cell anelles as cytoplasm endoplasmic reticulum Golgi apparatus and lipidrafts CoV2 enters cells through the ACE2 molecule which is abundantly expressed in the liver and in particular in bile epithelial cells [] Based on this expression the Direct cytopathic effect due to active viral replication in various liver cells SARScritically ill patients with COVID19 [] into hepatocytes and liver histology characterization It is also important to know in cells their capacity to efficiently produce both infectious and defective non 0cinfective whole virions There are not yet enough data to know the viral dynamics in the different tissues and the associated pathogenesis Anoxia respiratory failure is one of the main characteristics of COVID19 Anoxic hypoxic hepatitis is common in patients with severe symptoms [] Reactivation of preexisting liver disease Patients with preexisting chronic liver medications such as tocilizumab and baricitinib used to combat the adverse immune cholestatic liver disease various studies such as lopinavir ritonavir remdesivir chloroquine tocilizumab uminefovir traditional Chinese medicine so it is important to consider that they could be potentially hepatotoxic in some patients [] Druginduced liver damage DILI Initial clinical guidelines recommended antiviral agents for COVID19 so a variety of drugs have been administered in disease may be more susceptible to liver damage from SARSCoV2 Biological Journal Preproof Genetic factors Genetics may well be one of the determining factors in some reaction may also cause HBV reactivation [] and induce eventual impairment of liver function in those patients with HBV On the other hand it is still unknown whether SARSCoV2 infection exacerbates cholestasis in people with underlying patients who become seriously ill with COVID19 but until now we cannot be sure It is possible for example that the genetic variation that makes an individual more susceptible to high blood pressure or diabetes also makes him more vulnerable to the virus It will be important to find out what role genetic factors predisposing to liver steatosis have and their sensitivity to severe symptoms of COVID19 Ji and 0ccolleagues showed that subjects with metabolicassociated fatty liver disease MAFLD have a higher risk of COVID19 severity disease and abnormal liver blood tests than patients without MAFLD [] In contrast Louise Biquard [] demonstrated that MAFLD is not associated with changes in liver expression blood test abnormalities reported by Ji and colleagues is thus likely not explained by Concluding remarks The scenario is not yet complete which does not allow us to establish or understand the natural history of the disease and the participation or commitment of the liver in this disease Certainly the application of new technological platforms such as singlecell increased hepatic SARSCoV2 uptake Still several contradictory reports will help of genes implicated in SARSCoV2 infection The observed persistence of liver to find the real role of genetic factors in the evolution of this disease Journal Preprooftranscriptomic assays will allow us to quickly know the commitment of each cell type in affected ans and the meaning of viral dynamics in the various affected systems including the liver However as we have already learned from the old hepatotropic viruses history still is ongoing and we have much to learn and understand about the virologic characteristics of this emerging RNA virus that allow us to develop specific antivirals such as the case of HCV and the vaccine to decrease the impact of this œacute infection Declarations of interest none Ethical approval Not required 0c References [] Chen N Zhou M Dong X Qu J Gong F Han Y Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China httpsdoi101002path1570 [] Wang D Eraslan B Wieland T Hallstr¶m B Hopf T Zolg DP A deep proteome and transcriptome abundance atlas of healthy human tissues Mol Syst [] Hamming I Timens W Bulthuis MLC Lely AT Navis GJ van Goor H Tissue distribution of ACE2 protein the functional receptor for SARS coronavirus A first step in understanding SARS pathogenesis J Pathol “ a descriptive study Lancet “ httpsdoi101016S0140Journal Preproofa manifestation of COVID19 Rev Gastroenterol Mxico English Ed Biol 201915e8503 httpsdoi1015252msb20188503 Patients with COVID19 J Am Coll Cardiol httpsdoi101016jjacc202005001 [] Paranjpe I Fuster V Lala A Russak A Glicksberg BS Levin MA Association of Treatment Dose Anticoagulation with InHospital Survival Among Hospitalized [] Schmulson M D¡valos MF Berumen J Beware Gastrointestinal symptoms can be httpsdoi101016jrgmxen202004001 [] Cai Q Huang D Yu H Zhu Z Xia Z Su Y COVID19 Abnormal liver function tests J Hepatol httpsdoi101016jjhep202004006 0c[] Siddiqi HK Mehra MR COVID19 illness in native and immunosuppressed states A clinicaltherapeutic staging proposal J Hear Lung Transplant Off Publ Int Soc Hear Transplant “ httpsdoi101016jhealun202003012 [] Feng G Zheng KI Yan QQ Rios RS Targher G Byrne CD COVID19 and [] between markers of liver injury and mortality in COVID19 in China Hepatology httpsdoi101002hep31301 [] de la Rica R Bes M Aranda M del Castillo A Socias A Payeras A Low Transl Hepatol “ httpsdoi1014218JCTH202000018 albumin levels are associated with poorer outcomes in a case series of COVID19 patients in Spain a retrospective cohort study MedRxiv Liver Dysfunction Current Insights and Emergent Therapeutic Strategies J Clin Lei F Liu YM Zhou F Qin JJ Zhang P Zhu L Longitudinal association Journal Preproofmortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ httpsdoi101016S0140liver directly contributes to hepatic impairment in patients with COVID19 J Hepatol httpsdoi101016jjhep202005002 httpsdoi1011012020050720094987 [] Zhou F Yu T Du R Fan G Liu Y Liu Z Clinical course and risk factors for [] Wang Y Liu S Liu H Li W Lin F Jiang L SARSCoV2 infection of the [] Gerussi A Rigamonti C Elia C Cazzagon N Floreani A Pozzi R Coronavirus Disease COVID19 in autoimmune hepatitis a lesson from 0cimmunosuppressed patients Hepatol Commun 2020na httpsdoi101002hep41557 [] Richman D Whitley R Hayden F Clinical virology 4th ed ASM Press [] Ksiazek TG Erdman D Goldsmith CS Zaki SR Peret T Emery S A Novel “ httpsdoi101056NEJMoa030781 [] Kuiken T Fouchier RAM Schutten M Rimmelzwaan GF van Amerongen G van respiratory syndrome Lancet “ httpsdoi101016S0140[] Drosten C G¼nther S Preiser W Van der Werf S Brodt HR Becker S Identification of a novel coronavirus in patients with severe acute respiratory Riel D Newly discovered coronavirus as the primary cause of severe acute Coronavirus Associated with Severe Acute Respiratory Syndrome N Engl J Med Journal Preproofsyndrome N Engl J Med “ httpsdoi101056NEJMoa030747 outbreak associated with a new coronavirus of probable bat origin Nature “ httpsdoi101038s4158602020127 [] Mortola E Roy P Efficient assembly and release of SARS coronaviruslike ps by a heterologous expression system FEBS Lett “ httpsdoi101016jfebslet200409009 [] Fehr A Perlman S Coronaviruses Methods and Protocols Methods in Molecular Biology Chapter Coronaviruses an overview of their replication and [] Zhou P Yang XL Lou Wang XGG Hu B Zhang L Zhang W A pneumonia 0cpathogenesis Springer Berlin Heidelberg [] Belouzard S Millet JK Licitra BN Whittaker GR Mechanisms of coronavirus cell entry mediated by the viral spike protein Viruses “ httpsdoi103390v4061011 [] Vennema H Godeke GJ Rossen JW Voorhout WF Horzinek MC Opstelten DJ et [] Snijder EJ Decroly E Ziebuhr J The Nonstructural Proteins Directing Coronavirus [] Neuman BW Buchmeier MJ Supramolecular Architecture of the Coronavirus P Adv Virus Res “ httpsdoi101016bsaivir201608005 [] van der Hoeven B Oudshoorn D Koster AJ Snijder EJ Kikkert M Barcena M [] Neuman BW Kiss G Kunding AH Bhella D Baksh MF Connelly S A structural analysis of M protein in coronavirus assembly and morphology J Struct Biol “ httpsdoi101016jjsb201011021 expression of viral envelope protein genes EMBO J “ al Nucleocapsidindependent assembly of coronaviruslike ps by coJournal PreproofBiogenesis and architecture of arterivirus replication anelles Virus Res “ httpsdoi101016jvirusres201604001 RNA Synthesis and Processing vol 1st ed Elsevier Inc httpsdoi101016bsaivir201608008 [] Rabi F Al Zoubi M Kasasbeh G Salameh D AlNasser A SARSCoV2 and Coronavirus Disease what we know so far Pathogens [] Masters P The molecular biology of coronaviruses Adv Virus Res “ 0c [] Shang J Ye G Shi K Wan Y Luo C Aihara H Structural basis of receptor recognition by SARSCoV2 Nature “ httpsdoi101038s415860202179y [] Rabaan AA AlAhmed SH Haque S Sah R Tiwari R Malik YS SARSCoV“ [] Li W Moore MJ Vasilieva N Sui J Wong SK Berne MA Angiotensin“ httpsdoi101038nature02145 [] Hoffmann M KleineWeber H P¶hlmann S A Multibasic Cleavage Site in the [] Cohen FS How Viruses Invade Cells Biophys J “ httpsdoi101016jbpj201602006 SARSCoV and MERSCOV A comparative overview Le Infez Med converting enzyme is a functional receptor for the SARS coronavirus Nature Journal PreproofEM structure of the 2019nCoV spike in the prefusion conformation Science “ httpsdoi101126scienceabb2507 Spike Protein of SARSCoV2 Is Essential for Infection of Human Lung Cells Mol Cell 202078779784e5 httpsdoi101016jmolcel202004022 [] Ziegler CGK Allon SJ Nyquist SK Mbano IM Miao VN Tzouanas CN SARSCoV2 Receptor ACE2 Is an InterferonStimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues Cell “ httpsdoi101016jcell202004035 [] Wrapp D Wang N Corbett KS Goldsmith JA Hsieh CL Abiona O Cryo 0c[] Follis K York J Nunberg J Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell“cell fusion but does not affect virion entry Virology “ httpsdoi101016jvirol200602003 [] Millet JK Whittaker GR Host cell proteases Critical determinants of coronavirus [] Li MYY Li L Zhang Y Wang XSS Expression of the SARSCoV2 cell receptor httpsdoi101186s4024902000662x [] Xu H Zhong L Deng J Peng J Dan H Zeng X High expression of ACE2 receptor of 2019nCoV on the epithelial cells of oral mucosa Int J Oral Sci httpsdoi101038s413680200074x tropism and pathogenesis Virus Res “ httpsdoi101016jvirusres201411021 gene ACE2 in a wide variety of human tissues Infect Dis Poverty 20209NA Journal PreproofCoV2 protein interaction map reveals targets for drug repurposing Nature Infection of SARSCoV2 Gastroenterology 202015818311833e3 httpsdoi101053jgastro202002055 httpsdoi101038s4158602022869 [] Xu L Liu J Lu M Yang D Zheng X Liver injury during highly pathogenic human coronavirus infections Liver Int Off J Int Assoc Study Liver “ httpsdoi101111liv14435 [] Coomes EA Haghbayan H Interleukin6 in COVID19 A Systematic Review and [] Xiao F Tang M Zheng X Liu Y Li X Shan H Evidence for Gastrointestinal [] Gordon DE Jang GM Bouhaddou M Xu J Obernier K White KM A SARS 0cMetaAnalysis MedRxiv httpsdoi1011012020033020048058 [] Xu Z Shi L Wang Y Zhang J Huang L Zhang C Pathological findings of COVID19 associated with acute respiratory distress syndrome Lancet Respir Med “ httpsdoi101016S221326002030076X [] Zhang B Zhou X Qiu Y Feng F Feng J Jia Y Clinical characteristics of [] Chen G Wu D Guo W Cao Y Huang D Wang H Clinical and [] Chai X Hu L Zhang Y Han W Lu Z Ke A Specific ACE2 Expression in Invest “ httpsdoi101172JCI137244 death cases with COVID19 MedRxiv httpsdoi1011012020022620028191 immunological features of severe and moderate coronavirus disease J Clin Journal PreproofCholangiocytes May Cause Liver Damage After 2019nCoV Infection BioRxiv patients MedRxiv httpsdoi1011012020040120047381 httpsdoi10110120200203931766 [] Herold T Jurinovic V Arnreich C Hellmuth JC von BergweltBaildon M Klein M Level of IL6 predicts respiratory failure in hospitalized symptomatic COVID[] Grein J Ohmagari N Shin D Diaz G Asperges E Castagna A Compassionate Use of Remdesivir for Patients with Severe Covid19 N Engl J Med “ httpsdoi101056nejmoa2007016 [] U S Food and Drug Administration Fact sheet for health care providers emergency 0cuse authorization EUA of hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of COVID19 in certain hospitalized patients [] Varona Prez J Rodriguez Chinesta JM Riesgo de reactivacin de la hepatitis B asociado al tratamiento con corticoides frente a SARSCoV2 COVID19 Rev Cl­nica Espa±ola httpsdoi101016jrce202004012 [] Ji D Qin E Xu J Zhang D Cheng G Wang Y Nonalcoholic fatty liver httpsdoi101016jjhep202003044 SARSCoV2 in metabolicassociated fatty liver disease J Hepatol diseases in patients with COVID19 A retrospective study J Hepatol Journal Preproof[] Biquard L Valla D Rautou PE No evidence for an increased liver uptake of httpsdoi101016jjhep202004035 0cFigure legends Journal PreproofFig1 Proposed mechanisms of liver pathogenicity of SARSCoV2 in infected cells SARS CoV2 Infection2 Cytokinestorm3 Drugeffects4 Hypoxia5 PreviousliverdamageBiochemicallabmarkersWhite bloodcellsGenomereleaseReplicationTranslationVirionassemblyViral proteinsMaturevirus release\uf0e9AaDO2MitochondrialproteinsHypoxicisquemicliverinjuryLIVER DAMAGELopinavirRitonavirRemdesivirChloroquineTocilizumabOxidativeimbalanceSteatosisACE2S proteinCytopathiceffect\uf0e9GMCSF\uf0e9IL6\uf0e9IL1β\uf0e9IL2\uf0e9IL8\uf0e9CCL2\uf0e9CCL3\uf0e9CCL5\uf0e9CXCL \uf0e9ALT\uf0e9AST\uf0eaAlbumin\uf0e9PCR\uf0e9LDH\uf0e9Ddimer\uf0e9Ferritin\uf0e9Bilirubin 0c"

Thyroid_Cancer

A prediction model of outcome of a0SARS‘CoV‘ a0pneumonia a0based a0on a0laboratory a0findingsGang a0Wu1 a0Shuchang a0Zhou1 a0Yujin a0Wang1 a0Wenzhi a0Lv2 a0Shili a0Wang3 a0Ting a0Wang4 Xiaoming a0Li1The a0severe a0acute a0respiratory a0syndrome a0coronavirus a0 a0SARS‘CoV‘ a0has a0resulted a0in a0thousands a0of a0deaths a0in a0the a0world a0Information a0about a0prediction a0model a0of a0prognosis a0of a0SARS‘CoV‘ a0infection a0is a0scarce a0We a0used a0machine a0learning a0for a0processing a0laboratory a0findings a0of a0 a0patients a0with a0SARS‘CoV‘ a0pneumonia a0including a0 a0non‘survivors a0and a0 a0discharged a0patients a0The a0maximum a0relevance a0minimum a0redundancy a0mRMR a0algorithm a0and a0the a0least a0absolute a0shrinkage a0and a0selection a0operator a0logistic a0regression a0model a0were a0used a0for a0selection a0of a0laboratory a0features a0Seven a0laboratory a0features a0selected a0in a0the a0model a0were a0prothrombin a0activity a0urea a0white a0blood a0cell a0interleukin‘ a0receptor a0indirect a0bilirubin a0myoglobin a0and a0fibrinogen a0degradation a0products a0The a0signature a0constructed a0using a0the a0seven a0features a0had a0 a0[ a0] a0sensitivity a0and a0 a0[ a0] a0specificity a0in a0predicting a0outcome a0of a0SARS‘CoV‘ a0pneumonia a0Thus a0it a0is a0feasible a0to a0establish a0an a0accurate a0prediction a0model a0of a0outcome a0of a0SARS‘CoV‘ a0pneumonia a0based a0on a0laboratory a0findingsMost human coronavirus infections are mild However several betacoronaviruses can cause serious diseases or even death12 The mortality rates of severe acute respiratory syndrome coronavirus SARSCoV and Middle East respiratory syndrome coronavirus MERSCoV were and respectively SARSCoV2 is the pathogen for novel coronavirus disease COVID1934 which has resulted in thousands of deaths in the world since the beginning of The diagnosis of SARSCoV2 infection must be confirmed by the realtime reverse transcriptase polymerase chainreaction RTPCR or gene sequencing of specimens of patients56 Chest radiograph and laboratory findings are both important for accessing the severity of the disease7“ Critical patients should be admitted to Intensive Care Unit ICU of infectious disease hospital while mild patients could be kept and treated at isolation It is very important to effectively prioritize resources for patients with the highest risk because of the large number of infected people10ICU patients and nonICU patients differed significantly in some blood parameters including leukocytes neutrophils prothrombin time Ddimer total bilirubin TB lactate dehydrogenase high sensitivity cardiac troponin I and procalcitonin5711 Ruan et a0al12 retrospectively analyzed laboratory findings of nonsurvivors and discharged patients and found significant differences in lymphocytes platelets albumin TB urea nitrogen creatinine myoglobin Creactive protein and interleukin6 between the two groups These laboratory findings seemed useful in predicting outcome of SARSCoV2 infection However an advanced prediction model involving multiple laboratory parameters is urgently required to be applied in a clinicaldecision support system to improve the predictive and prognostic accuracyAs a branch of artificial intelligence machine learning ML helps establish accurate prediction model13“ However there are few publications reporting prediction of the outcome of SARSCoV2 pneumonia using ML methods based on laboratory findings Thus we retrospectively collected laboratory findings of discharged patients and nonsurvivors These data were dealt with a ML method similar to radiomics1617 We aim to establish a prediction model of outcome of SARSCoV2 pneumonia based on laboratory data1Department of Radiology Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Wuhan China 2Julei Technology Wuhan China 3Computational Biology Carnegie Mellon University Pittsburgh USA 4Department of Medical Ultrasound Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Wuhan China email 751884926qqcom lilyboston2002qqcomScientific RepoRtS 101038s41598020711147Vol0123456789wwwnaturecomscientificreports 0cMethodsAll methods were carried out in accordance with relevant guidelines and regulationsStudy a0design a0and a0participants a0 This study was approved by the Ethics Commission of Hospital TJ Written informed consent was waived by the Ethics Commission of hospitalThe author™s center was the designated hospital for severe and critical SARSCoV2 pneumonia Patients underwent repeated RTPCR tests to confirm SARSCoV2 Laboratory tests for SARSCoV2 pneumonia included blood routine test serum biochemical including glucose renal and liver function creatine kinase lactate dehydrogenase and electrolytes coagulation profile cytokine test markers of myocardial injury infectionrelated makers and other enzymes Repeated tests were done every “ a0days for monitoring the patient™s conditionOxygen support from nasal cannula to invasive mechanical ventilation was administered to patients according to the severity of hypoxaemia All patients were administered with empirical antibiotic treatment and received antiviral therapy Most of patients improved after treatment However a few critical patients continued to deteriorate and eventually diedData a0collection a0 fatal cases of SARSCoV2 pneumonia male median age a0years were collected by the electronic medical record system discharged patients with SARSCoV2 pneumonia whose age and gender matched the nonsurvivors were selected male median age a0years The admission date of these patients was from Feb to Mar We reviewed all laboratory findings for each patient Results of repeated tests were carefully compared to find the greatest deviation from normal value In general the greatest number in series of values was recorded However for platelets red blood cell lymphocytes hemoglobin calcium total protein albumin estimated glomerular filtration rate eGFR and prothrombin activity PTA the minimum was recorded Laboratory findings at the day of mortality were not used These recorded laboratory findings were considered as lab features of a patient A initial data set of patients nonsurvivor discharge was thus builtThere were patients who did not have the entire group of laboratory features thus their data were deleted from the dataset The remaining data of patients nonsurvivor discharge were analyzed by machine learningStatistical a0analysis a0and a0modeling a0 First all the variables were compared between nonsurvivors and discharged patients using the Mann“Whitney U test for nonnormally distributed features or the independent t test for normally distributed features1617 Features with P were considered significant variables and selected1617 Second Spearman™s correlation coefficient was used to compute the relevance and redundancy of the features1617 Third we applied the maximum relevance minimum redundancy mRMR algorithm to assess the relevance and redundancy of the features1617 The features were ranked according to their mRMR scores1617 Fourth the top features with highrelevance and lowredundancy were selected for least absolute shrinkage and selection operator LASSO logistic regression model The LASSO logistic regression model was adopted for further features selection1617 Some candidate features coefficients were shrunk to zero and the remaining variables with nonzero coefficients were finally selected1617 The model was used for calculating signature for each patient Mann“Whitney U test was used for comparing signature between two groups1617 Receiver operator characteristic ROC precision recall curve PRC analysis and Hosmer“Lemeshow test were used for further evaluation of modelThe statistical analyses were performed using R software version wwwrproje ct1617 The following R packages were used the œcorrplot package was used to calculate Spearman™s correlation coefficient the œmRMRe package was used to implement the mRMR algorithm the œglmnet was used to perform the LASSO logistic regression model and the œpROC package was used to construct the ROC curve1617ResultsNine laboratory features were eliminated in the first step of feature selection because of nonsignificance The remaining thirtyeight lab features were significantly different between two groups P and then mRMR scores were obtained for them There were seven features having nonzero coefficients after LASSO algorithm and were selected for the model Table a0 shows the fifteen features with the highest mRMR scores Figure a0 shows the correlation matrix heatmap of the thirtyeight significant features Figure a0 shows the feature selection process with LASSO algorithm Figure a0 shows the contribution of the seven features to the model Figure a0 shows the signatures of all patients as well as ROC Figure a0 shows the PRC for the modelNonsurvivors and discharged patients differed significantly in the signature derived from the model P The AUC was [ CI ] The sensitivity and specificity in predicting outcome of SARSCoV2 pneumonia were [ ] and [ ] respectively The area under precision recall curve AUPRC was Hosmer“Lemeshow test showed good calibration P for the modelThe seven features included in the prediction model were as follows PTA urea white blood cell WBC interleukin2 receptor IL2r indirect bilirubin IB myoglobin and fibrinogen degradation products FgDP All features had coefficients of positive number except PTA PTA and FgDP are from coagulation profile Urea and IB are from renal and liver function respectively WBC is from blood routine test Myoglobin is a marker of myocardial injury IL2r is related to immune response The signatures derived from the model could be positive or negative numbersScientific RepoRtS 101038s41598020711147Vol1234567890wwwnaturecomscientificreports 0cRankFeaturesPTAWBCUreaIL2rIBMyoglobinTBFgDPhsCRPFerritinLDHDdimereGFRNeutrophilsSodiummRMR scoreCoefficient after LASSOˆ’ Table The fifteen features with higher mRMR scores were selected for the step of LASSO logistic regression Some candidate features coefficients were shrunk to zero and the remaining variables with nonzero coefficients were selected mRMR maximum relevance minimum redundancy LASSO least absolute shrinkage and selection operator PTA prothrombin activity WBC white blood cell IL2r interleukin2 receptor IB indirect bilirubin TB total bilirubin FgDP fibrinogen degradation products hsCRP hypersensitive Creactive protein LDH lactate dehydrogenase eGFR estimated glomerular filtration rateFigure a0 Correlation matrix heatmap of significant features Spearman™s correlation coefficient was used to compute the relevance and redundancy of the featuresNonsurvivors and discharged patients did not differ in age or gender median age vs P percentage of males vs P The comparisons of laboratory findings between nonsurvivors and discharged patients are shown in Table a0Blood a0 routine a0 test a0 WBC and neutrophils were significantly higher in nonsurvivor group versus discharge group Lymphocyte platelets and red blood cells were significantly lower in nonsurvivors AUC for them were “Scientific RepoRtS 101038s41598020711147Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The fivefold crossvalidation A of the least absolute shrinkage and selection operator algorithm for feature selection process A vertical line was drawn at the optimal value Some candidate features coefficients were shrunk to zero B and the remaining seven variables with nonzero coefficients were finally selectedElectrolyte a0 Potassium chlorine and sodium were significantly higher in nonsurvivor group versus discharge group Calcium was significantly lower in nonsurvivors AUC for them were “Serum a0biochemical a0test a0 Glucose and globulin were significantly higher in nonsurvivor group versus discharge group Albumin and total protein were significantly lower in nonsurvivors AUC for them were “Renal a0 function a0 Urea and creatinine were significantly higher in nonsurvivor group versus discharge group The eGFR was significantly lower in nonsurvivors AUC for them were “Liver a0function a0 Total bilirubin direct bilirubin IB and glutamic oxaloacetic transaminase were significantly higher in nonsurvivor group versus discharge group AUC for them were “Scientific RepoRtS 101038s41598020711147Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Contribution of the features to the model The histogram shows the contribution of the seven features with nonzero coefficients The features are plotted on the yaxis and their coefficients are plotted on the xaxisFigure a0 Bar charts of the signature for patients The red bars indicate the signatures of discharged patients while the light green bars indicate the signatures of nonsurvivors The AUC was for the signatureCoagulation a0 profile a0 Prothrombin time activated partial thromboplastin time Ddimer international normalized ratio INR fibrinogen and FgDP were significantly higher in nonsurvivor group versus discharge group PTA was significantly lower in nonsurvivors AUC for them were “Cytokine a0them were “IL2r and IL6 were significantly higher in nonsurvivor group versus discharge group AUC for Infection‘related a0markers a0and a0myocardial a0injury a0markers a0 Procalcitonin high sensitive Creactive protein ferritin and Nterminal probrain natriuretic peptide NTproBNP were significantly higher in nonScientific RepoRtS 101038s41598020711147Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The precision recall curve for the model The area under precision recall curve was survivor group versus discharge group Myoglobin MB isoenzyme of creatine kinase and high sensitive cardiac troponin I were significantly higher in nonsurvivors AUC for them were “DiscussionNonsurvivors and discharged patients with SARSCoV2 pneumonia differed significantly in thirtyeight laboratory findings By using machine learning method we established a prediction model involving seven laboratory features The model was found highly accurate in distinguishing nonsurvivors from discharged patients The seven features selected by artificial intelligence also indicated that dysfunction of multiple ans or systems correlated with the prognosis of SARSCoV2 pneumoniaThe SARSCoV2 triggers a series of immune responses and induces cytokine storm resulting in changes in immune components518 When immune response is dysregulated it will result in an excessive inflammation even cause death719 Excessive neutrophils may contribute to acute lung damage and are associated with fatality20 Higher serum level of IL2r was found in nonsurvivors indicating excessive immune response In addition high leukocyte count in SARSCoV2 patients may be also due to secondary bacterial infection2122Liver injury has been reported to occur during the course of the disease2324 and is associated with the severity of diseases Increased serum bilirubin level was observed in fatal cases Acute kidney injury could have been related to direct effects of the virus hypoxia or shock2526 Blood urea level continued to increase in some cases Nonsurvivors had higher blood urea compared to survivors Myocardial injury was seen in nonsurvivors which was suggested by elevated level of myoglobin The mechanism of multiple an dysfunction or failure may be associated with the death of patients with SARSCoV2 pneumonia Some patients with SARSCoV2 infection progressed rapidly with sepsis shock which is well established as one of the most common causes of disseminated intravascular coagulation DIC27 The nonsurvivors in our cohort revealed significantly lower PTA compared to survivors At the late stages of SARSCoV2 infection level of fibrinrelated markers FgDP markedly elevated in most cases suggesting a secondary hyperfibrinolysis conditionA number of laboratory features were compared between nonsurvivors and discharged patients with SARSCoV2 pneumonia The two groups differed significantly in as many as thirtyeight lab features However none of the futures provided adequate accuracy in predicting the outcome of SARSCoV2 pneumonia Thus a novel prediction model involving multiple features was established in the study With machine learning methods previously used in radiomics a prediction model combining seven out of thirtyeight laboratory features was built for predicting the outcome of SARSCoV2 pneumoniaThe mRMR algorithm was used for assessing significant features to avoid redundancy between features The mRMR score of a feature is defined as the mutual information between the status of the patients and this feature minus the average mutual information of previously selected features and this feature172829 The top fifteen features with high mRMR scores were selected for the next step of modeling The least absolute shrinkage and selection operator logistic regression model was used to processing the features selected by mRMR algorithm LASSO is actually a regression analysis method that improves the model prediction accuracy and interpretability30 The signature calculated with the model can be positive or negative number corresponding with poor and good prognosis respectively Our results showed that the AUC of the signature was “ higher than that of a single featureThe modeling process is a black box however the choice of variables seems reasonable PTA can more accurately reflect the coagulation function compared to prothrombin time and can also reflect the degree of liver injury Urea is a good index to reflect the degree of renal function damage WBC can not only reflect immune Scientific RepoRtS 101038s41598020711147Vol1234567890wwwnaturecomscientificreports 0cLeucocyte 109LPlatelet 109LErythrocyte 1012LNeutrophils 109LLymphocyte 109LHemoglobin gLPotassium mmolLCalcium mmolLChlorine mmolLSodium mmolLGlucose mmolLTotal protein gLGlobulin gLAlbumin gLCreatinine μmolLUric acid μmolLTotal bilirubin μmolLDirect bilirubin μmolLIndirect bilirubin μmolLUrea mmolLEstimated glomerular filtration rate mlmin173 a0m2Glutamic oxaloacetic transaminase ULGlutamicpyruvic transaminase ULMyoglobin ngmLHigh sensitive cardiac troponin I pgmLMB isoenzyme of creatine kinase ngmLLactate dehydrogenase ULGlutamate dehydrogenase ULCreatine kinase ULProthrombin time sFibrinogen gLActivated partial thromboplastin time sThrombin time sD“D dimer μgmLProthrombin activityInternational standardized ratioFibrinogen degradation products μgmLProcalcitonin ngmLNterminal probrain natriuretic peptide pgmLFerritin μgLHypersensitive Creactive protein mgLInterleukin1β pgmLInterleukin2 receptor UmLInterleukin6 pgmLInterleukin10 pgmLNonsurvivors [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]Discharged patients [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]P Table Medians [interquartile range] of laboratory findings of patients with SARSCoV2 pneumonia were provided in the table Features were compared between nonsurvivors and discharged patients using the Mann“Whitney U test for nonnormally distributed features or the independent t test for normally distributed features SARSCoV2 severe acute respiratory syndrome coronavirus status but also secondary infection IL2r is an indicator of inflammation and immune response20 IB is related to liver function and possible hemolysis Myoglobin reflects the degree of myocardial injury The increase of FgDP is related to coagulation disorders including DIC Thus the current model involves multiple important systems related to prognosis In consideration of the high accuracy of the model it can be concluded that liver kidney myocardial damage coagulation disorder and excess immune response all contribute to the outcome of SARSCoV2 pneumoniaScientific RepoRtS 101038s41598020711147Vol0123456789wwwnaturecomscientificreports 0cIt is suitable to start to use this model after three repeated laboratory tests about a0weeks after admission because doctors may have enough data at that time Lots of laboratory findings are generated in hospitalization Which are most important for predicting outcome Our study at least answered such a problem Seven laboratory features could be used to construct a new signature with the model The new signature seems more useful than any single feature We encourage such a simpletouse model widely used in clinical practiceMost of clinical factors are not continuous variables such as underlying disease We used a machine learning method similar to radiomics which mainly deals with continuous features Our study focused on continuous laboratory variables We had to exclude noncontinuous clinical factor with the current machine learning method By using other methods a model that involves both continuous variables and category variables can be established Thus clinical factors raised as significant predictive factors such as respiratory status or radiological features could be included in the models However there are more than forty laboratory findings in our study making establishment of model difficult We felt it necessary to simplify laboratory features Thus we establish a submodel based on lab findings A new lab signature is thus created and is proved highly valuable In future study the signature may be combined with clinical factors to establish a more complex modelOur study has some limitations First this is a singlecenter retrospective study Multicenter largesample studies are required to validate our prediction model Second our model may not be directly used in other centers However they could easily establish a prediction model using their own data with machine learning method Third some patients who did not have all the lab findings were excluded Selection bias must be present due to patients exclusion Other studies with more strict design were thus required to reveal the bias Fourth statistical approach conducted in this study is not perfect As LASSO was used for variables or more patients were needed More patients should be collected in future studyIn conclusion it is feasible to establish a accurate prediction model of outcome of SARSCoV2 pneumonia based on laboratory findings Injury of liver kidney and myocardium coagulation disorder and excess immune response all correlate with the outcome of SARSCoV2 pneumoniaData a0availabilityAfter publication the data will be made available to others on reasonable requests to the corresponding authorReceived March Accepted August References Drosten C et al Identification of a novel coronavirus in patients with severe acuterespiratory syndrome N Engl J Med “ “ Zaki A M Boheemen S Bestebroer T M Osterhaus A D Fouchier R A Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia N Engl J Med “ Phelan A L Katz R Gostin L O The novel coronavirus originating in Wuhan China challenges for global health governance JAMA 101001jama20201097 Li Q et al Early transmission dynamics in Wuhan China of novel coronavirusinfected pneumonia N Engl J Med Huang C et al Clinical features of patients infected with novel coronavirus in Wuhan China Lancet “ Zhu N et al A novel coronavirus from patients with pneumonia in China N Engl J Med “ Wang D et al Clinical characteristics of hospitalized patients with novel coronavirusinfected pneumonia in Wuhan China JAMA “ Bernheim A et al Chest CT findings in coronavirus disease19 COVID19 relationship to duration of infection Radiology 101148radio l20202 Fang Y et al Sensitivity of chest CT for COVID19 comparison to RTPCR Radiology 101148radio l20202 General Office of the National Health Commission of China Diagnosis and treatment protocol for 2019nCoV 5th ed Beijing China National Health Commission of China Yang X et al Clinical course and outcomes of critically ill patients with SARSCoV2 pneumonia in Wuhan China a singlecentered retrospective observational study Lancet Respir Med “ Ruan Q Yang K Wang W Jiang L Song J Clinical predictors of mortality due to COVID19 based on an analysis of data of patients from Wuhan China Intensive Care Med “ Shiri I et al Nextgeneration radiogenomics sequencing for prediction of EGFR and KRAS mutation status in NSCLC patients using multimodal imaging and machine learning algorithms Mol Imaging Biol 101007s1130 Matsuzaka Y et al Prediction model of aryl hydrocarbon receptor activation by a novel QSAR approach deepSnapdeep learning Molecules Katić K Li R Zeiler W Machine learning algorithms applied to a prediction of personal overall thermal comfort using skin temperatures and occupants™ heating behavior Appl Ergon Jiang M et al Nomogram based on shearwave elastography radiomics can improve preoperative cervical lymph node staging for papillary thyroid carcinoma Thyroid “ Zhang P et al T2weighted imagebased radiomics signature for discriminating between seminomas and nonseminoma Front Qin C et al Dysregulation of immune response in patients with COVID19 in Wuhan China Clin Infect Dis Oncol 101093cidciaa2 Mahallawi W H Khabour O F Zhang Q Makhdoum H M Suliman B A MERSCoV infection in humans is associated with a proinflammatory Th1 and Th17 cytokine profile Cytokine “ Channappanavar R Perlman S Pathogenic human coronavirus infections causes and consequences of cytokine storm and immunopathology Semin Immunopathol “ Chen N et al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet “ Guan W et al Clinical characteristics of novel coronavirus infection in China N Engl J Med “ Scientific RepoRtS 101038s41598020711147Vol1234567890wwwnaturecomscientificreports 0c Tang N Li D Wang X Sun Z Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia J Thromb Haemost “ Xu L Liu J Lu M Yang D Zheng X Liver injury during highly pathogenic human coronavirus infections Liver Int Estenssoro E et al Pandemic influenza A in Argentina a study of patients on mechanical ventilation Am J Respir Crit Li K et al The clinical and chest CT features associated with severe and critical COVID19 pneumonia Investig Radiol “ Care Med “ “ Abe T et al Complement activation in human sepsis is related to sepsisinduced disseminated intravascular coagulation Shock Lin X Li C Ren W Luo X Qi Y A new feature selection method based on symmetrical uncertainty and interaction gain 101097SHK00000 Comput Biol Chem Wang J et al Machine learningbased analysis of MR radiomics can help to improve the diagnostic performance of PIRADS v2 in clinically relevant prostate cancer Eur Radiol “ Sauerbrei W Royston P Binder H Selection of important variables and determination of functional form for continuous predictors in multivariable model building Stat Med “ AcknowledgementsWe thank all patients and their families involved in the studyAuthor a0contributionsGW SZ and YW collected the epidemiological and clinical data TW WL and SW summarized all data GW XL drafted the manuscript TW and XL revised the final manuscriptCompeting a0interests The authors declare no competing interestsAdditional a0informationCorrespondence and requests for materials should be addressed to TW a0or a0XLReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Access This article is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this article are included in the article™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the article™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020711147Vol0123456789wwwnaturecomscientificreports 0c'

Thyroid_Cancer

"Type and diabetes confer an increased risk of pancreatic cancer PaC of similar magnitudesuggesting a common mechanism The recent finding that PaC incidence increases linearly with increasing fastingglucose levels supports a central role for hyperglycaemia which is known to cause carbonyl stress and advancedglycation endproduct AGE accumulation through increased glycolytic activity and nonenzymatic reactions Thisstudy investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanismsinvolvedMethods Pdx1CreLSLKrasG12D mice were interbred with mitosis luciferase reporter mice rendered diabetic withstreptozotocin and treated or not with carnosinol FL92616 a selective scavenger of reactive carbonyl speciesRCS and as such an inhibitor of AGE formation Mice were monitored for tumour development by in vivobioluminescence imaging At the end of the study pancreatic tissue was collected for histologyimmunohistochemistry and molecular analyses Mechanistic studies were performed in pancreatic ductaladenocarcinoma cell lines challenged with high glucose glycolysis and glycoxidationderived RCS their proteinadducts AGEs and sera from diabetic patientsResults Cumulative incidence of invasive PaC at weeks of age was in untreated diabetic vs in FL92616gtreated diabetic and in nondiabetic mice FL92616 treatment suppressed systemic and pancreaticcarbonyl stress extracellular signalregulated kinases ERK activation and nuclear translocation of Yesassociatedprotein YAP in pancreas In vitro RCS scavenging and AGE elimination completely inhibited cell proliferationstimulated by high glucose and YAP proved essential in mediating the effects of both glucosederived RCS andtheir protein adducts AGEs However RCS and AGEs induced YAP activity through distinct pathways causingreduction of Large Tumour Suppressor Kinase and activation of the Epidermal Growth Factor ReceptorERKsignalling pathway respectivelyContinued on next page Correspondence giuseppepuglieseuniroma1it Stefano Menini and Carla Iacobini contributed equally to this work1Department of Clinical and Molecular Medicine œLa Sapienza University Viadi Grottarossa Rome ItalyFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMenini Journal of Experimental Clinical Cancer Research Page of Continued from previous pageConclusions An RCS scavenger and AGE inhibitor prevented the accelerating effect of diabetes on PainINsprogression to invasive PaC showing that hyperglycaemia promotes PaC mainly through increased carbonyl stressIn vitro experiments demonstrated that both circulating RCSAGEs and tumour cellderived carbonyl stressgenerated by excess glucose metabolism induce proliferation by YAP activation hence providing a molecularmechanism underlying the link between diabetes and PaC and cancer in generalKeywords Pancreatic ductal adenocarcinoma Hyperglycaemia Reactive carbonyl species Methylglyoxal Advancedglycation endproducts Carnosine derivatives Yesassociated protein Large tumour suppressor kinase Epidermalgrowth factor receptor Extracellular signalregulated kinases BackgroundPancreatic cancer PaC is the tenth most common incident cancer but the seventh leading cause of cancerrelated death worldwide [] because of the poor 5yearsurvival outcomes [] Due to the rising prevalence ofrisk factors such as obesity and type diabetes PaC isexpected to become the second leading cause of cancerrelated death in the US by [] Type diabetes wasfound to be associated with a “7fold higher risk ofPaC in the first year after diabetes diagnosis and nearlytwofold thereafter [ ] Though type diabetes is themain contributor to this problem the entity and temporal trajectory of PaC risk were recently reported to besimilar in type diabetes [] suggesting a commonmechanism related to hyperglycaemia This concept issupported by the recent finding that PaC incidence increases linearly with increasing fasting glucose levelseven within the normal range []thus hindering the understanding ofPrevious studies have shown that type diabetes induced by a highfat diet promotes PaC [ ] Howeverthis experimental model of the metabolic syndrome doesnot allow assessing the role of hyperglycaemia independent of confounding factors such as obesity and hyperinsulinemiathemechanisms underlying the risk conferred by hyperglycaemia We have recently demonstrated that advancedglycation endproducts AGEs promote proliferation ofhuman pancreatic ductal adenocarcinoma PDA cell linesand that exogenous AGE administration markedly accelerates invasive tumour development in a mouse model ofKrasdriven PaC [] Accumulation of AGEs in diabetesis mainly due to increased formation of reactive carbonylspecies RCS derived from glucose autooxidation egglyoxal GO but also from cell metabolism of excess glucose through glycolysis eg methylglyoxal MGO []In turn RCS react with amino groups of proteins causingstructural and functional modifications The resulting irreversible adducts ie AGEs accumulate in tissues wherethey can exert further biological effects through interaction with specific receptors [ ]Carnosine betaalanylLhistidine is an endogenousinhibits AGEhistidinecontainingdipeptidethat[]carnosine derivativesformation by scavenging RCS [] Though LCarnosinewas proven to be effective in several carbonyl stressrelated disease conditionsincluding metabolicdisorders [“]its therapeutic use in humans ishampered by the presence of high levels of serum carnothus prompting the search for carnosinasesinase[“] The novelresistantbioavailable compound carnosinol ie 2S23aminopropanoylamino31Himidazol5ylFL [“] was shown to be highly effective in attenuating diabetesassociated vascular complications [] and obesityrelated metabolic dysfunctions [ ]Moreover it was recently shown that Lcarnosine is effective in counteracting glycolysisdependenttumourgrowth by quenching RCS []propanollesionsthe progression of preneoplasticThis study aimed at investigating whether hyperglycaemia associated with experimental type diabetes favourstomalignancy in a wellvalidated mouse model of PaC byincreasing carbonyl stress To this end mice weretreated with the RCS scavenger and inhibitor of AGEformation FL92616 An additional objective was toanalyse the effect of the diabetic milieu and of FL926 on the activity of Yesassociated protein YAP a keydownstream target of KRAS signalling required for progression of pancreatic intraepithelial neoplasias PanINsto invasive PaC [ ] and for MGOinduced tumourgrowth []MethodsIn vivo studyThe experimental protocols comply with the principles ofwwwnc3rsukarriveguidelines and were approved by the National Ethics Committee for Animal Experimentation ofof HealthAuthorization no 14702015PR The mice were housedin single cages with woodderived bedding material in aspecific pathogenfree facility with a 12h lightdark cycleunder controlled temperatures “ °C Mice werecared for in accordance with the Principles of LaboratoryAnimal Care National Institutes of Health publ no “ revised and with national laws and receivedItalian Ministrythe 0cMenini Journal of Experimental Clinical Cancer Research Page of water and food ad libitum The primary and secondaryendpoint were the development of invasive PaC and thedevelopmentprogression of PanINs respectivelyDesignThe effect of diabetes on PaC progression was investigatedin Pdx1CreLSLKrasG12D KC mice which develop autochthonous PaC in a pattern recapitulating human pathology with high fidelity by developing the full spectrum ofPaC progression from preneoplastic lesions PanINs toadenocarcinoma and metastasis [ ] KC mice wereinterbred with mitosis luciferase MITO“Luc reporter miceto obtain KCMito KCM mice [ ] The LSLKrasG12D lineage was maintained in the heterozygousstate Mice were screened by polymerase chain reactionPCR using tail DNA amplified by specific primers to theLoxP cassette flanking mutated KrasG12D wild type KrasCre recombinase and MITO genes as previously reported[ ] In the MITOLuc mouse an artificial minimal promoter derived from the cyclin B2 gene and induced by NFY drives the expression of the luciferase reporter specificallyin replicating cells Therefore both normal eg bone marrow and tumour actively proliferating cells may be localized by a bioluminescence imaging BLIbased screen [ ] We have previously shown that KCM mice developpreinvasive PanINs and invasive ductal PaC with thesame penetrance latency and histological features as thosedescribed for KC mice [] According to the Ethics Committee recommendations to limit the number of animalsthe experiments were stopped when it was sufficient toconfirm or reject the working hypothesis in a statisticallyand clinically meaningful mannerFigure shows the flowchart and timeline of study design Thirtythree KCM mice were rendered diabeticFig Flowchart and timeline of study design Please refer to the text for detailed description In dashed boxes groups of nondiabetic KCMmice Ctr that served as control for the effect of STZ STZnonDiab or FL92616 FL treatment Ctr FL on PaC development and progressionTo avoid unnecessary suffering three diabetic mice Diab and one Diab mouse treated with FL DiabFL were killed and weeks respectivelybefore the end of the study STZ streptozotrocin BLI bioluminescence imaging 0cMenini Journal of Experimental Clinical Cancer Research Page of with streptozotocin STZ and followed for weeksie up to weeks of age After an overnight fast weekold mice were intraperitoneally injected with mg·kgˆ’ STZ SigmaAldrich St Louis MO USA Success rate defined as the percentage of STZinjected micewith glucose levels mgdL for the entire studyperiod was Three days after injection diabetic mice were randomized to receive no treatmentDiab n or FL92616 gift of Flamma SpAChignolo d™Isola Italy [] at a dose of mg·kgˆ’ ˆ™dayˆ’ in the drinking water DiabFL n andinjected weekly with IU of insulin glargine to preventexcessive weight loss and ketoacidosis FL92616 wasshown to have a suitable absorption distribution metabolism excretion and toxicity ADMET profile and thegreatest potency and selectivity toward RCS among allother carnosine derivatives [] The FL92616 dosewas chosen based on previous results from our group[]showing high efficacy in preventing diabetesinduced renal injury and from other investigators indicating a good safety profile at the dose of “ g·kgˆ’ ·dayˆ’ [ ] Neither histological abnormalities of theliver kidney lung and heart nor functional abnormalities attributable to toxicity on these tissues wereobserved in this study or in a previous one [] STZtreated mice not fulfilling the criteria for diabetes diagnosis STZnonDiab n served as control for STZeffect on PaC seven of these mice failed to develophyperglycaemia whereas two had spontaneous recoveryfrom diabetes within weeks Vehicle salineinjectedKCM mice were used as nondiabetic controls and either left untreated Ctr n or treated with FL926 Ctr FL n to check for any drug effectMice were subjected to in vivo BLI every otherweek [ ] and daily manual palpation of the abdomen to check for tumour growth and avoid the lossof animals along with the need to cope with the related ethicalissues ie compliance with the 3Rsprinciples Briefly min after administration of Dluciferin mgkg body weightintraperitoneal Perkin Elmer Hopkinton MA USA photon emissionfrom the different body areas was acquired for minand analysed with a CCD camera Xenogen IVIS Lumina System Perkin Elmer A specific region ofinterest ROI corresponding to the abdominal areaoccupied by the pancreas was manually selected andthe total photon flux ps from this ROI was evaluated with Living Image software Caliper Life Sciences Perkin Elmer [ ]At the end of the study mice were anaesthetizedwith ketamine mg·kgˆ’ Imalgene ip and xylazine mg·kgˆ’ Rompum ip and killed by cervical dislocation According to the Ethics Committeerecommendations to avoid suffering three Diab andone DiabFL mice presenting with both positive BLIand a palpable abdominal mass or poor general condition were killed and weeks respectively beforethe end of the study The lungs and the middle partof the gastrointestinal tractincluding the pancreasand the liver were dissected and exposed to theCCD camera for min for photon emission assessment The pancreas was dissected photographed andthen one part was stored at ˆ’ °C forweightedmolecular analysis whereas the other part was processed for histologicalimmunohistochemical analysis[] At time of collection a technician CC seeAcknowledgementstoallow blinded analysisrecoded biologicalsamplesMetabolic parametersBody weight and blood glucose were monitored weeklyAt the end of the study the levels of haemoglobin HbA1c an indicator of longterm glycaemic control wereassessed by using the Mouse HbA1c Assay Kit Crystal Chem Zaandam Netherlands and serum AGEsand total protein carbonyls PCOs two carbonyl stressmarkers were measured by ELISA OxiSelect„¢ Advanced Glycation EndProduct Competitive ELISA Kitno STA817 and OxiSelect„¢ Protein Carbonyl ELISAKit no STA310 respectively Cell Biolabs Inc SanDiego CA USAPancreas histologySix 4μmthick nonserial pancreatic sections stainedwith haematoxylin and eosin were examined to confirm the presence of invasive PaC Pancreas withoutinvasive PaC were analysed to grade dysplastic ductsie PanINs according to previously established criteria [] The numbers oflowgrade PanIN1ABand highgrade PanIN23 dysplastic ducts werecounted and expressed as a percentage of total ductsin the specimen []Pancreatic AGEsERK phosphrylation status nuclear YAP and itstarget gene connective tissue growth factor CTGFLevels of AGEs pERK and CTGF protein in homogenates and of active nonphosphorylated YAP1 innuclear extracts of pancreas of mice were assessed byWestern blot Human PDA tissues n were obtained from the Pathology Unit of Sant™Andrea HospitalRome Italyin agreement with the ethical guidelinesestablished by the locally appointed Ethics CommitteePancreatic tissue distribution of AGEs and activatedYAP1 in mouse and human specimens were evaluatedby dual label immunofluorescence and immunoperoxidase respectively [ ] For immunofluorescence agoat polyclonalrabbitantiAGE antibodyand a 0cMenini Journal of Experimental Clinical Cancer Research Page of sections weremonoclonal antibody to active nonphosphorylatedYAP1 were used as primary antibodies followed by appropriate secondary fluorescent antibodies see Supplementary Table S1 for antibodies in Additional file Sections were analysed at a fluorescence microscopeZeiss AXIO A1 equipped with an Axiocam colorcamera Carl Zeiss Italy Milan Italy For immunoperoxidase formalinfixed paraffin embedded sections μm thick were rehydrated and treated with H2O2in PBS for min to block endogenous peroxidase activity Heat mediated antigen retrieval was performed withœAntigen Unmasking Solution Citric Acid Based H Vector Laboratories Burlingame CA USA forAGE staining or TrisEDTA buffer pH for YAPstaining both for min Nonspecific binding wasblocked by incubation in Protein block serum free AgilentDako Santa Clara CA USA for min at roomtemperature Thenincubated withAvidinBiotin blocking Kit SP2002 Vector Laboratories for min an antiAGE antibody Abcam Cambridge UK ab23722 or an antibody directed to theactive nonphosphorylated YAP1 Abcam ab205270at °C overnight and the appropriate biotinylated secondary antibody at room temperature for min seeSupplementary Table S1 for antibodies in Additional file Finally sections were stained with UltraTek Horseradish Peroxidase ABL015 ScyTek Laboratories UTUSAfor min followed by 33diaminobenzidineDABH2O2 ChromogenSubstrate Kit High ContrastACV500 ScyTek Laboratories until the reaction product was visualized min and counterstained withhematoxylin AGE positive staining and nuclear expression of YAP were measured in random fields of eachsection at a final magnification of 250X and 400X respectively by means of the interactive image analyzerImagePro Premier ImmaginiComputer MilanItaly AGE positivity was expressed as the mean percentage of field™s area occupied by the specific stain Expression status of active YAP in tumor specimens wasassessed by counting the number of nuclei positive forYAP and expressed as the mean ratio of YAPpositive nuclei to total nucleiand AGEstheIn vitro studyThe in vitro study investigated the putative role ofRCStumourpromoting effect of high glucose HG and the protective effect of the carbonylsequestering agent andAGE inhibitor FL92616as mediators ofDesignHuman MIA PaCa2 Catalogue No Lot No14A02 and Panc1 Catalogue No Lot No10G011 cells SigmaAldrich were used for assessing theeffects of HG and FL92616 on cell proliferation Experiments aimed at investigating the molecular mechanismsunderlying the glucosemediated effects and the protection by FL92616 were conducted on MIA PaCa2 cellsMycoplasma contamination in cell cultures was regularlytested by PCR MycoSPY Kit Biontex Laboratories GmbHMunchen Germany Human PDA cells were maintainedin DMEM supplemented with FBS and incubated indifferent conditions for three daysie normoglycaemia normal glucose mM hyperglycaemia HG mM treated with MGO or GO μM SigmaAldrich two RCS and AGE precursors or the preformed AGE Nεcarboxymethyllysine CML μgmLprepared as previously reported [ ] with or withoutFL92616 mM and exposed to DMEM lowglucose medium containing of pooled sera from nondiabetic or diabetic individuals before and after AGE removalfrom diabetic serum by an immunoadsorptionmethod see below with or without FL92616 mMInformed consent was obtained from nondiabetic anddiabetic individuals Moreover both YAP and EpidermalGrowth Factor Receptor EGFR were silenced to assessthe role of YAP and EGFR pathway in RCS and AGEinduced cell proliferation see belowRemoval of AGEs from diabetic serumAGEs were removed from diabetic serum using animmunoadsorption method To immunoprecipitateAGEmodified proteins μl of diabetic serum wasincubated for h with μl of Pierce NHSactivatedmagnetic beads Thermofisher Scientific covalentlyconjugated with μg of antiAGE antibody Abcamsee Supplementary Table S1 for antibodies in Additional file according to the manufacturer instruction To confirm the efficiency of AGE depletionAGE concentration in both treated unbound serumfraction and untreated diabetic serum was evaluatedin triplicate by ELISA OxiSelect„¢ Advanced GlycationEndProduct Competitive ELISA Kit no STA817Cell Biolabs Inc San Diego CA USA Followingthis procedure the concentration of AGEs in diabeticserum was reduced by about reaching a concentration similar to that of the nondiabetic serum seethe œResults sectionYAP and EGFR silencingYAP and EGFR were silenced using smallinterferingRNAs siRNAs and irrelevant scrambled siRNAs as control Thermo Fisher Scientific Waltham MA USAValidated predesigned siRNA oligonucleotides and related TaqMan assays are detailed in SupplementaryTable S2 see Additional file Lipofectamine RNAiMAX Thermo Fisher Scientific transfections were performed using nM of each siRNA 0cMenini Journal of Experimental Clinical Cancer Research Page of Cell survival and proliferationCell viability and proliferation were evaluated by Cytoselect WST1 Cell Proliferation Assay Cell Biolabs following the manufacturer instructionsYAP1 its upstream regulators large tumour suppressor Kinase 1LATS1 and EGFRERK pathway and itsmolecular targets CTGF WTN5A and EMP2 in inhuman PDA cells Cells were extracted in SDS buffer containing protease and phosphatase inhibitorsSigma Aldrich Nuclear protein extracts were obtainedfrom cell monolayers with the Nuclear Extract Kit Active Motif Corp Carlsbad CA USA Protein concentrations were determined using the Bradford Assay KitBioRad Hercules CA USA Nuclear protein levels ofYAP1 and cellular protein levels of total and EGFRphosphorylated at Tyr1068 pEGFR total and pERK and LATS1 a key kinase of the Hippo pathway []were assessed by Western blotting see SupplementaryTable S1 for antibodies in Additional file KRAS activity was evaluated by the KRAS activation Assay Kit noSTA400K Cell Biolabs Inc according to the manufacturer™s protocol Briefly mg of lysate was subjected topulldown and μg of lysate was used to measure totalKRAS Pulldown and totallysates were subjected toWestern blotting procedure using the primary antibodyagainst KRAS provided by the kit The mRNA levels ofCTGFCCN2 WTN5A and EMP2 three recognized molecular targets of YAP [ ] were assessed by realtime PCR RTPCR using a StepOne RealTime PCRSystem and TaqMan Gene Expression assays ThermoFisher Scientific [] listed in Supplementary Table S3see Additional file Statistical analysisResults are expressed as mean ± SD mean ± SEM orpercentage Differences between cell typestreatmentsor animal groups were assessed by oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisons or twoway ANOVA followed bythe Bonferroni posttest as appropriate Betweengroup differencesin PaC incidence were assessedusing the Chisquared test and Fisher™s exact test tocompute a Pvalue from a contingency table A Pvalue of was considered to be significant Allstatisticalincluding linear regression analysiswere performed on raw data using GraphPad Prismversion for Windows GraphPad Software SanDiego CA USAtestsResultsIn vivo studyMetabolic parametersSTZtreated KCM mice developed hyperglycaemiastartingandabout h postinjection Fig 2ashowed a slight decline in the growth curve vs Ctrmice which reached statistical significance only at and weeks of age Fig 2b Despite no differencein body weight Fig 2c blood glucose Fig 2d andHbA1c levels Fig 2e FL92616 treatment preventedthe diabetesassociated increase in circulating AGEsFig 2f and total PCOs Fig 2g as assessed at theend of the studyInvasive PaC development Representative BLI imagesat the end of the study period and total photon fluxinduction from pancreas at and weeks of ageare shown in Fig 3a At sacrifice pancreas weightwas significantly P increased in Diab ± g vs Ctr ± g and vs DiabFL ± g KCM mice Pancreasbody weight percent ratiowas almost tripled in Diab vs Ctr mice whereas nostatistical difference was observed between DiabFLand Ctr mice Fig 3b and Table As assessed byhistology Fig 3c cumulative incidence ofinvasivePaC at weeks of age was in Diab mice vs in DiabFL and in Ctr mice Fig 3d and Table Representative BLI images and pancreas histologyfrom Ctr Diab and DiabFL are shown in Fig 3cdNeither the Ctr FL nor the STZnonDiab groupshowed significant differences in the incidence invasive PaC and pancreasbody weight percent ratio vsthe Ctr group Table Furthermore no betweengroup differences were observed in tumour invasiveness except for an apparent reduction in DiabFL vsDiab group Table However the few cases of PaCin DiabFL n and Ctr n mice prevent toperform statistical comparisons among groupsformetastatic disease Representative ex vivo BLI andhistology images of liver and lung metastases are presented in Supplementary Fig S1 in Additional file Grading of dysplastic ducts in mice free of invasivePaC Table showed significant differences betweenDiabFL and Diab mice for the percentage of normalducts which was higher and of highgrade PanINswhich was lowerin the FL92616 treated arm Inaddition Ctr FL mice presented with higher normalducts and lowerlowgrade PanINs vs Ctr micewhereas no difference was observed between STZnonDiab and Ctr micePancreatic AGEs ERK phosphrylation status nuclear YAP and connectivegrowth factorCTGF Pancreatic accumulation of AGEs Fig 4aand levels pERK Fig 4b CTGF Fig 4c awellestablished transcriptional target of YAP [ ] and nuclear YAP1 Fig 4d were increased inDiab vs Ctr mice and increments were prevented bytissue 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig Glucose and HbA1c levels body weight and hyperglycaemiaassociated carbonyl stress Blood glucose levels and body weight during thestudy period a and b and at the end of the study period weeks of age1 c and d and HbA1c levels e and serum levels of AGEs f andtotal PCOs g at the end of the study period weeks of age1 in control Ctr Ctr treated with FL92616 Ctr FL diabetic Diab and Diabtreated with FL92616 DiabFL KCM mice Statistical significance between groups for time course of blood glucose a and body weight c wascalculated using twoway ANOVA followed by the Bonferroni posttest Each time point represents mean ± SD of animals until the 17th weekof age and “ animals from the 18th to the 22nd week of age Statistical significance for blood glucose c body weight d serum levels ofAGEs e and PCOs f at weeks of age1 was assessed using oneway ANOVA followed by the StudentNewmanKeuls test for multiplecomparisons Each dot represents one case and bars represent mean ± SEM P or P vs Ctr    P vs Diab 1Except for threeDiab and one Diab FL mice which were killed and weeks respectively before the end of the study see œResults section for further detailslabelFL92616 treatment Dualimmunofluorescenceanalysis confirmed the association between AGEs andnuclear YAP1 in PaC lesions from Diab mice Fig4e A significant positive relationship between AGEaccumulation and nuclear YAP1 levels was also observed in human PDA Fig 4fgIn vitro studyProliferation of human PDA cellsHG concentration mimicking diabetic hyperglycaemiapromoted PDA cell growth and this effect was prevented by FL92616 Fig 5ab The AGE precursors RCS MGO and GO and the preformed AGE 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig In vivo BLI and gross and microscopic examination of pancreas Representative BLI at the end of the study period and total photon fluxps induction from pancreas at and weeks of age1 a pancreasbody weight percent ratio b representative pancreas histology coriginal magnification 100X scale bar μm and cumulative incidence of PaC d in control Ctr diabetic Diab and Diab treated with FL DiabFL KCM mice at the time of sacrifice Statistical significance between groups for pancreasbody weight percent ratio a wascalculated using oneway ANOVA followed by the StudentNewmanKeuls test for multiple comparisons Each dot represents one case and barsrepresent mean ± SEM Statistical significance for PaC incidence b was assessed using the Chisquared test and Fisher™s exact test P vsCtr  P vs Diab Is islet invasive PaC arrows PanINs 1Except for three Diab and one Diab FL mice which were killed and weeksrespectively before the end of the study see œResults section for further detailsCML also stimulated PDA cell proliferation FL926 was able to inhibit cell proliferation induced byMGO and GO but not CML Fig 5c Treatmentwith CML but not with MGO induced ERK activation and FL92616 was ineffective in counteractingphosphorylation status Fig 5d However the proliferating effect of both the RCS MGO and the AGECML was associated with YAP1 nuclear persistenceand activity Again FL92616 efficiently preventedeffectCMLtheofonERKthe nuclear translocation of YAP1 induced by MGObut failed to counteract the effect of CML Fig 5eConsistently FL92616 treatment reversed the MGOinduced upregulation of gene expression of CTGFWnt Family Member 5A WNT5A and EpithelialMembrane Protein EMP2three wellrecognizedYAP target genes [ ] Conversely FL92616was ineffective in preventing the modulatory effect ofCML on the mRNA level of these genes Supplementary Fig S2 in Additional file 0cMenini Journal of Experimental Clinical Cancer Research Page of CtrDiab1Diab FL1Ctr FL ± ±     ± Table Pancreatic cancer PaC incidence PancreasBodyweight Wt percent ratio and metastasisPaC NtotPancreasBody Wt Metastasis Ntot PaC ± STZnonDiab ± Cumulative incidence of PaC and PancreasBody weight Wt percent ratio incontrol Ctr diabetic Diab Diab treated with FL92616 DiabFL Ctr treatedwith FL92616 Ctr FL and streptozotocintreated nondiabetic STZnonDiab KCM mice at the end of the study weeks of diabetes weeks ofage1 The number of KCM mice with metastasis liver and or lung on thetotal number of PaC cases is also shown KCM LSLKrasG12D Pdx1Cre MITONtot number of casestotal number of mice Ntot PaC number of casestotal number of PaC PaC ductal adenocarcinoma and hepatic andor lungmetastasis were confirmed by histology P or P vs Ctr   P or  P vs Diab Statistical significance between groups forPancreasBody Weight percent ratio was calculated using oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisonsStatistical significance for PaC rate was assessed using the Chisquared testand Fisher™s exact test Except for three Diab and one Diab FL mice which were killed and weeks respectively before the end of the studyMechanisms underlying RCS and AGEinduced YAPactivationSilencing of YAP1 using two independent siRNAssiYAP1 and Fig 6a significantly inhibitedthe transcription activity of YAP target genes induced by both MGO and CML in PDA cells Fig6b In MGOtreated cells YAP induction was associated with a decrease in protein levels of LATS1 awellestablished negative regulator of YAP activity[] whereas CML treatmentfailed to modulateLATS1 Fig 6c Instead treatment with CML butnot with MGO was found to induce EGFR phosphorylation pEGFR Fig 6d EGFR silencing Fig6e almost completely reversed YAP1 nuclear translocation Fig 6f KRAS activation and ERK phosphorylation Supplementary Fig S3ABinduced by CMLEffects of serum from diabetic patients on proliferation ofhuman PDA cellsThe levels of AGEs were ± μgmL in thepooled sera from diabetic patients and ± μgmLin pooled sera from nondiabetic individuals The diabetic serum induced a 3fold increase in PDA cell proliferation compared to the nondiabetic serum This effectwas greatly reduced by prior selective AGE removalfrom the diabetic serum AGE levels ± μgmLand almost completely reversed by combining AGE removal from serum and FL92616 treatment of PDAcells Fig DiscussionDespite the epidemiological evidence of increased PaCrisk in both type [] and [ ] diabetestheunderlying mechanisms still remains to be elucidatedHere we showed that STZinduced type diabeteswhich is characterized by marked hyperglycaemia andinsulin ia without weight gain [] significantlyaccelerated tumour progression in a mouse model ofKras“driven PaC The absence of obesity and insulinresistance argues in favour of the hypothesis that thePaCpromoting effect of diabetes is directly related tothe adverse effects of hyperglycaemiaIn additionRCS trapping and AGE inhibition by FL92616 efficiently prevented the acceleration of PanIN progression to invasive PaC induced by diabetes Thedifference in the incidence of PaC between the twodiabetic groups ie untreated and treated with FL occurred despite similar increases of bloodglucose levels supporting the conceptthat glucosemetabolites but not glucose per se were responsiblefor PaC promotion STZtreated mice that failed todevelop or reversed hyperglycaemia showed the samePaC incidence as the Ctr group thus ruling out aneffect of STZ on invasive PaC development in DiabmiceOur finding of an association between AGE accumulation and YAP induction in PaC in

Thyroid_Cancer

progression of breast cancerare greatly affected by the immune environment Neutrophils are the most abundantleucocytes in circulation and act as the spearhead in ‚ammationincluding inbreast cancer Circulating neutrophils are closely related to the prognosis of breastcancer patients and tumorltrating neutrophils have varied functions at differentstages of breast cancer such as antitumor or tumorpromoting neutrophils which aretermed N1 and N2 neutrophils respectively In this review we will discuss the utilityof circulating neutrophils for predicting prognosis and therapeutic efficacy and theunderlying mechanisms of their chemotaxis the dynamic regulation of their antitumoror protumor functions and their different spatial distributions in tumor microenvironmentFinally we also discuss the possibility of targeting neutrophils as a therapeutic strategyin breast cancerKeywords breast cancer immunotherapy neutrophils neutrophiltolymphocyte ratio tumor microenvironmentSpecialty sectionThis was submitted toCancer Immunity and ImmunotherapyINTRODUCTIONa section of the journalFrontiers in ImmunologyReceived May Accepted July Published August CitationZhang W Shen Y Huang H Pan SJiang J Chen W Zhang T Zhang Cand Ni C A Rosetta Stone forBreast Cancer Prognostic Value andDynamic Regulation of Neutrophil inTumor MicroenvironmentFront Immunol 103389fimmu202001779Breast cancer BC is the most common malignancy in women worldwide Although BCis classified as a malignant disease with low immunogenicity recent evidence has revealeda promising outcome of therapies with blocking immune checkpoints in both early andadvanced stages “ The efficacy of immunotherapy is closely related to the tumor immunemicroenvironment especially to ltrating immune cells To date macrophages and Tcells are the most wellstudied immune cells in BC whereas increasing evidence has indicatedthat neutrophils are also key in the oncogenesis and metastasis of BC in addition circulatingneutrophils have been reported to have great prognostic prediction value Neutrophils are themost abundant leucocytes in blood and usually act as the first line of host defense against pathogens However due to their short life span an average of “ h in blood it is difficult to employthis subset of cells for experiments which has resulted in a poor understanding of their role in solidtumors In addition some contradictory results reported in vitro studies or animal experimentshave suggested a dual eï¬ect of neutrophils in tumor developmentFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerNeutrophils can present both antitumorigenic œN1 andprotumorigenic œN2 phenotypesin various cancers orspecific circumstances The term neutrophil in several studiesalso includes both mature neutrophils and myeloidderivedsuppressor cells MDSCs MDSCs are described as a subsetof neutrophils with immunosuppressive functions that expressCD11b and Gr1 and can be divided into monocyticM CD11bLy6C MDSCs and GPMN CD11bLy6GMDSCs and GPMN MDSCs usually share a commonset of markers and similar morphologicalfeatures withneutrophils To avoid confusion we mainly focus on the biologicalfunction of mature neutrophils and related therapeutic strategiesfor targeting them in BC We provide a comprehensive reviewofthe prognostic value of circulating neutrophils and themechanisms of how tumorassociated neutrophils TANs exertantitumor or tumorpromoting functions in BC and in theend we also discuss the potential of targeting neutrophils as atherapeutic strategy in cancerPROGNOSTIC VALUE OF THENEUTROPHILTOLYMPHOCYTE RATIONLRTumors can be thought of as wounds that will not heal andare characterized by chronic ‚ammation Neutrophils are themost rapidly responding immune cells to ‚ammation andmany studies have found that the NLR is closely related to theprognosis and treatment response in patients bearing BC A recent metaanalysis of studies including patientswith both early and advanced BC revealed that patients witha higher NLR before treatment had poorer diseasefree survivalDFS than those with a lower NLR before treatment but theNLR was not related to overall survival OS the subgroupanalysis found that the NLR was associated with prognosisonly in earlystage patients but not in patients with metastasisAbbreviations BC Breast cancer MDSCs Myeloidderived suppressor cellsTANs Tumorassociated neutrophils NLR Neutrophiltolymphocyte ratio DFSDiseasefree survival OS Overall survival ALC Absolute lymphocyte count NCTNeoadjuvant chemotherapy pCR Pathological complete response PLR Platelettolymphocyte ratio TAMs Tumorassociated macrophages CTCs Circulatingtumor cells NETs Neutrophil extracellular traps MPO MyeloperoxidaseGCSF Granulocyte colonystimulating factor ECs Endothelial cells PMNsPolymorphonuclear neutrophils ICAM1 Intercellular adhesion molecule MMP9 Matrix metalloproteinases9 ROS Reactive oxygen species HMGB1Highmobility group box TLR4 Tolllike receptor TNBC Triplenegativebreast cancer MES Macrophageenriched subtype NES Neutrophilenrichedsubtype H2O2 Hydrogen peroxide TNFα Tumor necrosis factorα HOCIHypochlorous acid TRPM2Transientcation channelsubfamily M member ADCC Antibodydependent cellular cytotoxicityNK Natural killer NE Neutrophil elastase NRP1 Neuropilin1 IRS1 Insulinreceptorsubstrate1 PI3K Phosphatidylinositol 3kinase VEGF Vascularendothelial growth factor TIMP1 Tissue inhibitor of matrix metalloproteaseTGF Transforming growth factor 27HC 27hydroxycholesterol PAD4Peptidyl arginine deiminase TINs Tumorltrating neutrophils CRTConventionalradiotherapy MRT Microbeam radiation therapy DAMPsDamageassociated molecular patterns ICB Immune checkpoint blockadeLDNs Lowdensity neutrophils HDNs Highdensity neutrophils NAMPTNicotinamideadeninedinucleotide GTX granulocyte transfusiontransferase NAD Nicotinamidereceptor potentialphosphoribosyl Since similar metaanalyses were not based on individualpatient data which may cause significant bias we reviewed andcompared the individual reports and found some issues worthdiscussing here Widmann first reported the correlationbetween the NLR and BC prognosis in patients and itwas found that a higher NLR ‰¥ before treatment was anadverse factor for both short and longterm mortality Themajority of retrospective studies thereafter have drawn similars “ and the NLR was found to be consistentamong diï¬erent BC subtypes at baseline However aprospective substudy of GEICAM9906 which comprised patients did not find any prognostic value of the NLR afteradjustment for clinicopathological factors in addition a highNLR was independently associated with worse DFS in only highrisk patients the hormone receptornegativeHER2 populationand in patients with ‰¥ lymph node metastases Anotherstudy with early BC patients also found that the NLR beforesurgery was not associated with DFS indicating that thepresurgery NLR may be valuable only in patients with a hightumor burdenseveralIn addition to the above studiesstudies alsoexplored the prognostic value of the NLR posttreatment or withcontinuous assessment A retrospective study comparing theabsolute lymphocyte count ALC and the NLR eight consecutivetimes before and after chemotherapy found that patients whodied had lower ALC and higher NLR values than patients whoremained alive throughout the treatment course additionallyamong the patients who died a steady increase in the NLRover the baseline measurement was observed at subsequenttime points Another retrospective study included BCpatients with DFS values of more than years and it interestinglyfound that NLR sampled during followup rather than beforeany treatment was an independent prognostic factor for laterecurrence However there is still no compelling explanationfor the abovementioned inconsistent results In addition sincelymphocytes are critical in cancer immune surveillance andneutrophils have been reported to play a protumor role in moststudies low lymphocytes and high neutrophils in circulationmay also suggest immunosuppression status and studiesfocused on the relationship between neoadjuvant chemotherapyNCT and the NLR might support the above hypothesis Acomprehensive review of the existing reports shows that moststudies have found that a low NLR indicates a higher NCTresponse and pathological complete response pCR rate “in addition the NLR has showed predictive value not only inall molecular types of BC but also in both operable and locallyadvanced BC Interestingly although Suppan et aldid not find a significant correlation between the initial NLR andprognosis the same cohort revealed a low NLR as a significantparameter for predicting chemotherapy response p A low NLR was also reported to be associated with a higherresponse rate to primary endocrine therapy for locally advancedor metastatic BC Although increasing evidence suggests a close associationbetween the NLR and prognosis in BC several issues remain thatmake clinical application difficult One of the most importantreasons is the lack of a consensus cutoï¬ value As we listhere Table the cutoï¬ values for the NLR in the publishedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerTABLE Characteristics of the studies related to neutrophiltolymphocyte ratioReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSignificance of NLRNR ysHigh NLR indicates lowersurvival rate p Surgery NCT moSurgery ysNR moUnivariate analysis indicateshigh NLR related to lower RFSp and OS p High NLR indicates lower5year OSMultivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Adjuvanttranstuzumab moHigh pretreatment NLRindicates shorter DFSNoh Korea“Koh Korea“Yao China“Luminal ABHER2enrichedTNBCERPRpositiveHER2enrichedLuminal ABERPRpositiveHER2enrichedTNBCPistelli Italy“TNBCUlas Turkey“HER2enrichedJia China“ER PRpositiveHER2enrichedTNBCBozkurt Turkey“TNBCAsano Japan“TNBCn NLR n ‰¤ NLR ‰¥ n n NLR ‰¤ n NLR n n NLR NLR n NLR ‰¤ n NLR n n NLR n NLR n n NLR n NLR ‰¤ n n NLR ‰¤ n NLR n n NLR n NLR n NCT surgery mo moSurgeryadjuvantchemotherapyandradiotherapyNCT ysMultivariate analysis indicateslow NLR is related to superiorDFS p and p Multivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Univariate analysis indicateslow NLR is related to favorableprognosis in TNBC patientswho achieved pCR p hazard ratio High pretreatment NLRindicates poor allcausemortality with a multivariableHR of CI“High NLR upon recurrenceindicates shorter OSrecurrence rates p Low NLR indicates higher OSp Rimando USA“Nonmetastatic BC n NLR ‰¤ n NLR n Radiotherapychemotherapy moIwase Japan“TNBCn NRL n NLR ChemotherapyNRNCT surgery moHernandez et alSpain“Miyagawa Japan“Ferroni Italy“Luminal ABERPRpositiveHER2enrichedTNBCLocally Advanced orMetastatic BCLuminal ABHER2enrichedTNBCn NLR n NLR ‰¥ n n NLR ‰¤ n NLR n Qiu China“NonmetastaticTNBCn NLR n NLR ‰¥ n EribulinNRLow NLR indicates better PFSp NCTchemotherapyendocrinetherapytrastuzumabregimensSurgery NCTchemotherapy moHigh pretreatment NLRindicates worse DFS HR and OS HR moLow NLR indicates higher OSp and DFS p ContinuedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alTABLE ContinuedNeutrophils in Breast CancerReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSignificance of NLRIimori Japan“Luminal ABHER2enrichedTNBCMando Argentina “Early stage BCLee Korea“TNBCXuan China“TNBCFujmoto Japan“With high counts oflymphocytesImamura Japan“HER2enrichedn NLR n NLR ‰¥ n n NRL n NLR ‰¤ n NLR n n NLR n NLR ‰¥ 293n n NLR n NLR n n NLR n NLR ‰¥ n Endocrinetherapy moSurgery moNCTNRSurgeryNRNRSurgeryadjuvantchemotherapiesendocrinetherapiesTrastuzumabemtansineNRLow NLR indicates aprolongation of PFS p and OS p High NLR indicates lower DFSp Low NLR indicates superiorOS p and DFS p Low NLR indicates longer DFSp Low NLR indicates better RFSp Low NLR at baseline indicatesbetter PFS p andOS p NLR Neutrophiltolymphocyte ratio ER Estrogen receptor PR Progesterone receptor HER2 Human epidermal growth factor receptor Mo Months Ys Years DFS Diseasefreesurvival OS Overall survival PFS Progressionfree survival RFS Relapse free survival pCR Pathological complete response TNBC Triplenegative breast cancer NCT Neoadjuvantchemotherapy NR Not recordedstudies were between and In addition based on individualstudies the sensitivity of the NLR fluctuates greatly “and the specificity is much lower “ Therefore some researchers have tried to determine a betteralternative parameter In addition to the NLR the platelettolymphocyte PLR ratio has also been investigated and comparedwith the NLR in BC A single central retrospective study with hormone receptornegative nonmetastatic BC patients reportedthat both elevated NLR and PLR were associated with poor OShowever the multivariate analysis revealed that only the NLR p but not the PLR p was a significant indicatorfor both DFS and OS Additionally since the absolutelymphocyte count has also been reported as a prognostic factorthe predictive values of the PLR and NLR were evaluated afteradjusting for the total lymphocyte count The results showed thatthe PLR was no longer a significant predictor for 5year mortalityand the NLR remained a significant predictor irrespective ofthe lymphocyte count Furthermore it was revealed thatthe combination of the NLR and PLR could further improvethe predictive value Two retrospective studies found that thehighest rate of pCR was in the group of patients withan NLRlowPLRlow profile and the lowest rate was inthe group with an NLRhighPLRhigh profile in additionwhen the cutoï¬ values for the NLR and PLR were applied thespecificity of predicting a pCR increased from to Howeverthe causal relationship between the NLR andpoor prognosis in malignant disease has yet to be illuminatedAccording to an assessment with paired peripheral bloodand pancreatic cancer specimens Takakura found thata high NLR was associated with increased tumorassociatedTAMsanditseemsdecreased Thereforetumorassociatedmacrophageslymphocytes but was not significantly related to CD66bltrating neutrophilsthat anincrease in neutrophils in peripheral blood is not necessarilyrelated to the number of TANs Several basic studies havesuggested a unique mechanism ofthe protumor functionof circulating neutrophils protecting circulating tumor cellsCTCs Circulating neutrophils can cluster around tumor cellsand induce tumor cell aggregation aiding tumor cell survivalby hiding them from immune surveillance Neutrophilextracellular traps NETs are webs of decondensed chromatbers conjugated together with histones myeloperoxidaseMPO elastase and other cytoplasmic proteins Recentstudies also found that neutrophils could form many NETs bothin circulation and in tumor lesions and could coordinate withplatelets to capture CTCs and facilitate cancer metastasis In addition neutropenia is very common in cancer patientsundergoing chemotherapy and supportive treatment withgranulocyte colonystimulating factor GCSF can inducea neutrophilic response as a consequence neutrophils areprimed toward a proNETotic phenotype and may suppress thecytotoxic activity of T cells as well as impair immune surveillance On the other hand lymphocytes have the propensityto mount an adaptive antitumor response in malignant disease and decreased lymphocyte numbers are considered to berelated to an insufficient immunologic reaction which mayincrease the risk of tumor relapse or metastasis Clearlya general association between prognosis and the NLR exists inBC but large prospective studies and rigorous research are stillrequired to determine its clinical significanceFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerMECHANISM OF NEUTROPHILCHEMOTAXIS TO THE TUMORMICROENVIRONMENTNeutrophils are considered the main immune cells that provideprotection against invading pathogens which can be induced bytrauma infection and malignant disease The recruitmentof neutrophils is greatly dependent on certain chemokinesincluding interleukin IL8 also known as CXCL8 CXCL and CXCL2 IL8 is a pro‚ammatory cytokineand acknowledged as the most important chemoattractant forneutrophils in the tumor microenvironment IL8 mainlycomes from endothelial cells ECs and monocytes in the tumormicroenvironment upon certain stimulation such as physicalinjury hypoxia chemotherapy or radiotherapy and other celltypes including fibroblasts and keratinocytes can secrete IL8 aswell In addition to its chemotactic eï¬ect it was revealedthat IL8 could provoke neutrophils to release NETs to assistcancer cell migration By livecell fluorescence microscopyGupta confirmed that activated ECs could induceNETosis characterized by typical extracellular DNA latticeswhen cocultured with polymorphonuclear neutrophils PMNsand activated ECs In addition activated ECs produceother ‚ammatory cytokines such as Pselectin Eselectinand intercellular adhesion molecule ICAM1 to facilitateneutrophil adhesion to ECs and migration Furthermoretumorpromoting neutrophils in BC cells are also characterizedby high expression of matrix metalloproteinases9 MMP9 which was found to cleave CXCL5 potentiating itsaction in neutrophil recruitment as a positive feedback functionin tumors IL17 was also found to control neutrophilrecruitmentin lung metastasis of BC in a mouse modelCD3CD4 and γδ T cells were the major sources of IL17 and it was interesting to find that the absence of γδT cells or neutrophils markedly reduced pulmonary and lymphnode metastases without ‚uencing primary tumor progressionwhich suggested a collaborative relationship between γδ T cellsand neutrophils in promoting BC lung metastasis Howeverin an orthotopic hepatocellular carcinoma model Sofia et alreported that TANs exert an overt antitumor role by suppressingγδ T17 cells via reactive oxygen species ROS contraryto the phenomenon that within the 4T1derived BC modelCD11bLy6G neutrophils that ltrate and surround livermetastases were found to be tumor promoting Thesecontroversial results suggest both promoting and suppressiveroles of TANs in diï¬erent circumstancesHighmobility group box HMGB1 usually acts as adamageassociated molecular pattern that is released by dyingcells or stressed cells to initiate ‚ammation and was laterfound to be an important chemoattractant for neutrophils Epithelial cellderived HMGB1 was found to recruit neutrophilsto the necrotic site through its receptor RAGE Enrichmentof platelets has been reported in the microenvironment ofmultiple cancers including BC and ltrating plateletscould be activated by the large amounts of adenosine phosphatereleased by necrotic cells as a result of chemotherapy Activated plateletderived HMGB1 known as the major mediatorof injuryinduced thrombosis in vivo can also stimulateNETosis through Tolllike receptor TLR4 and RAGE onneutrophils and as a positive feedback mechanism releasedNETs strongly induce a prothrombotic state and activate platelets Meanwhile tumor cellderived exosomal HMGB1 wasalso found to activate neutrophils through the TLR4NFκB pathway which promotes its survival by increasing theautophagic response and polarizing TANs to a protumor type It is noteworthy that various reports imply the coreposition of the NFκB pathway in the activation and recruitmentof neutrophils In addition to HMGB1 tumor cellsincluding BC cells have been reported to secrete other peptidessuch as a2 isoform VATPase a2V to activate the NFκBpathway in neutrophils thereby promoting their recruitment andinhibiting their apoptosis Additionally breast involutionafter weaning is characterized by acute ‚ammation and anincrease in estrogen It was found that estrogen could inducethe mammary ltration of neutrophils and upregulate theexpression of protumor cytokineschemokines such as COX2and MMPs in mammary ltrating neutrophils similarIn additionto lymphocytes and macrophagesneutrophils are more likely to localize in tumors of triplenegativebreast cancer TNBC than to tumors of other BC subtypes Recently Zhang identified neutrophils and macrophages asthe most frequent ltrating immune cells in various BC murinemodels and BC could be classified into a macrophageenrichedsubtype MES and a neutrophilenriched subtype NES It wasinteresting to find that there were only a few neutrophils inthe MES but a large number of macrophages in the NES This mutual repelling phenomenon in the MES and NES mayresult in spatial segregation within the same tumor The authorsspeculated that a possible mechanism could be the factors derivedfrom macrophages that inhibit the IL8dependent chemotaxis ofneutrophils ANTITUMOR FUNCTION OF TANs IN BCThe polarization of neutrophils can be diï¬erentially regulatedin the tumor microenvironment In a mouse model Fridlender found that TANs from the early tumor stage were liketumorkilling cells which produce high levels of hydrogenperoxide H2O2 tumor necrosis factor TNFα and NOand that TANs are more likely to obtain a protumorigenicphenotype with tumor progression Although few studieshave directly compared the phenotype and function of TANsbetween early and latestage tumors there are still some clues tosupport this hypothesis A phenotypical and functional analysisof TANs in earlystage lung cancer found an activated phenotypeCD62lowCD54high that was able to stimulate T cell proliferationand IFNγ release which suggested a pro‚ammatory ratherthan immunosuppressive state of TANs in earlystage lungcancer MPO is an enzyme characteristic of mature œN1type neutrophils which are able to convert H2O2 to cytotoxichypochlorous acid HOCI Recently a retrospectivestudy of BC cases revealed that MPOpositive neutrophilsFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerin additiondefined as ‰¥ cellstissue punch were found in of evaluablecases while the luminal ERPR and Her2 Her2enriched andtriplenegative types had positive rates of and respectivelyltrationby MPOpositive neutrophils was a significant independentfavorable indicator for both OS and DFS Notably almost all ofthe patients included in this study had earlystage disease T1 N01 and the data suggested that MPOpositiveneutrophils were much more abundant in BC cases with low Tand N stages than in advanced cases in univariate analysesIn addition a direct tumor killing function of neutrophils hasalso been reported One of the classical factors working againsttumor cells is ROS Recent research in mouse BC models revealedthat ROSmediated cell lysis was dependent on Ca2 channelsand mediated by transient receptor potential cation channelsubfamily M member TRPM2 expression on tumor cells Although TCGA analysis revealed a high expression ofTRPM2 in BC cells httpgepia2cancerpkucnindex activeNOX1 catalase and SOD were also increased in the membraneof cancer cells forming a complex mechanism by which tumorcell apoptosis induced by ROS is prevented In additiontumor cells are characterized by enhanced metabolic activity andhigh levels of intracellular ROS which indicates that directcytotoxic eï¬ects of neutrophilproduced ROS are not sufficientIn addition to the direct cytotoxic eï¬ect TANs containing ROShave been found to strongly suppress IL17producing γδ Tcells which are critical for shaping the immune suppressivemicroenvironment in various solid tumors “ and have alsobeen reported to promote BC cell extravasation and metastasis In addition neutrophils could also express Fc receptors andexert antibodydependent cellular cytotoxicity ADCC eï¬ectssimilar to those of T cells and macrophagesleading to atrogocytosis eï¬ect to destroy cancer cells However somestudies have indicated that neutrophils are more likely to bedistributed at the periphery of tumors at the initiation stage which may make controlling tumor growth with thesecellcell contactdependent mechanisms ineï¬ectivePROTUMOR EFFECTS OF TANsMore studies suggest that neutrophils facilitate tumor promotionand metastasis in BC than antitumor eï¬ect Overexpression ofthe chemokines CCL2 and CCL17 is a recognized feature ofN2 neutrophils Richmond found that exogenousCCL2 enhances the killing eï¬ect of neutrophils against BCcells in vitro while this antitumor activity was not observedin vivo Instead intranasal delivery of CCL2 to BALBc micemarkedly enhanced lung metastasis of BC cells and increasedthe recruitment of CD4 T cells and CD8 central memory Tcells CCL17 secretion from TANs was found to support tumrowth by recruiting CD4 Treg cells and macrophages In addition to recruiting immunesuppressive cells TANs werereported to promote the accumulation of BC cells in the lungand directly inhibit natural killer NK cellmediated clearanceof tumor cells Human NK cells can be divided intoCD56dim antitumor and CD56bright protumor subsets andCD56bright NK cells are enriched in the tumor microenvironmentand draining lymph nodes Early reports revealed thatROS and arginase1 from neutrophils impair the maturation andcytotoxic function of NK cells but CD56brightCD16ˆ’ NKcell are resistant to neutrophilderived ROS perhaps due to theirhigh antioxidative capacity Meanwhile NK cells couldbe recruited by TANs via CCL2 and CCL5 which may explainthe preferential accumulation of CD56bright NK cells in tumormicroenvironments with high ROS levels Extracellular arginine is crucial to signal local CD8 cellsand increase their CD3ζ expression which is key for T cellsto survey antigens presented on MHC class I molecules andit was also found to be necessary for T cell activation andsurvival Tumor cellderived IL8 could lead to TANdegranulation resulting in arginase1 release and conversion ofextracellular arginine to ornithine and urea thereby dampeningthe survival and cytotoxic eï¬ect of CD8 T cells Neutrophil elastase NE is also released by TANs and canbe endocytosed by tumor cells via neuropilin1 NRP1 thisresults in the crosspresentation of PR1 which is an NEderivedHLAA2restricted peptide that may be an immunotherapeutictarget Besides upon endocytosis NE is to bind insulinreceptor substrate1 IRS1 which removes the inhibitory eï¬ectof IRS1 on phosphatidylinositol 3kinase PI3K to enhance theproliferation of cancer cells Recentleukocytesreports highlighted thethe important characteristics of malignantespeciallyneutrophils preferentially uptake tumor derived extracellularvesicles or named exosomes Hypercoagulability is oneoftumors andhas been reported associated with NETs Breast cancer cell4T1derived exosomes induced NETs formation in neutrophilsbesides tumorderived exosomes also interacted with NETsto significantly accelerate venous thrombosis in vivo Furthermore several reports also indicated the cancer derivedexosomes prolonged lifespan of neutrophils and also polarizedneutrophils toward protumor type increasingevidence hasIn addition to direct modulation ofthe protumormicroenvironmentfound thatneutrophils promote tumor cell migration and the formation ofa metastatic niche Tumor angiogenesis is regardedas a prerequisite for tumor metastasis and TANs have beenrecognized as an important source of vascular endothelialgrowth factor VEGF upon specific stimulation in the tumormicroenvironment Neutrophils were also found to beone of the main sources of MMP9 and the link betweenMMP9 and VEGF has been reported previously The absenceof MMP9 has been reported to have a similar function as theinhibition of VEGF signaling indicating that MMP9 serves asan angiogenic switch during tumorigenesis by inducing VEGFrelease from the matrix “ In addition Gabriele et alalso found that MMP9 was expressed by a small number ofcells in close proximity to the vasculature such as ltrating‚ammatory cells rather than tumor cells In additionseveral serine proteases are also produced by TANs such asNE cathepsin G and proteinase3 which have been reported toactivate MMP2 to promote tumor invasion and proliferation In addition although neutrophils were reportedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerto produce little tissue inhibitor of matrix metalloproteaseTIMP1 Wang observed that BC cells with CD90positiveexpression could induce the TIMP1 secretion by TANs and asa reciprocal eï¬ect TIMP1 induced EMT and metastasis in BC Other neutrophilderived cytokines such as IL1 IL6and IL17α have been reported to initiate EMT of cancer cells byactivating JAK2STAT3 and ERK signaling Inadditiontheprimaryto modulatingtumormicroenvironment neutrophils can also assist the formationof the cancer premetastatic niche in distant ans CTCsare precursors for metastatic lesion formationintravascularNETs were found to protect CTCs from attack by circulatingimmune cells and dysregulated NETs were found to induce‚ammatory vascular injury EC shrinkage and tissue damage “ Moreover in vitro and in vivo experiments foundthat activated neutrophils promote the adherence of CTCs toECs and facilitate their lung and liver metastasis RecentlyAceto provided strong evidence that neutrophils escortCTCs in BC to assist metastasis With detection of cellsurface markers and Wright Giemsa staining they identified thatmost CTCassociated white blood cells were N2like neutrophilsIn addition singlecell RNA sequencing revealed higher Ki67expression in disseminated tumor cells from CTC neutrophilclusters than in standalone CTCs In the same study TNFαoncostatin M IL1 and IL6 were frequently expressed byCTCassociated neutrophils and matched by the receptors oncorresponding CTCs on the other hand CTCs from the CTCneutrophil clusters expressed high gene levels encoding GCSFtransforming growth factor TGF3 and IL15 which havebeen reported to activate neutrophils “ illuminating amechanism of neutrophilCTC cluster formationIn addition to escorting CTCs in circulation several studieshave found that neutrophil accumulation is a prerequisitefor cancer metastasis For both orthotopic transplantationand spontaneous BC models neutrophils were suggestedto accumulate in the distant an before cancer cellsltration Obesity and elevated cholesterol arerisk factors for BC development and poor prognosis Interestingly 27hydroxycholesterol 27HC increased thenumber of polymorphonuclearneutrophils and γδ T cells atdistal metastatic sites and neutrophils were required for themetastatic eï¬ects of 27HC Egeblad developeda confocal intravital lung imaging system and found that NETswere formed early in the lung and continued to form forthe next few days after tail vein injection of BC cells Inaddition based on immunofluorescence staining of humanprimary BC and matched metastatic lung lesions they foundthat the abundance of NETs was highest in TNBC but NETswere absent or very rare in luminal BC samples which mayexplain the higher metastatic ability of TNBCs than luminalBCs In ovarian cancer an ‚ux of neutrophils in the omentumwas also observed before metastasis and blockade of NETformation with peptidyl arginine deiminase PAD4 an enzymethat is essentia

Thyroid_Cancer

pulmonary disease COPD is due to structural changes and narrowing of small airways and parenchymaldestruction loss of the alveolar attachment as a result of pulmonary emphysema which all lead to airflow limitation Extracorporeal shock waves ESW increase cell proliferation and diï¬erentiation of connective tissue fibroblasts To date no studiesare available on ESW treatment of human bronchial fibroblasts and epithelial cells from COPD and control subjects We obtainedprimary bronchial fibroblasts from bronchial biopsies of patients with mildmoderate COPD and control smokers with normallung function 16HBE cells were also studied Cells were treated with a piezoelectric shock wave generator at low energy mJmm2 pulses After treatment viability was evaluated and cells were recultured and followed up for and h Cellgrowth WST1 test was assessed and proliferation markers were analyzed by qRTPCR in cell lysates and by ELISA tests in cellsupernatants and cell lysates After ESW treatment we observed a significant increase of cell proliferation in all cell types CKitCD117 mRNA was significantly increased in 16HBE cells at h Protein levels were significantly increased for cKit CD117 at h in 16HBE p and at h in COPDfibroblasts p � for PCNA at h in 16HBE p � for y1 CD90 at and h in CSfibroblasts p � and p � for TGF1 at h in CSfibroblasts p � for procollagen1 at h inCOPDfibroblasts p � and for NFκBp65 at and h in 16HBE p � and p � In the peripheral lung tissueof a representative COPD patient alveolar type II epithelial cells TTF1 coexpressing cKit CD117 and PCNA were occasionally observed ese data show an increase of cell proliferation induced by a low dosage of extracorporeal shock waves in16HBE cells and primary bronchial fibroblasts of COPD and control smoking subjects Backgrounde progressive chronic airflow limitation in chronic obstructive pulmonary disease COPD is due to two majorpathological processes i remodeling and narrowing ofsmall airways and ii destruction of the lung parenchymawith loss of the alveolar attachments as a result of pulmonaryemphysema [] Chronic ‚ammation in the lung plays a 0cCanadian Respiratory Journaltherapycentral role in both the small airway remodeling and thepulmonary emphysema [“] Lung volume reductionsurgery and lung transplantation while possible in endstageCOPD are restricted to just a few selected patients []httpwwwgoldcopdcom RegenerativeforCOPD includes mesenchymal stromal cell MSC or tissueengineering therapies But while bone marrow MSC oradipose tissue MSC treatments showed promising results inmice with induced emphysema [] clinical trials performedin COPD patients have been discouraging [ ] ere are alarge number of animal studies in which lung regenerationhas been successfully stimulated For instance in a rat modelof elastaseinduced emphysema administration of alltransRA ATRA stimulated alveolar regeneration [] keratinocyte growth factor KGF FGF7 administered afterpneumonectomy augmented alveolarization [] administration of HGF stimulated alveolar regeneration enhancedlung vascularization and improved exercise tolerance andgas exchange [] intratracheal administration of bFGF torats and dogs with elastaseinduced emphysema improvedalveolar dimensions and lung microvessel density [] andVEGF administration enhanced postpneumonectomy alveolar growth in mice [] But again the attempts tostimulate lung regeneration in COPD patients with emphysema with orally administered ATRA yielded no differences in computed tomography CT lung function orquality of life scores between treatment groups [ ] andRARc selective agonist administration also showed nodiï¬erences in CT scores or lung function in treated vsnontreated COPD patients [ ] However the therapeutic potential of regenerative pharmacology is still at thebeginning of its development And many authors haveshown that the human lung also in adulthood retains asignificant regenerative potential from the large to the smallairways and in terminal and respiratory bronchioles [] andthat tissue regeneration is achieved in two ways by proliferation of common diï¬erentiated cells andor by deployment of specialized stemprogenitor cells [ ]Extracorporeal shock wave therapy ESWT is applied inmany musculoskeletal diseases and in regenerative medicinebased on its capability to induce neoangiogenesis osteogenesis regeneration and remodeling through stem cellstimulation [] ESW in combination with tenogenicmedium improved the diï¬erentiation of human adiposederived stem cells hASCs into tenoblastlike cells []ESW combined with osteogenic medium increased the osteogenic diï¬erentiation of treated hASCs [] while stemcell diï¬erentiation into myofibroblasts was partially reducedby ESW treatment [] But to our knowledge no data areavailable on ESW treatment of primary bronchial fibroblastsof patients with COPD and control healthy smokers orbronchial epithelial cells 16HBEMarkers of cell proliferation include CD117 cKit orSCFR a receptor tyrosine kinase protein that binds to stemcell factor SCF expressed on hematopoietic stem cells Itcan also be expressed by mast cells melanocytes in the skininterstitial cells of Cajal in the digestive and urogenital tract[] cardiac pericytes [] amniotic fluid stem cells []stemprogenitor cells in conducting airway epithelium ofporcine lung [] and dendritic cells in the lung []Another marker of cell proliferation is proliferating cellnuclear antigen PCNA It is expressed in the nuclei of cellsand is involved in DNA replication DNA repair andchromatin remodeling [ ] In the lung of COPD patients alveolar type II epithelial cells and endothelial cells[] and small airway bronchiolar epithelium [] expressdecreased PCNA levels compared with related nonCOPDcontrol groups A third marker of cell proliferation is CD90y1thymocyte diï¬erentiation antigen1 a glycophosphatidylinositol cell surface protein expressed by thymocytes CD34 cells mesenchymal stem cells endothelialcells and cardiac fibroblasts It is also considered a marker ofmultipotent mesenchymal stem cells when expressed inassociation with other markers CD29 CD44 CD73CD105 [ ]We aimed in this study to analyze the proliferative eï¬ectof shock waves when applied as an external challenge toprimary bronchial fibroblasts of COPD patients and controlsmokers and to immortalized bronchial epithelial cells16HBE To this end we investigated cell markers expression related to this proliferative stimulus Methods Ethics Statement Collection and processing of bronchialbiopsies at the Institute of Veruno NO and collection andprocessing of the peripheral lung tissues at the UniversityHospital of Orbassano during lung resection for a solitaryperipheral neoplasm were approved by the ethics andtechnical committees ofthe Istituti Clinici ScientificiMaugeri CTS p102 and San Luigi Hospital OrbassanoTO CE N Italy the study complied withthe Declaration of Helsinki and written informed consentwas obtained from each participant Cell Culture and Treatments We used the SV40 large Tantigentransformed 16HBE cellline which retains thediï¬erentiated morphology and function of normal humanbronchial epithelial cells NHBE [] and primary humanbronchial fibroblasts obtained from bronchial biopsies ofpatients with COPD n � and control smoking subjectsn � with normal lung function Primary bronchial fibroblasts were obtained from bronchial biopsies obtainedfor diï¬erent protocol studies [] Bronchial biopsies weretreated with type II collagenase min at °C InvitrogenGIBCA and cultured in DMEM until confluentprimary fibroblasts were obtained 16HBE cells and primarybronchial fibroblasts were maintained in Dulbecco™s modified minimum essential medium DMEM supplementedwith vv fetal bovine serum FBS IUmL penicillin μgmL streptomycin 1x nonessential amino acids mMsodium pyruvate and mM glutamine °C CO2 []When cells were “ confluent the complete mediumwas replaced with DMEM with FBS for starvation time h e shockwave generator utilized for the in vitroexperiments was a piezoelectric device Piezoson Richard Wolf Knittlingen Germany designed for clinical 0cCanadian Respiratory Journaluse in orthopedics and traumatology Aliquots of mL ofcell suspension adjusted to × cellsmL were placed in mm polypropylene tubes completely filled with culturemedium e shock wave unit was kept in contact with thecellcontaining tube by means of a waterfilled cushionCommon ultrasound gel was used as a contact mediumbetween the cushion and tube ESW treatment was as follows energy flux density EFD � mJmm2 pulsesfrequency � shockss is EFD is a mediumhigh energywe already used for previous in vitro diï¬erentiation studiesin tendons [] After treatment cell viability was evaluatedby trypan blue exclusion and primary fibroblasts werepassaged in DMEM complete for hours 16HBEcells were cultured for and h because of their lowerresistance to starvation T0 corresponds to hours post ESWtreatment for all experiments reported Nontreated fibroblasts or 16HBE cells were used as controls Cell growth wasevaluated by the colorimetric test WST1 All experimentswere performed in quadruplicate ie four independentexperiments for each type of treatment ESW or noESWand each time exposure Extraction and Quantification of RNA and qRTPCRfrom Primary Bronchial Fibroblasts and 16HBE Total RNAfrom treated and nontreated cells was purified and isolatedusing an RNAspin Mini RNA Isolation kit GE HealthcareLife Sciences Pittsburgh USA following the manufacturer™sinstructions Total RNA was resuspended in μL nucleasefree water RNA concentration was determined using aUVvisible spectrophotometer λ260280 nm EppendorfBioPhotometer plus and stored at ˆ’°CQiagenQT00000728e expression of genes of interest was measured usingSYBR Green Qiagen UK for qPCR in a Corbett RotorGene Corbett Cambridge UK system Onestep realtime PCR was carried out by amplifying mRNA using theQuantiFast„¢ SYBR Green RTPCR kitITaccording to the manufacturer™s instructions and the genespecific primers Qiagen IT We detected the expression ofcKit or SCFR CD117 Cat QT01844549 Qiagen PCNACat QT00024633 y1 CD90Cat QT00023569TGF1CatProcollagenIQT01005725 and NFκBp65 Cat QT01007370 Weperformed independent experiments and quantitative PCRmeasurements in quadruplicate for each type of treatmentESW or noESW and each time exposure Briefly the PCRreaction mix prepared in a total volume of μL was run onthe Rotor Gene Q Qiagen IT and the following PCR runprotocol was used °C for min reverse transcription°C for min PCR initial activation step amplificationcycles of °C for s denaturation and °C for scombined annealingextension followed by melting curveanalysis to ensure the specificity of PCR amplificationGlyceraldehyde phosphate dehydrogenase GAPDHQT01192646 Qiagen was used as the reference gene forevery target gene per sample and the data were normalizedagainst the respective GAPDH signaling Cycle thresholdCT values were determined using the Rotor Gene Qsoftware RotorGene Q Series Software eCatexpression levels of all genes studied were normalized toGAPDH levels in each sample to determine the expressionbetween treated and nontreated cells using the ˆ’ΔCt method[] for primary bronchial fibroblasts and the ˆ’ΔΔCt for16HBE cells [] ELISA Tests in the Supernatants or Cell Lysates of ESWTreated and Nontreated Cells Protein extraction andquantification in the supernatants or cell lysates of ESWtreated and nontreated cells were performed as reported inTable Suppliers Cat Numbers dilution conditions anddetection limits of the ELISA kits used are also reported eELISA kits WST1 cell proliferation kit and MPERmammalian protein extraction kit were used according tothe manufacturer™s instructions Table CKit CD117PCNA and NFκBp65 were quantified in cell lysates CD90TGF1 and procollagen1 were quantified in the cellsupernatants Immunohistochemistry of the Lung Parenchyma of Patients with COPD Samples were frozen in liquid nitrogenprecooled is tane after embedding in OCT and used forcryostat sectioning and immunostaining of some cellproliferationrelated molecules Single immunostainings offrozen sections were performed with mouse anti“thyroidtranscription factor1 TTF1 sc53136 Santa Cruz rabbitanticKit CD117 ARG51826 ARGBIO and rabbit antiPCNA PAS27214 ermo Fisher primary antibodiesAntibody binding was demonstrated with secondary antibodies antimouse Vector BA and antirabbitVector BA followed by ABC kit AP AK5000VECTASTAIN and FastRed Substrate red color Doublestainings were performed using also ABC kit Elite PK6100VECTASTAIN and diaminobenzidine substrate browncolor for identification of TTF1 positive alveolar type IIepithelial cells [] coexpressing cKit CD117 or PCNAantigens Slides were included in each staining run usinghuman tonsil nasal polyp or breast cancer as positivecontrols For the negative control slides normal nonspecificmouse or rabbit immunoglobulins Santa Cruz Biotechnology Santa Cruz CA USA were used at the same proteinconcentration as the primary antibodiesmean± standardthe unpaired ttest Probability values of p were Statistical Analysis Group data were expressed asorinterquartile range IQR for morphologichistologic dataDiï¬erences between treatment groups were analyzed usingor mediandeviationrangeconsidered significant Data analysis was performed usingthe Stat View SE Graphics program Abacus Concepts IncBerkeley CA USA Results ESW Eï¬ects on Cell Proliferation ESW treatment at adosage of mJmm2 pulses frequency � shockssof primary bronchial fibroblasts from COPD patients n � 0cCanadian Respiratory JournalPackyearsExsmokercurrent smokerTable Clinical characteristics of chronic obstructive pulmonary disease COPD patients and control smokers who provided bronchialfibroblasts for œin vitro experimentsSubjectsCOPD1COPD2COPD3± Mean± SD± Mean± SDIndividual and mean± standard deviation SD data M male F female FEV1 forced expiratory volume in s BD bronchodilator FVC forced vitalAge years MFMMM”MMM”± ± ± ± ± ± ± CurrentCurrentCurrentFEV1 postBDFEV1 preBDCurrentCurrentNDNDND”FEV1FVCCS1CS2CS3Ex””capacity ND not determined Patients were classified according to the Global Initiative for Chronic Obstructive Lung disease httpgoldcopdorg levels ofseverity for COPD For COPD patients FEV1FVC are postbronchodilator values ANOVA test FEV1 p � FEV1FVC p � No significantdiï¬erences were observed for age p � and packyears p � smokedTable List of ELISA tests cell proliferation and protein extraction kits used For ELISA tests dilution of the supernatants or cell lysatesamples used and detection limits are also reportedTargetcKit or SCFR CD117PCNAy1 CD90TGF1Procollagen1NFκBp65WST1 cell proliferationMPER mammalian protein extraction ermo Scientific ngmL “ ngmL ngmL “ ngmL pgmL “ pgmL pgmL “ pgmL pgmL “ pgmL17pgmL “ pgmLCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpSEA121 HuSEA591MiSEB404 HuSEA124 HuSEA957 HuKHO0371KA1384Dilution PBS PBSDetection limit range diluent buï¬erInvitrogenAbnovaNo dilNo dilNo dilSupplierCat ashowed a significantly increased proliferation index at and h after treatment compared with nontreated bronchial fibroblasts Figure 1a ESWtreated primary bronchial fibroblasts from control smokers with normal lungfunction n � also showed a significant increase of theproliferation index at and h aftertreatmentFigure 1b Treated bronchial epithelial cells 16HBEshowed significantly increased proliferation index values at and h after treatment when compared with nontreated16HBE cells Figure 1c ESW Eï¬ects on mRNA and Protein Levels of Cell Proliferation and Cell Remodeling Markers Primary bronchialfibroblasts from COPD patients n � control smokersn � and human bronchial epithelial cells 16HBE werestimulated with extracorporeal shock waves at a dosage of mJmm2 pulses and compared with paired nonstimulated primary bronchial fibroblasts and 16HBE cellsCKit mRNA was significantly increased in ESWtreated16HBE cells at h p � and decreased in CSfibroblasts at h p � compared with nontreated cellsFigures 2b and 2c Furthermore a tendency to increased cKit mRNA levels was observed after ESW treatment for COPDfibroblasts Figure 2a CKit protein wassignificantly increased in the cell lysates at h after ESWtreatment in primary bronchial fibroblasts of COPD patientsp � Figure 2d and in 16HBE cells p at h after ESW treatment Figure 2f No significantchanges were observed for cKit protein in ESWtreatedprimary bronchial fibroblasts from control smokers CSbronchialfibroblastswith normal lung function Figure 2e PCNA mRNAlevels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared with nontreatedcells Figures 3a“3c PCNA protein in the cell lysatesshowed a tendency to be increased in primary bronchialfibroblasts of CS p � at h after ESW treatmentFigure 3e and a significant increase was observed at hT0 in 16HBE cells p � after ESW treatmentFigure 3f No significant changes were observed inprimaryof COPD patientsFigure 3d y1 CD90 mRNA levels were not significantly diï¬erent in ESWtreated fibroblasts and 16HBE cellscompared with nontreated cells Figures 4a“4c y1CD90 protein in the cell supernatants was significantlyincreased in primary bronchial fibroblasts of CS at hp � after ESW treatment Figure 4e No significant changes were observed in primary bronchial fibroblastsof COPD patients or in 16HBE cells Figures 4d and 4fTGF1 mRNA levels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared withnontreated cells Figures 5a“5c TGF1 protein in thecell supernatants was significantly increased in primarybronchial fibroblasts of CS at h p � after ESWtreatment Figure 5e No significant changes were observed in primary bronchial fibroblasts of COPD patients orin 16HBE cells Figures 5d and 5f Procollagen1 mRNAlevels were not significantly diï¬erent in ESWtreated fibroblasts and 16HBE cells compared with nontreated cellsFigures 6a“6c Procollagen1 protein in the cellsupernatants wasincreased in primarysignificantly 0cCanadian Respiratory JournalabIncreased cell proliferation was observed in all cellular types studied after challenge with ESW Ttestˆ—ˆ—p andˆ—ˆ—ˆ—p Figure WST1 test for evaluation of cell proliferation after extracorporeal shock wave ESW stimulation of primary bronchial fibroblastsof COPD patients n � a primary bronchial fibroblasts of control smokers n � b and bronchial epithelial cells 16HBE ccbronchial fibroblasts of COPD patients at h p � after ESW treatment Figure 6d No significant changeswere observed in primary bronchial fibroblasts of CS or in16HBE cells Figures 6e and 6f NFκBp65 mRNAlevels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared with nontreatedcells Figures 7a“7c NFκBp65 protein in the cell lysates was decreased in primary bronchial fibroblasts ofCOPD patients at h p � after ESW treatmentFigure 7d and increased in 16HBE cells at h p � and h p � after ESW treatment Figure 7f Nosignificant changes were observed in primary bronchial fibroblasts of CS Figure 7e Immunohistochemistry in the Lung Parenchyma of COPDPatients of Alveolar Type II Epithelial Cells Expressing cKitand PCNA In the lung parenchyma of COPD patientsalveolar type II epithelial cells were identified by the use ofanti“thyroid transcription factor1 TTF1antibodyImmunopositivity for cKit CD117 and PCNA was alsooccasionally observed in alveolar septa Figure Doublestaining used for identification of TTF1 cells coexpressingcKitFigures 9a and 9b and PCNAFigures 9c and 9d showed that alveolar type II epithelial cells coexpressing cKit and PCNA were present eventhough rarely observedCD117 Discussionis study shows that extracorporeal shock waves induce cellproliferation of bronchial epithelial cells 16HBE and primary bronchial fibroblasts of COPD patients and controlsmokers As far as markers of cell proliferation are concerned cKit CD117 was increased in bronchial epitheliumat both mRNA and protein levels h after ESW treatmentand it was also increased in primary bronchial fibroblasts at h after ESW challenge Other markers indicative of cellproliferation were also increased PCNA protein increased inCOPDWST1NO ESWESWT24T48T72T0012345ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—CST0T24T48T72NO ESWESWWST1012345ˆ—ˆ—ˆ—ˆ—ˆ—16HBET0T24T48NO ESWESWWST1012345ˆ—ˆ—ˆ—ˆ—ˆ—ˆ— 0cCanadian Respiratory JournaladbecfFigure CKit CD117 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBEcKit increased at mRNA c and protein f levels In primary bronchial fibroblasts of COPD patients cKit increased at protein level d Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Proliferating cell nuclear antigen PCNA mRNA a b c and protein d e f expression after ESW treatment in primary bronchialfibroblasts of COPD patients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchialepithelium 16HBE PCNA increased at protein f level Ttest was used for comparative purposes and p values are reported in the graphsT0T24T48T7201234102030COPD CNCOPD ESWcKit CD117 “ˆ† Ct0180021103960767CS CNCS ESWcKit CD117 “ˆ† CtT0T24T48T7202461020304050002016HBE CN16HBE ESWcKit CD117 “ˆ†ˆ† CtT0T24T480246204060800435041800320010020030006839038680037305624COPD CNCOPD ESWcKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESW000500100015002000250006727038680877507636cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW16HBE CN16HBE ESW020406080P 000010791501364cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT0T24T48T72PCNA “ˆ† Ct0005101520COPD CNCOPD ESWT0T24T48T7205101520PCNA “ˆ† CtCS CNCS ESWT0T24T48000510152025PCNA “ˆ†ˆ† Ct16HBE CN16HBE ESW01450027520139305288COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00102030PCNA ngmL00002004006008010000512084270367304489PCNA ngmLCS CNCS ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW0123004620190109820PCNA ngmL16HBE CN16HBE ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SW 0cCanadian Respiratory JournalacebdfFigure y1 CD90 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts ofcontrol smokers y1 increased at protein level at and h e Ttest was used for comparative purposes and p values are reported in thegraphsbronchial epithelial cells at h after ESW challenge y1CD90 protein increased in CS“primary bronchial fibroblasts at and h after ESW treatment molecules morerelated to remodeling such as TGF1 protein were increased in CS“primary bronchial fibroblasts at h afterESW treatment and procollagen1 protein increased at hfollowed by a decrease at h in COPD“primary bronchialfibroblasts after ESW treatment A marker of ‚ammationtranscription factor NFκBp65 protein was decreased inCOPD“primary bronchial fibroblasts at h after ESWtreatment but it was increased in CS“primary bronchialfibroblasts and in bronchial epithelial cells after ESWtreatment Markers of cell proliferation such as cKit andPCNA were observed in the peripherallung of COPDT0T24T48T72COPD CNCOPD ESWThy1 CD90 “ˆ† Ct0005101520CS CNCS ESWThy1 CD90 “ˆ† CtT0T24T48T72012316HBE CN16HBE ESWThy1 CD90 “ˆ† ˆ†CtT0T24T4801020304009523087570853209221COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00200040006000800010000Thy1 CD90 pgmLCS CNCS ESW01500003150239300410T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000200004000060000Thy1 CD90 pgmL16HBE CN16HBE ESW035960811001447T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW010203040Thy1 CD90 pgmL 0cCanadian Respiratory JournaladbecfFigure TGF1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPD patients ad primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts of controlsmokers TGF1 increased at protein level at h e Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Procollagen1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts ofCOPD patients procollagen1 increased at protein level d at h T0 followed by a decrease at h panel d Ttest was used forcomparative purposes and p values are reported in the graphsT0T24T48T72TGF 1 “ˆ† Ct0005101520COPD CNCOPD ESWT0T24T48T72TGF 1 “ˆ† Ct00051015CS CNCS ESWT0T24T48TGF 1 “ˆ† Ct0005101516HBE CN16HBE ESW07196046450373903445T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESWTGF 1 pgmL005010015004487044930863500385T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESWTGF 1 pgmL0005001000150004757089490102101199T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW16HBE CN16HBE ESWTGF 1 pgmL010203040T0T24T48T72Procollanen1 “ˆ† Ct000510152025COPD CNCOPD ESWT0T24T48T72Procollagen1 “ˆ† Ct000510152025CS CNCS ESWT0T24T48Procollagen1 “ˆ†ˆ† Ct00051015202516HBE CN16HBE ESW00220057350024202359T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESW00100020003000Procollagen1 pgmL00541053750944605958T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000100002000030000Procollagen1 pgmLCS CNCS ESW010340898407490T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW050100150200Procollagen1 pgmL16HBE CN16HBE ESW 0cCanadian Respiratory JournaladbecfFigure NFκBp65 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPD patientsa d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBE NFκBp65 increased at protein panel f level at and h of exposure In primary bronchial fibroblasts of COPD patients NFκBp65 decreased atprotein level d at h In primary bronchial fibroblasts of control smokers NFκBp65 increased at protein level e at h Ttest wasused for comparative purposes and p values are reported in the graphspatients and both these markers were occasionally coexpressed by alveolar epithelial type II cells TTF1 in thesepatientsExtracorporeal shock wave therapy is applied in regenerative medicine since it is capable of inducing neoangiogenesis osteogenesis and remodeling through stemcell stimulation [] On the other hand while regenerativetherapy applied to mice with induced emphysema has shownpromising results [] clinical trials performed in COPDpatients were discouraging [ ] Since the human lung alsoin adulthood maintains a significant regenerative potential[“] due to proliferation of diï¬erentiated of stemprogenitor cells andor by their stimulation [ ] we hereinvestigated the proliferative action of ESW at low dosage inbronchial epithelial cells and in primary bronchial fibroblasts of control smokers CS and patients with COPD Ourdata show that all the cell types studied significantly increased their proliferation index WST1 test after ESWtreatment in agreement with data previously obtained formuscle cells or tendon fibroblasts [] Interestingly thecKit CD117 receptor tyrosine kinase protein and mRNAwere increased in 16HBE cells and cKit protein also increased in primary bronchial fibroblasts of COPD patientsafter ESW stimulation It is not clear however if this cellresponse represents an intermediate dediï¬erentiation step ora simple proproliferative stimulus for stimulated 16HBEcells and COPD“primary bronchial fibroblasts Since weexposed welldiï¬erentiated cells we believe that this transitory increment may be interpreted as a proproliferativestimulus induced by ESW exposureIn bronchial epithelial cells 16HBE proliferating cellnuclear antigen PCNA considered a marker of cellproliferation was increased after ESW stimulation confirming again the proproliferative role of ESW exposurefor these lung structure cells is finding in view of thedecreased PCNA levels reported in the lung of COPDpatients [ ] compared with control subjects is particularly relevant since ESW stimulation may contrastthese lower PCNA levels characterizing the damaged lungof these patientse increased y1 CD90 protein level shown afterESW exposure in CS“primary bronchial fibroblasts was notobserved in ESWtreated COPD“primary bronchial fibroblasts or in 16HBE treated cells PCNA protein alsotended to be higher in CS“primary bronchial fibroblastsafter ESW treatment but not in COPD“primary bronchialfibroblasts ese diï¬erences in the response to ESWchallenge of COPD“ and CS“primary bronchial fibroblastsmay in part be due to the reduced proliferation capacity ofthese cells derived from COPD lungs as previously reported [ ] In our welldiï¬erentiated ESWexposedfibroblasts we interpret the increment of y1 protein NFκBp65 “ˆ† Ct012345T24T48T72T0COPD CNCOPD ESW NFκBp65 “ˆ† Ct01234T24T48T72T0CS CNCS ESW NFκBp65 “ˆ†ˆ† Ct000510152025T24T48T016HBE CN16HBE ESW00805026820020907045NFκBp65 pgmL0050000100000150000T72 CNT72 SWT24 SWT48 CNT48 SWT0 SWT24 CNT0 CNCOPD CNCOPD ESWNFκBp65 pgmL036510427702233003830020000400006000080000100000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CNT72 SWT72 CNCS CNCS ESWNFκBp65 pgmL0015500002062865000100001500002004006008001000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CN16HBE CN16HBE ESW 0cCanadian Respiratory JournalFigure Photomicrographs showing thyroid transcription factor1 TTF1 expression panels a b cKit CD117 c d and proliferatingcell nuclear antigen PCNA e f in the peripheral lung tissue of a representative patient with chronic obstructive pulmonary diseaseCOPD Arrows indicate positively stained cells mainly located in the alveolar septa Bars � micronsafter ESW treatment”like that of cKit”as a proproliferative stimulus induced by the treatmentWe found increased levels of secreted TGF1 inCS“primary bronchial fibroblasts h after ESW stimulation TGF signaling pathways are involved in the regulationof many cell functions and in the maintenance of cellularhomeostasis [] We recently reported a decrease of TGF1and TGF3 in bronchiolar epithelium and alveolar macrophages of COPD patients compared with CS [] and thisdecrease may favor the increase of autoimmunity responsesin these patients [] We speculate that the inductionthrough ESW challenge of an increase of TGF in bronchialfibroblasts may play a role in the TGF repositioning andgain in homeostatic function of this important protein in thelungs of COPD patientsTGF induced extracellular matrix and procollagen1production has been reported in pulmonary fibroblasts[] even though it was also reported that the increase ofprofibrotic markersincluding procollagen1 in humanlung fibroblasts may be NLRP3 ‚ammasome dependentand TGF independent [] and associated with increased‚ammation ofthe lung [] We here observed aTTF a0Lung COPDCD a0Lung COPDPCNA a0Lung COPDabcdef 0cCanadian Respiratory JournalFigure Photomicrographs showing alveolar type II epithelial cells TTF1 cells red color coexpressing cKit CD117 brown color ab and PCNA brown color c d in the peripheral lung tissue of a representative patient with COPD Positive doublestained cells can berecognized in the alveolar septa even though their presence was only rarely observed Arrows indicate positively stained cells located in thealveolar septa Bars � micronstransitory increase of procollagen1 protein in COPD“primary bronchial fibroblasts at h after ESW

Thyroid_Cancer

pulmonary disease COPD is due to structural changes and narrowing of small airways and parenchymaldestruction loss of the alveolar attachment as a result of pulmonary emphysema which all lead to airflow limitation Extracorporeal shock waves ESW increase cell proliferation and diï¬erentiation of connective tissue fibroblasts To date no studiesare available on ESW treatment of human bronchial fibroblasts and epithelial cells from COPD and control subjects We obtainedprimary bronchial fibroblasts from bronchial biopsies of patients with mildmoderate COPD and control smokers with normallung function 16HBE cells were also studied Cells were treated with a piezoelectric shock wave generator at low energy mJmm2 pulses After treatment viability was evaluated and cells were recultured and followed up for and h Cellgrowth WST1 test was assessed and proliferation markers were analyzed by qRTPCR in cell lysates and by ELISA tests in cellsupernatants and cell lysates After ESW treatment we observed a significant increase of cell proliferation in all cell types CKitCD117 mRNA was significantly increased in 16HBE cells at h Protein levels were significantly increased for cKit CD117 at h in 16HBE p and at h in COPDfibroblasts p � for PCNA at h in 16HBE p � for y1 CD90 at and h in CSfibroblasts p � and p � for TGF1 at h in CSfibroblasts p � for procollagen1 at h inCOPDfibroblasts p � and for NFκBp65 at and h in 16HBE p � and p � In the peripheral lung tissueof a representative COPD patient alveolar type II epithelial cells TTF1 coexpressing cKit CD117 and PCNA were occasionally observed ese data show an increase of cell proliferation induced by a low dosage of extracorporeal shock waves in16HBE cells and primary bronchial fibroblasts of COPD and control smoking subjects Backgrounde progressive chronic airflow limitation in chronic obstructive pulmonary disease COPD is due to two majorpathological processes i remodeling and narrowing ofsmall airways and ii destruction of the lung parenchymawith loss of the alveolar attachments as a result of pulmonaryemphysema [] Chronic ‚ammation in the lung plays a 0cCanadian Respiratory Journaltherapycentral role in both the small airway remodeling and thepulmonary emphysema [“] Lung volume reductionsurgery and lung transplantation while possible in endstageCOPD are restricted to just a few selected patients []httpwwwgoldcopdcom RegenerativeforCOPD includes mesenchymal stromal cell MSC or tissueengineering therapies But while bone marrow MSC oradipose tissue MSC treatments showed promising results inmice with induced emphysema [] clinical trials performedin COPD patients have been discouraging [ ] ere are alarge number of animal studies in which lung regenerationhas been successfully stimulated For instance in a rat modelof elastaseinduced emphysema administration of alltransRA ATRA stimulated alveolar regeneration [] keratinocyte growth factor KGF FGF7 administered afterpneumonectomy augmented alveolarization [] administration of HGF stimulated alveolar regeneration enhancedlung vascularization and improved exercise tolerance andgas exchange [] intratracheal administration of bFGF torats and dogs with elastaseinduced emphysema improvedalveolar dimensions and lung microvessel density [] andVEGF administration enhanced postpneumonectomy alveolar growth in mice [] But again the attempts tostimulate lung regeneration in COPD patients with emphysema with orally administered ATRA yielded no differences in computed tomography CT lung function orquality of life scores between treatment groups [ ] andRARc selective agonist administration also showed nodiï¬erences in CT scores or lung function in treated vsnontreated COPD patients [ ] However the therapeutic potential of regenerative pharmacology is still at thebeginning of its development And many authors haveshown that the human lung also in adulthood retains asignificant regenerative potential from the large to the smallairways and in terminal and respiratory bronchioles [] andthat tissue regeneration is achieved in two ways by proliferation of common diï¬erentiated cells andor by deployment of specialized stemprogenitor cells [ ]Extracorporeal shock wave therapy ESWT is applied inmany musculoskeletal diseases and in regenerative medicinebased on its capability to induce neoangiogenesis osteogenesis regeneration and remodeling through stem cellstimulation [] ESW in combination with tenogenicmedium improved the diï¬erentiation of human adiposederived stem cells hASCs into tenoblastlike cells []ESW combined with osteogenic medium increased the osteogenic diï¬erentiation of treated hASCs [] while stemcell diï¬erentiation into myofibroblasts was partially reducedby ESW treatment [] But to our knowledge no data areavailable on ESW treatment of primary bronchial fibroblastsof patients with COPD and control healthy smokers orbronchial epithelial cells 16HBEMarkers of cell proliferation include CD117 cKit orSCFR a receptor tyrosine kinase protein that binds to stemcell factor SCF expressed on hematopoietic stem cells Itcan also be expressed by mast cells melanocytes in the skininterstitial cells of Cajal in the digestive and urogenital tract[] cardiac pericytes [] amniotic fluid stem cells []stemprogenitor cells in conducting airway epithelium ofporcine lung [] and dendritic cells in the lung []Another marker of cell proliferation is proliferating cellnuclear antigen PCNA It is expressed in the nuclei of cellsand is involved in DNA replication DNA repair andchromatin remodeling [ ] In the lung of COPD patients alveolar type II epithelial cells and endothelial cells[] and small airway bronchiolar epithelium [] expressdecreased PCNA levels compared with related nonCOPDcontrol groups A third marker of cell proliferation is CD90y1thymocyte diï¬erentiation antigen1 a glycophosphatidylinositol cell surface protein expressed by thymocytes CD34 cells mesenchymal stem cells endothelialcells and cardiac fibroblasts It is also considered a marker ofmultipotent mesenchymal stem cells when expressed inassociation with other markers CD29 CD44 CD73CD105 [ ]We aimed in this study to analyze the proliferative eï¬ectof shock waves when applied as an external challenge toprimary bronchial fibroblasts of COPD patients and controlsmokers and to immortalized bronchial epithelial cells16HBE To this end we investigated cell markers expression related to this proliferative stimulus Methods Ethics Statement Collection and processing of bronchialbiopsies at the Institute of Veruno NO and collection andprocessing of the peripheral lung tissues at the UniversityHospital of Orbassano during lung resection for a solitaryperipheral neoplasm were approved by the ethics andtechnical committees ofthe Istituti Clinici ScientificiMaugeri CTS p102 and San Luigi Hospital OrbassanoTO CE N Italy the study complied withthe Declaration of Helsinki and written informed consentwas obtained from each participant Cell Culture and Treatments We used the SV40 large Tantigentransformed 16HBE cellline which retains thediï¬erentiated morphology and function of normal humanbronchial epithelial cells NHBE [] and primary humanbronchial fibroblasts obtained from bronchial biopsies ofpatients with COPD n � and control smoking subjectsn � with normal lung function Primary bronchial fibroblasts were obtained from bronchial biopsies obtainedfor diï¬erent protocol studies [] Bronchial biopsies weretreated with type II collagenase min at °C InvitrogenGIBCA and cultured in DMEM until confluentprimary fibroblasts were obtained 16HBE cells and primarybronchial fibroblasts were maintained in Dulbecco™s modified minimum essential medium DMEM supplementedwith vv fetal bovine serum FBS IUmL penicillin μgmL streptomycin 1x nonessential amino acids mMsodium pyruvate and mM glutamine °C CO2 []When cells were “ confluent the complete mediumwas replaced with DMEM with FBS for starvation time h e shockwave generator utilized for the in vitroexperiments was a piezoelectric device Piezoson Richard Wolf Knittlingen Germany designed for clinical 0cCanadian Respiratory Journaluse in orthopedics and traumatology Aliquots of mL ofcell suspension adjusted to × cellsmL were placed in mm polypropylene tubes completely filled with culturemedium e shock wave unit was kept in contact with thecellcontaining tube by means of a waterfilled cushionCommon ultrasound gel was used as a contact mediumbetween the cushion and tube ESW treatment was as follows energy flux density EFD � mJmm2 pulsesfrequency � shockss is EFD is a mediumhigh energywe already used for previous in vitro diï¬erentiation studiesin tendons [] After treatment cell viability was evaluatedby trypan blue exclusion and primary fibroblasts werepassaged in DMEM complete for hours 16HBEcells were cultured for and h because of their lowerresistance to starvation T0 corresponds to hours post ESWtreatment for all experiments reported Nontreated fibroblasts or 16HBE cells were used as controls Cell growth wasevaluated by the colorimetric test WST1 All experimentswere performed in quadruplicate ie four independentexperiments for each type of treatment ESW or noESWand each time exposure Extraction and Quantification of RNA and qRTPCRfrom Primary Bronchial Fibroblasts and 16HBE Total RNAfrom treated and nontreated cells was purified and isolatedusing an RNAspin Mini RNA Isolation kit GE HealthcareLife Sciences Pittsburgh USA following the manufacturer™sinstructions Total RNA was resuspended in μL nucleasefree water RNA concentration was determined using aUVvisible spectrophotometer λ260280 nm EppendorfBioPhotometer plus and stored at ˆ’°CQiagenQT00000728e expression of genes of interest was measured usingSYBR Green Qiagen UK for qPCR in a Corbett RotorGene Corbett Cambridge UK system Onestep realtime PCR was carried out by amplifying mRNA using theQuantiFast„¢ SYBR Green RTPCR kitITaccording to the manufacturer™s instructions and the genespecific primers Qiagen IT We detected the expression ofcKit or SCFR CD117 Cat QT01844549 Qiagen PCNACat QT00024633 y1 CD90Cat QT00023569TGF1CatProcollagenIQT01005725 and NFκBp65 Cat QT01007370 Weperformed independent experiments and quantitative PCRmeasurements in quadruplicate for each type of treatmentESW or noESW and each time exposure Briefly the PCRreaction mix prepared in a total volume of μL was run onthe Rotor Gene Q Qiagen IT and the following PCR runprotocol was used °C for min reverse transcription°C for min PCR initial activation step amplificationcycles of °C for s denaturation and °C for scombined annealingextension followed by melting curveanalysis to ensure the specificity of PCR amplificationGlyceraldehyde phosphate dehydrogenase GAPDHQT01192646 Qiagen was used as the reference gene forevery target gene per sample and the data were normalizedagainst the respective GAPDH signaling Cycle thresholdCT values were determined using the Rotor Gene Qsoftware RotorGene Q Series Software eCatexpression levels of all genes studied were normalized toGAPDH levels in each sample to determine the expressionbetween treated and nontreated cells using the ˆ’ΔCt method[] for primary bronchial fibroblasts and the ˆ’ΔΔCt for16HBE cells [] ELISA Tests in the Supernatants or Cell Lysates of ESWTreated and Nontreated Cells Protein extraction andquantification in the supernatants or cell lysates of ESWtreated and nontreated cells were performed as reported inTable Suppliers Cat Numbers dilution conditions anddetection limits of the ELISA kits used are also reported eELISA kits WST1 cell proliferation kit and MPERmammalian protein extraction kit were used according tothe manufacturer™s instructions Table CKit CD117PCNA and NFκBp65 were quantified in cell lysates CD90TGF1 and procollagen1 were quantified in the cellsupernatants Immunohistochemistry of the Lung Parenchyma of Patients with COPD Samples were frozen in liquid nitrogenprecooled is tane after embedding in OCT and used forcryostat sectioning and immunostaining of some cellproliferationrelated molecules Single immunostainings offrozen sections were performed with mouse anti“thyroidtranscription factor1 TTF1 sc53136 Santa Cruz rabbitanticKit CD117 ARG51826 ARGBIO and rabbit antiPCNA PAS27214 ermo Fisher primary antibodiesAntibody binding was demonstrated with secondary antibodies antimouse Vector BA and antirabbitVector BA followed by ABC kit AP AK5000VECTASTAIN and FastRed Substrate red color Doublestainings were performed using also ABC kit Elite PK6100VECTASTAIN and diaminobenzidine substrate browncolor for identification of TTF1 positive alveolar type IIepithelial cells [] coexpressing cKit CD117 or PCNAantigens Slides were included in each staining run usinghuman tonsil nasal polyp or breast cancer as positivecontrols For the negative control slides normal nonspecificmouse or rabbit immunoglobulins Santa Cruz Biotechnology Santa Cruz CA USA were used at the same proteinconcentration as the primary antibodiesmean± standardthe unpaired ttest Probability values of p were Statistical Analysis Group data were expressed asorinterquartile range IQR for morphologichistologic dataDiï¬erences between treatment groups were analyzed usingor mediandeviationrangeconsidered significant Data analysis was performed usingthe Stat View SE Graphics program Abacus Concepts IncBerkeley CA USA Results ESW Eï¬ects on Cell Proliferation ESW treatment at adosage of mJmm2 pulses frequency � shockssof primary bronchial fibroblasts from COPD patients n � 0cCanadian Respiratory JournalPackyearsExsmokercurrent smokerTable Clinical characteristics of chronic obstructive pulmonary disease COPD patients and control smokers who provided bronchialfibroblasts for œin vitro experimentsSubjectsCOPD1COPD2COPD3± Mean± SD± Mean± SDIndividual and mean± standard deviation SD data M male F female FEV1 forced expiratory volume in s BD bronchodilator FVC forced vitalAge years MFMMM”MMM”± ± ± ± ± ± ± CurrentCurrentCurrentFEV1 postBDFEV1 preBDCurrentCurrentNDNDND”FEV1FVCCS1CS2CS3Ex””capacity ND not determined Patients were classified according to the Global Initiative for Chronic Obstructive Lung disease httpgoldcopdorg levels ofseverity for COPD For COPD patients FEV1FVC are postbronchodilator values ANOVA test FEV1 p � FEV1FVC p � No significantdiï¬erences were observed for age p � and packyears p � smokedTable List of ELISA tests cell proliferation and protein extraction kits used For ELISA tests dilution of the supernatants or cell lysatesamples used and detection limits are also reportedTargetcKit or SCFR CD117PCNAy1 CD90TGF1Procollagen1NFκBp65WST1 cell proliferationMPER mammalian protein extraction ermo Scientific ngmL “ ngmL ngmL “ ngmL pgmL “ pgmL pgmL “ pgmL pgmL “ pgmL17pgmL “ pgmLCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpSEA121 HuSEA591MiSEB404 HuSEA124 HuSEA957 HuKHO0371KA1384Dilution PBS PBSDetection limit range diluent buï¬erInvitrogenAbnovaNo dilNo dilNo dilSupplierCat ashowed a significantly increased proliferation index at and h after treatment compared with nontreated bronchial fibroblasts Figure 1a ESWtreated primary bronchial fibroblasts from control smokers with normal lungfunction n � also showed a significant increase of theproliferation index at and h aftertreatmentFigure 1b Treated bronchial epithelial cells 16HBEshowed significantly increased proliferation index values at and h after treatment when compared with nontreated16HBE cells Figure 1c ESW Eï¬ects on mRNA and Protein Levels of Cell Proliferation and Cell Remodeling Markers Primary bronchialfibroblasts from COPD patients n � control smokersn � and human bronchial epithelial cells 16HBE werestimulated with extracorporeal shock waves at a dosage of mJmm2 pulses and compared with paired nonstimulated primary bronchial fibroblasts and 16HBE cellsCKit mRNA was significantly increased in ESWtreated16HBE cells at h p � and decreased in CSfibroblasts at h p � compared with nontreated cellsFigures 2b and 2c Furthermore a tendency to increased cKit mRNA levels was observed after ESW treatment for COPDfibroblasts Figure 2a CKit protein wassignificantly increased in the cell lysates at h after ESWtreatment in primary bronchial fibroblasts of COPD patientsp � Figure 2d and in 16HBE cells p at h after ESW treatment Figure 2f No significantchanges were observed for cKit protein in ESWtreatedprimary bronchial fibroblasts from control smokers CSbronchialfibroblastswith normal lung function Figure 2e PCNA mRNAlevels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared with nontreatedcells Figures 3a“3c PCNA protein in the cell lysatesshowed a tendency to be increased in primary bronchialfibroblasts of CS p � at h after ESW treatmentFigure 3e and a significant increase was observed at hT0 in 16HBE cells p � after ESW treatmentFigure 3f No significant changes were observed inprimaryof COPD patientsFigure 3d y1 CD90 mRNA levels were not significantly diï¬erent in ESWtreated fibroblasts and 16HBE cellscompared with nontreated cells Figures 4a“4c y1CD90 protein in the cell supernatants was significantlyincreased in primary bronchial fibroblasts of CS at hp � after ESW treatment Figure 4e No significant changes were observed in primary bronchial fibroblastsof COPD patients or in 16HBE cells Figures 4d and 4fTGF1 mRNA levels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared withnontreated cells Figures 5a“5c TGF1 protein in thecell supernatants was significantly increased in primarybronchial fibroblasts of CS at h p � after ESWtreatment Figure 5e No significant changes were observed in primary bronchial fibroblasts of COPD patients orin 16HBE cells Figures 5d and 5f Procollagen1 mRNAlevels were not significantly diï¬erent in ESWtreated fibroblasts and 16HBE cells compared with nontreated cellsFigures 6a“6c Procollagen1 protein in the cellsupernatants wasincreased in primarysignificantly 0cCanadian Respiratory JournalabIncreased cell proliferation was observed in all cellular types studied after challenge with ESW Ttestˆ—ˆ—p andˆ—ˆ—ˆ—p Figure WST1 test for evaluation of cell proliferation after extracorporeal shock wave ESW stimulation of primary bronchial fibroblastsof COPD patients n � a primary bronchial fibroblasts of control smokers n � b and bronchial epithelial cells 16HBE ccbronchial fibroblasts of COPD patients at h p � after ESW treatment Figure 6d No significant changeswere observed in primary bronchial fibroblasts of CS or in16HBE cells Figures 6e and 6f NFκBp65 mRNAlevels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared with nontreatedcells Figures 7a“7c NFκBp65 protein in the cell lysates was decreased in primary bronchial fibroblasts ofCOPD patients at h p � after ESW treatmentFigure 7d and increased in 16HBE cells at h p � and h p � after ESW treatment Figure 7f Nosignificant changes were observed in primary bronchial fibroblasts of CS Figure 7e Immunohistochemistry in the Lung Parenchyma of COPDPatients of Alveolar Type II Epithelial Cells Expressing cKitand PCNA In the lung parenchyma of COPD patientsalveolar type II epithelial cells were identified by the use ofanti“thyroid transcription factor1 TTF1antibodyImmunopositivity for cKit CD117 and PCNA was alsooccasionally observed in alveolar septa Figure Doublestaining used for identification of TTF1 cells coexpressingcKitFigures 9a and 9b and PCNAFigures 9c and 9d showed that alveolar type II epithelial cells coexpressing cKit and PCNA were present eventhough rarely observedCD117 Discussionis study shows that extracorporeal shock waves induce cellproliferation of bronchial epithelial cells 16HBE and primary bronchial fibroblasts of COPD patients and controlsmokers As far as markers of cell proliferation are concerned cKit CD117 was increased in bronchial epitheliumat both mRNA and protein levels h after ESW treatmentand it was also increased in primary bronchial fibroblasts at h after ESW challenge Other markers indicative of cellproliferation were also increased PCNA protein increased inCOPDWST1NO ESWESWT24T48T72T0012345ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—CST0T24T48T72NO ESWESWWST1012345ˆ—ˆ—ˆ—ˆ—ˆ—16HBET0T24T48NO ESWESWWST1012345ˆ—ˆ—ˆ—ˆ—ˆ—ˆ— 0cCanadian Respiratory JournaladbecfFigure CKit CD117 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBEcKit increased at mRNA c and protein f levels In primary bronchial fibroblasts of COPD patients cKit increased at protein level d Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Proliferating cell nuclear antigen PCNA mRNA a b c and protein d e f expression after ESW treatment in primary bronchialfibroblasts of COPD patients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchialepithelium 16HBE PCNA increased at protein f level Ttest was used for comparative purposes and p values are reported in the graphsT0T24T48T7201234102030COPD CNCOPD ESWcKit CD117 “ˆ† Ct0180021103960767CS CNCS ESWcKit CD117 “ˆ† CtT0T24T48T7202461020304050002016HBE CN16HBE ESWcKit CD117 “ˆ†ˆ† CtT0T24T480246204060800435041800320010020030006839038680037305624COPD CNCOPD ESWcKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESW000500100015002000250006727038680877507636cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW16HBE CN16HBE ESW020406080P 000010791501364cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT0T24T48T72PCNA “ˆ† Ct0005101520COPD CNCOPD ESWT0T24T48T7205101520PCNA “ˆ† CtCS CNCS ESWT0T24T48000510152025PCNA “ˆ†ˆ† Ct16HBE CN16HBE ESW01450027520139305288COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00102030PCNA ngmL00002004006008010000512084270367304489PCNA ngmLCS CNCS ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW0123004620190109820PCNA ngmL16HBE CN16HBE ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SW 0cCanadian Respiratory JournalacebdfFigure y1 CD90 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts ofcontrol smokers y1 increased at protein level at and h e Ttest was used for comparative purposes and p values are reported in thegraphsbronchial epithelial cells at h after ESW challenge y1CD90 protein increased in CS“primary bronchial fibroblasts at and h after ESW treatment molecules morerelated to remodeling such as TGF1 protein were increased in CS“primary bronchial fibroblasts at h afterESW treatment and procollagen1 protein increased at hfollowed by a decrease at h in COPD“primary bronchialfibroblasts after ESW treatment A marker of ‚ammationtranscription factor NFκBp65 protein was decreased inCOPD“primary bronchial fibroblasts at h after ESWtreatment but it was increased in CS“primary bronchialfibroblasts and in bronchial epithelial cells after ESWtreatment Markers of cell proliferation such as cKit andPCNA were observed in the peripherallung of COPDT0T24T48T72COPD CNCOPD ESWThy1 CD90 “ˆ† Ct0005101520CS CNCS ESWThy1 CD90 “ˆ† CtT0T24T48T72012316HBE CN16HBE ESWThy1 CD90 “ˆ† ˆ†CtT0T24T4801020304009523087570853209221COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00200040006000800010000Thy1 CD90 pgmLCS CNCS ESW01500003150239300410T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000200004000060000Thy1 CD90 pgmL16HBE CN16HBE ESW035960811001447T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW010203040Thy1 CD90 pgmL 0cCanadian Respiratory JournaladbecfFigure TGF1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPD patients ad primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts of controlsmokers TGF1 increased at protein level at h e Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Procollagen1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts ofCOPD patients procollagen1 increased at protein level d at h T0 followed by a decrease at h panel d Ttest was used forcomparative purposes and p values are reported in the graphsT0T24T48T72TGF 1 “ˆ† Ct0005101520COPD CNCOPD ESWT0T24T48T72TGF 1 “ˆ† Ct00051015CS CNCS ESWT0T24T48TGF 1 “ˆ† Ct0005101516HBE CN16HBE ESW07196046450373903445T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESWTGF 1 pgmL005010015004487044930863500385T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESWTGF 1 pgmL0005001000150004757089490102101199T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW16HBE CN16HBE ESWTGF 1 pgmL010203040T0T24T48T72Procollanen1 “ˆ† Ct000510152025COPD CNCOPD ESWT0T24T48T72Procollagen1 “ˆ† Ct000510152025CS CNCS ESWT0T24T48Procollagen1 “ˆ†ˆ† Ct00051015202516HBE CN16HBE ESW00220057350024202359T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESW00100020003000Procollagen1 pgmL00541053750944605958T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000100002000030000Procollagen1 pgmLCS CNCS ESW010340898407490T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW050100150200Procollagen1 pgmL16HBE CN16HBE ESW 0cCanadian Respiratory JournaladbecfFigure NFκBp65 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPD patientsa d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBE NFκBp65 increased at protein panel f level at and h of exposure In primary bronchial fibroblasts of COPD patients NFκBp65 decreased atprotein level d at h In primary bronchial fibroblasts of control smokers NFκBp65 increased at protein level e at h Ttest wasused for comparative purposes and p values are reported in the graphspatients and both these markers were occasionally coexpressed by alveolar epithelial type II cells TTF1 in thesepatientsExtracorporeal shock wave therapy is applied in regenerative medicine since it is capable of inducing neoangiogenesis osteogenesis and remodeling through stemcell stimulation [] On the other hand while regenerativetherapy applied to mice with induced emphysema has shownpromising results [] clinical trials performed in COPDpatients were discouraging [ ] Since the human lung alsoin adulthood maintains a significant regenerative potential[“] due to proliferation of diï¬erentiated of stemprogenitor cells andor by their stimulation [ ] we hereinvestigated the proliferative action of ESW at low dosage inbronchial epithelial cells and in primary bronchial fibroblasts of control smokers CS and patients with COPD Ourdata show that all the cell types studied significantly increased their proliferation index WST1 test after ESWtreatment in agreement with data previously obtained formuscle cells or tendon fibroblasts [] Interestingly thecKit CD117 receptor tyrosine kinase protein and mRNAwere increased in 16HBE cells and cKit protein also increased in primary bronchial fibroblasts of COPD patientsafter ESW stimulation It is not clear however if this cellresponse represents an intermediate dediï¬erentiation step ora simple proproliferative stimulus for stimulated 16HBEcells and COPD“primary bronchial fibroblasts Since weexposed welldiï¬erentiated cells we believe that this transitory increment may be interpreted as a proproliferativestimulus induced by ESW exposureIn bronchial epithelial cells 16HBE proliferating cellnuclear antigen PCNA considered a marker of cellproliferation was increased after ESW stimulation confirming again the proproliferative role of ESW exposurefor these lung structure cells is finding in view of thedecreased PCNA levels reported in the lung of COPDpatients [ ] compared with control subjects is particularly relevant since ESW stimulation may contrastthese lower PCNA levels characterizing the damaged lungof these patientse increased y1 CD90 protein level shown afterESW exposure in CS“primary bronchial fibroblasts was notobserved in ESWtreated COPD“primary bronchial fibroblasts or in 16HBE treated cells PCNA protein alsotended to be higher in CS“primary bronchial fibroblastsafter ESW treatment but not in COPD“primary bronchialfibroblasts ese diï¬erences in the response to ESWchallenge of COPD“ and CS“primary bronchial fibroblastsmay in part be due to the reduced proliferation capacity ofthese cells derived from COPD lungs as previously reported [ ] In our welldiï¬erentiated ESWexposedfibroblasts we interpret the increment of y1 protein NFκBp65 “ˆ† Ct012345T24T48T72T0COPD CNCOPD ESW NFκBp65 “ˆ† Ct01234T24T48T72T0CS CNCS ESW NFκBp65 “ˆ†ˆ† Ct000510152025T24T48T016HBE CN16HBE ESW00805026820020907045NFκBp65 pgmL0050000100000150000T72 CNT72 SWT24 SWT48 CNT48 SWT0 SWT24 CNT0 CNCOPD CNCOPD ESWNFκBp65 pgmL036510427702233003830020000400006000080000100000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CNT72 SWT72 CNCS CNCS ESWNFκBp65 pgmL0015500002062865000100001500002004006008001000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CN16HBE CN16HBE ESW 0cCanadian Respiratory JournalFigure Photomicrographs showing thyroid transcription factor1 TTF1 expression panels a b cKit CD117 c d and proliferatingcell nuclear antigen PCNA e f in the peripheral lung tissue of a representative patient with chronic obstructive pulmonary diseaseCOPD Arrows indicate positively stained cells mainly located in the alveolar septa Bars � micronsafter ESW treatment”like that of cKit”as a proproliferative stimulus induced by the treatmentWe found increased levels of secreted TGF1 inCS“primary bronchial fibroblasts h after ESW stimulation TGF signaling pathways are involved in the regulationof many cell functions and in the maintenance of cellularhomeostasis [] We recently reported a decrease of TGF1and TGF3 in bronchiolar epithelium and alveolar macrophages of COPD patients compared with CS [] and thisdecrease may favor the increase of autoimmunity responsesin these patients [] We speculate that the inductionthrough ESW challenge of an increase of TGF in bronchialfibroblasts may play a role in the TGF repositioning andgain in homeostatic function of this important protein in thelungs of COPD patientsTGF induced extracellular matrix and procollagen1production has been reported in pulmonary fibroblasts[] even though it was also reported that the increase ofprofibrotic markersincluding procollagen1 in humanlung fibroblasts may be NLRP3 ‚ammasome dependentand TGF independent [] and associated with increased‚ammation ofthe lung [] We here observed aTTF a0Lung COPDCD a0Lung COPDPCNA a0Lung COPDabcdef 0cCanadian Respiratory JournalFigure Photomicrographs showing alveolar type II epithelial cells TTF1 cells red color coexpressing cKit CD117 brown color ab and PCNA brown color c d in the peripheral lung tissue of a representative patient with COPD Positive doublestained cells can berecognized in the alveolar septa even though their presence was only rarely observed Arrows indicate positively stained cells located in thealveolar septa Bars � micronstransitory increase of procollagen1 protein in COPD“primary bronchial fibroblasts at h after ESW

Thyroid_Cancer

"metastasis is a major cause of death in cancer patients new antimetastatic strategies are needed to improvecancer therapy outcomes Numerous pathways have been shown to contribute to migration and invasion ofmalignant tumors Aspartate hydroxylase ASPH is a key player in the malignant transformation of solid tumorsby enhancing cell proliferation migration and invasion ASPH also promotes tumor growth by stimulation ofangiogenesis and immunosuppression These effects are mainly achieved via the activation of Notch and SRCsignaling pathways ASPH expression is upregulated by growth factors and hypoxia in different human tumors andits inactivation may have broad clinical impact Therefore small molecule inhibitors of ASPH enzymatic activity havebeen developed and their antimetastatic effect confirmed in preclinical mouse models ASPH can also be targetedby monoclonal antibodies and has also been used as a tumorassociated antigen to induce both cluster ofdifferentiation CD and CD4 T cells in mice The PAN3011 vaccine against ASPH has already been tested in aphase clinical trial in patients with prostate cancer In summary ASPH is a promising target for antitumor andantimetastatic therapy based on inactivation of catalytic activity andor immunotherapyKeywords ASPH Small molecule inhibitor Metastasis ImmunotherapyBackgroundCancer is a multifactorial disease with an approximate million fatalities in Worldwide it is the secondleading cause of death [] The complex modifications inthe genome affected by the interactions between hostand environment lead to cancer development and progression Despite advancements in characterizing themolecular mechanisms of oncogenesis tumor progression and metastasis [] delayed cancer detection limitedsurgical options therapeutic resistance and tumor recurrence are serious obstacles in decreasing the prevalence and fatality rate of cancer Since metastasis is theprimary cause of deaths from cancer the design oftherapeutictarget mechanisms oftumorcell migration and invasiveness is essential In thisapproachesthat Correspondence smahelmnaturcunicz1Department of Genetics and Microbiology Faculty of Science CharlesUniversity BIOCEV Vestec Czech RepublicFull list of author information is available at the end of the regard a growing number of investigations of signalingpathways involving products of oncogenes and tumorsuppressor genes in human carcinomas has helped toelucidate the mechanisms underlying malignant transformation of cells and facilitated the development ofnew and more efficient therapeutic methodsAspartate hydroxylase ASPH has been identified asone of the cell surface proteins associated with malignant transformation of tumor cells [ ] ASPH belongsamong the most important biological targets to controlmigration and invasion of tumor cells as its overexpression has been observed in “ of human solid tumors [“] The overexpressed ASPH is transportedfrom the endoplasmic reticulum to the plasma membrane which results in exposure of the Cterminal regionto the extracellular environment where it is accessible toantibody binding Recently molecular targeted therapyhas been developed against this target using small molecule inhibitors SMI that can inhibit the catalytic site The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKanwal Journal of Experimental Clinical Cancer Research Page of in the Cterminal region Moreover as antigenic epitopes that reside on the ASPH protein can efficientlystimulate cluster of differentiation CD and CD8 Tcell responses unique to tumor cells harboring ASPHthis enzyme can be used as a tumor associated antigenTAA in immunotherapy [ ]ThedioxygenasesStructure of the ASPH gene and isoformsASPH is a type II transmembrane protein of approxito the family of αmately kDa that belongsketoglutaratedependenthydroxylated products of ASPH hydroxylation were firstdetected in blood coagulation proteins [“] ASPHwas initially identified in the bovine liver as an enzymeresponsible for catalyzing the hydroxylation of aspartyland asparaginyl residues in calcium binding epidermalgrowth factor cbEGFlike domains of various proteins[] Fig Thereafter the human ASPH gene wascloned and characterized [] This gene spanning base pairs long region of genomic DNA and containing exons is located at the position q123 of thehuman chromosome The ASPH sequence is highlyconserved in mammalian evolution The sequence of thehuman protein is from about identical to the sequences of rat and mouse analogs and the catalytic siteis quite conserved among proteins of these three species[] The whole ASPH protein consists of five domainsan Nterminal cytoplasmic a universal transmembranea positively charged luminal a calcium binding and a Cterminal catalytic domain [] Tissue specific transcription is directed from two putative promoters P1 and P2which differ in their regulation sequences [ ] Whilethe transcription from the P1 promoter was observed inmost human tissues the P2 promoter is activated by thecalciumdependent transcription factor myocyte enhancer factor MEF2 particularly in muscle tissues []The ASPH gene undergoes extensive alternative splicingresulting in four protein isoforms ie ASPH humbugjunctate and junctin [ ] These proteins vary in theCterminal region which affects their function [ ]The two longest ASPH transcript variantsthat aretranscribed from the P1 and P2 promoters and differ inthe length of the ²untranslated region encode the fulllength ASPH protein This protein contains the catalyticCterminal domain that catalyzes the posttranslationalhydroxylation in the cbEGFlike domains of numerousproteins Supplementary Fig including receptors receptor ligands and extracellular adhesion moleculesthat influence cell motility and invasiveness [ ] Thetruncated isoforms humbug junctate and junctin sharethe Nterminal part with the ASPH protein but lackcatalytic function They are involved in calcium homeostasis [] Humbug has a potential role in cell adhesionand calcium flux and similar to ASPH its overexpressionhas been correlated with aggressive tumorcell behavior[]reticulummembranebound protein that is known for its functionin the regulation of the intracellular Ca2 concentrationJunctin is a structural membrane protein and as an integral part of the complex consisting of the ryanodine receptor calsequestrin and triadin influences calciumrelease from the sarcoplasmic reticulum [ ]sarcoendoplasmicJunctateisaLocalization in cells tissue distribution andexpression regulationASPH is predominantly a cellsurface protein [] that isalso localized in the endoplasmic and sarcoplasmicreticulum [] Furthermore a recent study identifiedmitochondriallocalization of ASPH in hepatocellularcarcinoma HCC In that study ASPH overexpressioncorrelated with an instability of mitochondrial DNA andmitochondrial dysfunction that may lead to more aggressive pathological outcomes in HCC []ASPH is abundantly expressed in proliferating placental trophoblastic cells [ ] and in decidua and endometrial glands [] and has a potential role in placentalimplantation and fetal growth [] On the contrary theASPH expression in normal adult tissues is relativelylow or negligible However ASPH expression is inappropriately activated during oncogenesis when ASPH is required for generation of malignant and metastaticphenotypes The elevated expression of ASPH at bothFig ASPH catalytic reaction Aspartyl and asparaginyl residues in cbEGFlike domains are hydroxylated 0cKanwal Journal of Experimental Clinical Cancer Research Page of transcription and translation levels has been shown in awide range of transformed cell lines as well as humancarcinoma tissues including hepatocellular pancreaticcolon prostate lung breast ovarian and cervical carcinoma cholangiocarcinoma neuroblastoma and gastriccancer Table The first study that demonstrated thesignificantly higher expression of both ASPH mRNAand protein in HCC and cholangiocarcinoma relative totheir normal adjacent tissue counterparts was by Lavaissiere [] Subsequently they verified the role of upregulated ASPH protein production and its enzymaticfunction in the malignant transformation on biliary epithelium the NIH3 T3 cell line and animal models []The level of ASPH also correlated with cell motility andinvasiveness in in vitro experiments [ ] In thestudy by Maeda [] the overexpression of theASPH protein was in accordance with worse clinical andhistopathological characteristics of the intrahepatic cholangiocarcinomas and prognosis of patients Similar findings were obtained in other studies for hepatocellular[ ] nonsmall cell lung [] and colon carcinomas[] and glioblastoma multiforme [] Recentlytheprognostic significance of 2oxoglutaratedependent oxygenase expression was demonstrated by analysis of expression profile datasets of tumor samples and nontumor samples ASPH has been identified asone of the genes which upregulated expression couldserve for risk stratification of patients with cancertypes [] In glioblastoma the prognostic significance ofASPH was suggested by profiling of alternative mRNAsplicing []ASPH gene expression is upregulated via Wntcatenin [] and insulininsulinlike growth factor IGF1insulin receptor substrate IRS1 signaling [ ]through extracellular signalregulated kinaseERKmitogenactivated protein kinase MAPK andphosphatidylinositol3kinaseprotein kinase B PI3KAkt pathways Fig for review see ref [] InsulinIGF1IRS1 signaling affects cell growth and survival andcan be involved in oncogenesis in various human tumorsTable Summary of the studies which have identified the elevated ASPH expression in human tumor tissuesStudyPositive cases of studied samples nnTumor tissuesDetectionmethodIHCAntibody recognized region ofASPH proteinFB50 Ab NterminusFB50 Ab NterminusFB50 Ab NterminusFB50 Ab NterminusFB50 Ab Nterminus or 15C7 Abcatalytic domainFB50 Ab NterminusmAb G3 hybridomaPolyclonalFB50 mAb NterminusIHCIHCIHCIHCRTqPCRIHCRTqPCRIHCIHCIHCRTqPCRIHCFB50 Ab NterminusLavaissiere []HepatocellularCholangiocarcinomaBreastColonPalumbo []Pancreatic adenocarcinomaSepe et al[]Primitive neuroectodermalmedulloblastoma neuroblastomaMaeda et al[]Cantarini []CholangiocarcinomaHepatocellularMonte et al[]HepatocellularYang et al[]Wang et al[]Dong et al[]Tang et al[]Lin et al[] types of tumor tissuesaHepatocellularPancreatic cancerHepatocellularBreast 75fold higher level of mRNAcompared to normal tissue 7fold higher level of mRNAcompared to normal tissuePancreatic ductal adenocarcinomaOgawa []aLiver kidney breast cervical ovarian Fallopian tube laryngeal lung thyroid pancreatic thymic prostate bladder esophagus gastric gall bladder colon andrectum cancer and cholangiocarcinomaFB50 Ab NterminusIHC 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig Regulation of ASPH expression and ASPH involvement in signaling pathways The expression of the ASPH protein can be regulated atseveral levels The ASPH gene can be amplified in tumor cells and its transcription activated by INIGF1 and Wnt catenin pathways or inducedby hypoxia At the posttranscriptional level miR200a and miR135a can downregulate ASPH expression Stability of the ASPH protein can bereduced by phosphorylation with GSK3 Conversely ASPH can enhance GSK3 activity by inhibition of its phosphorylation with AKT and p38kinases ASPH also supports cell proliferation epithelialmesenchymal transition migration invasion and angiogenesis and consequently tumrowth and metastasis by hydroxylation of the Notch receptor and ligands ex JAG and interaction with pRb vimentin and ADAMs Finallyinactivation of NK cells by ASPH has been demonstrated Green arrow activation signal red bar inhibitory signal[] The catenindependent Wnt pathway regulatescell proliferation motility and differentiation and is oneof the most frequently modified pathways in humanmalignancies Upon aberrant activation of Wnt signalingcatenin is accumulated in the cytoplasm and subsequently translocated to the nucleus [] where an interaction between catenin and Tcellfactorlymphoidenhancerbinding factor TCFLEF proteins forms atranscriptional regulatory complex which enhances theexpression of Wnt target genes including IRS1 []ASPH was proposed as a common link between Wntcatenin and insulinIGF1IRS1 pathways and downstream signaling []The regulation of ASPH gene expression in tumorsmight also be affected by a copy number variation Inthe study by Kadota [] the ASPH gene locus hasbeen identified as one of the DNA regions with focalamplification in primary breast cancer In colorectal cancer ASPH gain or amplification was found in ofsamples [] Next a suppressant role of the microRNAmiR200a in posttranscription regulation of the ASPHexpression in hepatoma cells has been found [] MiR200a belongs to miR200 family which plays significantrole in preventing cancer initiation and metastasis forreview see ref[] Similarly miR135a has beenshown to suppress ASPH in endometrial cancer []Moreover consistent with the protein sequence analysis that recognized numerous prospective phosphorylation sites of glycogen synthase kinase3 GSK3casein kinase CK2 protein kinase A PKA and protein kinase C PKC on ASPH [] several studies demonstrated that phosphorylation can regulate the ASPHprotein expression [ “] Inhibition of the GSK3activity did not modify mRNA expression but increased 0cKanwal Journal of Experimental Clinical Cancer Research Page of the ASPH protein level [] Direct phosphorylation ofASPH by GSK3 probably decreases ASPH stability andthus reduces cell mobility [] ASPH protein expressionwas also increased by inhibitors of PKA PKC and CK2[] Mutational analysis of potential sites of phosphorylation demonstrated complex and nonuniform effects ofASPH phosphorylation on protein expression enzymaticactivity and subcellular localization [ ] ThereforeASPH phosphorylation probably regulates the functionof this protein by various mechanismsASPH expression can also be regulated by hypoxia andoxidative stress In human neuronal cells this effect wasmediated by hypoxia inducible factor alpha HIF1αthat is stabilized under hypoxiaoxidative stress whenthe prolyl hydroxylase domain PHD proteins and factorinhibiting HIF FIH are inactivated Consequently theHIF1 heterodimer made up of subunits HIF1α andHIF1 functions as a transcription factor likely enhancing ASPH expression by binding to hypoxiaresponsiveelements [] In hypoxic regions of glioblastoma bothHIF1α and ASPH were highly expressed particularly inmore aggressive mesenchymal subtype of glioblastomasuggesting a possible involvement of ASPH in mesenchymal transition [] Brewitz showed reducedASPH hydroxylation activity at low oxygen concentrations and suggested an ASPH role in oxygen hypoxiasensing ASPH upregulation induced by hypoxia couldcompensate for reduced enzymatic activity [] Moreover a recent study reported an oxidative stress state ofthe castrationresistant prostate cancer cells upon ASPHoverexpression which was reversed by silencing ASPHexpression or generating hypoxic conditions resulting inimpaired cell proliferation and invasion []ASPH protein interactions and signaling pathwaysThe ASPH hydroxylation consensus sequence is confined within cbEGFlike domains that are found in proteins of diverse function including Notch receptors andligands clotting factors structural proteins of the extracellular matrix and ligands of the tyro3Axl family ofreceptor tyrosine kinases []The Notch signaling cascade is a remarkably conservedpathway Notch proteins Notch1 Notch4 are singlepasscell surface receptors that mediate communication betweencells and their expression is crucial for proper embryonicdevelopment [] Notch signaling mainly results in cell differentiation but also plays a significant role in proliferationapoptosis and the maintenance and selfrenewal of stemcells Dysregulation of the Notch pathway is directly linkedto cancer vascular disorders and congenital defects [] In mammals Notch signaling activated by binding ofone of two families of canonical Notch ligandsjaggedJAG1 and JAG2 and delta like DLL1 DLL3 and DLL4leads to the generation of the cleaved Notch intracellulardomain NICD fragment and its nuclear translocation Inthe nucleus the NICD fragment interacts with the DNAbinding complex CSL CBF1RBPjκ SuH Lag1 Thiscomplex is then converted from a repressor into an activator leading to increased transcription of target genes suchas hes family bHLH transcription factor HES1 HESwith YRPW motif HEY1 CD44 epithelial cell adhesionmolecule EPCAM cmyc protooncogene matrix metallopeptidase MMP29 cyclin D1 cyclooxygenase vascular endothelial growth factor VEGF and proliferatingcell nuclear antigen PCNA [ ]Upregulation of ASPH results in enzymatic modification of the cbEGFlike repeats in the Notch receptorextracellular domain and its ligands which promotes thereceptor interaction with the ligands and the activationof Notch signaling [ ] Furthermore the interactionof ASPH with a disintegrin and metallopeptidase domainADAM stabilizes this complex and enhances theS2 cleavage ofthe Notch receptors and subsequentNICD fragment release [] The activation of the targetgenes in malignant cells increases cell proliferation migration and invasion []through the epithelialtomesenchymal transition EMT that is probably upregulated by the interaction of ASPH with vimentin []Consequentlythis activation supports tumor growthand metastasis The ASPHNotch axis also stimulatesthe release of exosomes that transfer proteins involvedin invasion metastasis metabolism and immunosuppression [ ]The SRC kinase pathway is another important pathwayin malignant cell transformation that regulates a complex signaling network promoting angiogenesis invadopodia formation and maturation and metastasis []ASPH has been identified as an SRC pathway activatorOverexpressed ASPH directly interacts with ADAM12 and strengthens the SRC activation by these proteinswhich promotes MMPmediated extracellular matrixdegradation and tumor invasiveness []ASPH can also contribute to malignant phenotype ofcells by interaction with other proteins Iwagami et alrevealed the interaction of ASPH with GSK3 that prevents GSK3 inactivation by phosphorylation with upstream kinases [] This mechanism was confirmed ina castrationresistant prostate cancer model [] GSK3 is a multifunctional kinase that is involved in variousprocesses including glycogen metabolism cell divisionand cell fate determination Some types of tumors aresensitive to GSK3 inhibitors [] Recently Huang elucidated a direct binding of ASPH with retinoblastoma protein pRb leading to pRb phosphorylation[] They also showed that this effect was mediated byincreased binding of cyclindependent kinase CDK CDK4 and cyclins D1 and E with pRb and wasdependentAsASPH enzymaticonactivity 0cKanwal Journal of Experimental Clinical Cancer Research Page of phosphorylation of pRb inactivates its tumorsuppressorfunction ASPH can contribute to the progression of cellcycle via interaction with pRbEffect of ASPH on an immune systemTumor generation and progression are influenced bycancer immunoediting that involves immunosurveillanceand escape from a host immune system [] In theseprocesses various mechanisms of both innate and adaptive immunity are included [] Immune cells that infiltrate developing tumors are initially antitumorigenicbut in tumor microenvironment they can be modifiedinto cells with protumorigenic properties []As potential targets of ASPH hydroxylation are alsoexpressed on immune cells this enzyme could affect thefunction of immune system particularly in tumor microenvironment when ASPH is overexpressed on cancercells Indeed such effect was demonstrated for humannatural killer NK cells by using recombinant ASPHwhich reduced viability and cytotoxicity of these cells viaenhancing caspase signaling and decreasing the surfaceexpression of activating receptorsrespectively []Antibodies against ASPH inhibited these effectsInteraction of ASPH with other immune cells has notbeen studied However we suppose possible influence ofASPH on different tumorinfiltrating cells This assumption comes from the involvement of Notch signaling indifferentiation and function of various immune cells fibroblasts mesenchymal cells and endothelial cells Forinstance Notch activation contributed to stimulation ofproinflammatoryantitumorigenic M1 polarization inboth bone marrowderived primary macrophages [] and tumorassociated macrophages[] WhenNotch signaling was abrogated protumorigenic M2polarization was induced even by stimulators of M1polarization [] miR125a has been identified as adownstream mediator of Notch signaling in macrophages [] Similarly the Notch pathway plays an important role in differentiation of other types of myeloidcells and probably all subsets of CD4 and CD8 T cells[] Different Notch receptors and their interactionwith different ligands contribute to these processes []Moreover noncanonical Notch signaling is implicatedin regulation of immune cells [] While activation ofNotch signaling in some cells eg T helper cells cytotoxic CD8 T cells and M1 macrophages supports induction ofincluding antitumorimmunity in other cells particularly regulatory T cellsit leads to immunosuppression [] Thus immunostimulatory effect of Notch signaling is often inhibited intumor microenvironment to enable the tumor cells toescape from the host immunity [] Therapeutics affecting Notch signaling in malignant diseases are being developed and tested in clinical trials but their effects onimmune reactionsimmune reactions and possible combination with immunotherapy have not been properly studiedASPH as a therapeutic targetOncogenic abilities of ASPH have been experimentallydemonstrated using tumor cell lines and mouse and ratmodels of different types of human tumors with ASPHoverexpression including cholangiocarcinoma [ ] hepatocellular carcinoma [ ]neuroblastoma [] pancreatic cancer [ ] glioma [] breast carcinoma [] castrationresistant prostatecancer [] and colorectal cancer [] In studies analyzingASPH function various approaches were utilized to revealsignaling pathways affected by ASPH Particularly ASPHexpression was diminished by using small interfering RNAs[ ] short hairpin RNAs [ ] or theCRISPRCas9 system [ ] The importance of ASPHenzymatic activity in these processes was shown by the sitedirected mutagenesis [ ] or treatment by SMIs [ ] In vitro assays showed ASPH involvement in cell proliferation migration and invasion Cellularalterations included EMTinhibition of apoptosis andstemness acquisition Tumor growth and invasivenesscould further be supported by ASPHinduced extracellularmatrix degradation angiogenesis and transendothelial migration Notch and SRC signaling are probably major pathways influenced by ASPH Fig and contributing toincreased aggressiveness of tumor cells that was verified inin vivo models Thus these studies also demonstrated thatASPH is a suitable target for cancer treatment especially bySMIs or immunotherapySmall molecule inhibitorsSMIs of ASPH Fig have been developed and used totest the role of ASPH in a wide range of cancer modelsincluding subcutaneous orthotopic and patient derivedxenograft in vivo models [ ] A small orallybioavailable inhibitor has severalintrinsic advantagesover immunotherapy approaches Not only can these inhibitors inhibit the catalytic activity of ASPH unlike conventional antibodies that simply bind to the protein butthey can also penetrate into the cell and inhibit ASPHcatalytic activity in the endoplasmic reticulum Differentcancers have different ASPH expression patterns andwhile surface expression is quite common in pancreaticcancer and hepatocellular carcinoma intracellular overexpression patterns have also been observed []The first ASPH SMIs published were the tetronimidesMOI500 and MOI1100 Tetronimides were originallysynthesized in by Dahn [] and are redoxactivemimics of ascorbic acid and 2oxoglutarate MOI500 isa mixed inhibitor that inhibits both ASPH and the FatMass and Obesity protein FTO [] and is not onlyorally bioavailable but also can penetrate the blood 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig Small molecule inhibitors of ASPHthereand kinasesbrain barrier MOI1100 is a more potent inhibitor ofASPH and is also more selective [] Despite investigation against a wide range ofirondependent dioxygenasesare no other knownenzymatic targets for MOI1100 Enhanced activity wasobserved by replacing the chlorine with a trifluoromethylgroup [] as in MOI1151 and even a greater improvement in in vivo activity was found by replacing the trifluoromethyl group with a carboxymethyl group as inMOI1182 although it is not yet clear if the nature ofthis enhancement is due to increased inhibitory activityorenhanced solubility parameters MOI1182 isreported to have the ability to suppress invasive activityat a concentration of nM [] SMIs of ASPH have acharacteristic in vitro concentration dependent profilewhere the activity of the SMI plateaus at value around viability [] emphasizing the noncytotoxic properties of this class of inhibitorsNatural products and inhibitors of other enzymes thathave been repurposed as ASPH inhibitors have alsorecently been reported in the patent literature includingbosutinibcepharanthineCN2019101414327 and guaianolides related to nortrilobolideCN2019104575886CN2019101414219CN2019101414187 0cKanwal Journal of Experimental Clinical Cancer Research Page of sesquiterpene with complex anticancerBosutinib is a wellknown inhibitor of BCRABL and SRCtyrosine kinases approved for the treatment of chronic myelogenous leukemia [] Cepharanthine is a natural productactivityincluding AMPactivated protein kinase AMPK activationand nuclear factor kappa B NFκB inhibition [] Nortrilobolide and related compounds are reported to be potentcytotoxic agents with subnanomolar sarcoendoplasmicreticulum calcium ATPase SERCA inhibition [] Recently a family of potent pyridine dicarboxylates have alsobeen published [] utilizing a mass spectrometrybasedinhibition assay [] These compounds are related toknown irondependent dioxygenase inhibitors 23pyridinedicarboxylate 24pyridine dicarboxylate and 26pyridinedicarboxylate The synthesized pyridine dicarboxylateswere assayed for activity against a range of other enzymesto include PHD2 FIH and lysinespecific demethylase 4EKDM4E in addition to ASPH with varying degrees of selectivity However while cellbased activities have not beenevaluated the dicarboxylate nature of the compounds maybe useful for cell surface ASPH inhibitors that may nothave cell penetrating activity []immunity As a target of humoralImmunotherapyASPH can be used not only as a target of the inhibitors inactivating its enzymatic activity but also as a target of immune reactions leading to destruction of tumor cells andtumor growth suppression Since ASPH is cell surface displayed on tumor cells it represents a tumorassociatedantigen that can be targeted by both cellmediated andhumoralimmunityASPH on the surface of cancer cells can be bound by antibodies that mediate antibodydependent cellular cytotoxicity ADCC complement dependent cytotoxicity CDCor antibodydependent cellular phagocytosis ADCP []When the ASPH antigen is processed in tumor cells orantigen presenting cells antigenic peptides are presentedon these cells by human leukocyte antigen HLA class Ior class II molecules and recognized by CD8 or CD4 Tlymphocytes respectively [] that can be stimulated byimmunization breaking tolerance to selfantigens []Induction of ASPHspecific CD4 and CD8 T cells wasexamined in blood samples of HCC patients Using synthetic peptides derived from ASPH after prediction of HLAclass I and HLA class IIrestricted epitopes it has beenfound that ASPH is a highly immunogenic protein that activates both types of analyzed T cells [] Thus efficientantitumor reactions could be stimulated by immunizationThe first vaccine against ASPH was based on matureddendritic cells DC loaded with the ASPH protein andtested in an orthotopic rat model of intrahepatic cholangiocarcinoma [] This study showed that vaccinationstimulated cytotoxicity against cancer cells in an in vitroassay and decreased tumor growth and metastasis BothCD8 and CD4 cells contributed to an antitumor effectinduced in a mouse model of HCC by immunizationwith ASPHloaded DCs []The next antiASPH vaccine was based on a bacteriophage lambda display system The viral capsid proteingpD was fused with the N or Cterminus of ASPH andimmunogenicity ofthese nanopforming constructs was verified in two mouse tumor models []The vaccine PAN3011 containing these constructs hasalready been examined in a phase clinical trial in patients with biochemically relapsed prostate cancer[] This study demonstrated safety and immunogenicity of PAN3011 and indicated an antitumor effect interms of the reduction of prostate specific antigen PSAor PSA doubling time ASPHspecific immune responseswere mediated by both antibodies and T lymphocytesAs ASPH is a type II transmembrane protein its Cterminus carrying the enzymatic domain is exposed outside cells and can be bound by antibodies that can be usedfor diagnostic and therapeutic purposes Development ofASPHspecific antibodies has been described in several s [“] The human IgG1 PAN622 recognizesthe catalytic domain of ASPH This antibody is not directly cytotoxic for tumor cells but is internalized and candeliver cytotoxic moieties into cells [] In the subsequent study with a mouse model of metastatic breast cancerandradioimmunotherapy with promising results [] MouseIgG1 monoclonal antibody binding to the CterminalASPH domain mediated ADCC by human NK cells []Recently a secondgeneration antibody approach hasbeen disclosed The prepared antibody binds to the extreme Cterminus of ASPH US that is involved in specific substrate recognition [] Thereforethis antibody has direct ASPH inhibitory activity anddoes not require any radioisotope or cytotoxic payloadfor potential therapeutic activityPAN622 wasbioimagingusedforConclusionsASPH is an important enzyme in malignant transformationof cells It stimulates tumor cell proliferation migration andinvasion but it can also affect other cells in tumor microenvironment Two main pathways Notch and SRCthrough which ASPH promotes the tumor growth havebeen identified It has also been shown that ASPH expression is induced by some growth factors and hypoxia and isregulated at various levels The overexpression of ASPHand its downstream targets has been detected in numeroushuman malignancies Since ASPH is not expressed in appreciable level in normal adult tissues and the catalytic domain is localized on the cell surface it has been proposedas one of the most exciting potential therapeutic targetsFig Small inhibitory molecules orally bioavailable havebeen developed and successfully tested in several cancer 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig ASPH as a therapeutic target ASPH expression is upregulated by growth factors and hypoxia Its enzymatic activity can be inhibited bySMIs or monoclonal antibodies which results in reduction of cell proliferation angiogenesis immunosuppression and cell migration and invasionConsequently tumor growth and metastasis are also reduce

Thyroid_Cancer

"Follicular dendritic cell sarcoma FDCS is a rare mesenchymal tumor that mostly occurs in systemiclymph nodes FDCS in the uterine cervix has not yet been reportedCase presentation A 49yearold woman was referred to our department with a cervical tumor which washistologically suspected to be undifferentiated carcinoma She underwent hysterectomy salpingooophorectomyand pelvic lymphadenectomy after neoadjuvant chemotherapy with paclitaxel and carboplatin The resectedspecimen contained high numbers of spindle cells and was immunohistochemically confirmed to be FDCS Thetumor was completely resected and recurrence was not detected at a 16month followupConclusion FDCS is an extremely rare malignant tumor in the uterine cervix and an accurate diagnosis andcomplete resection are essential for a good prognosisKeywords Follicular dendritic cell sarcoma Cervical cancerBackgroundFollicular dendritic cell sarcoma FDCS is a rare mesenchymal tumor that was initially reported in []Three types of tumors are derived from dendritic cellsFDCS derived from follicular dendritic cells that presentantigens to B lymphocytes in lymph follicles [] interdigitating dendritic cell sarcoma derived from the Tcellzones of lymphoid ans such as the paracortex anddeep cortex of the lymph nodes and fibroblastic reticular cell sarcoma derived from a reticular network oflymphoid ans [] An accurate diagnosis is challenging without an appropriate series ofimmunohistochemistry therefore some tumors may be diagnosed asundifferentiated carcinoma The accuracy of a diagnosisinfluences the prognosis of patients because of the lowresponse rate to established chemotherapy and radiotherapy [“] Correspondence myoshihara1209mednagoyauacjp2Department of Obstetrics and Gynecology Nagoya University GraduateSchool of Medicine Tsurumacho Showaku Nagoya Aichi JapanFull list of author information is available at the end of the Although previous studies described FDCS in the cervical lymph nodes liver stomach and tonsils FDCS inthe uterine cervix has not yet been reported We hereinpresent a 49yearold woman diagnosed with FDCS inthe uterine cervix which was successfully treated bycomplete surgery without postoperative adjuvant therapybased on a precise pathological diagnosis This is thefirst case of FDCS arising from the uterine cervix andwe described the time course of this patient from herinitial admission to diagnosis and treatment We alsoemphasized the importance of considering FDCS as adifferential diagnosis for cervical tumors because an accurate diagnosis and complete resection are essential fora good prognosisCase presentationA 49yearold woman gravida para with abnormalgenital bleeding was referred to our department with acervical mass She had a previous history of uterine myomectomy and cervical polypectomy which were both benign diseases Pelvic magneticimagingrevealed a — mm solid tumor developing from theresonance The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cNakamura BMC Women's Health Page of findingsrevealed aIntraoperativeand carboplatin AUC5 mgbody Massive genitalbleeding occurred months after the admission therefore the patient underwent extended total hysterectomybilateral salpingooophorectomy and pelvic lymphadenectomysmallamount of bloody ascites and the resected specimen hada necrotic mass at the posterior side of the cervix Fig Tumor cells exhibited the same histopathological characteristics as those in the previous biopsy Fig 3a Additionalimmunohistochemistry revealed cells that werepositive for both CD68 and FDC Fig 3b which wasconsistent with the diagnosis of FDCS The tumor wascompletely resected and lymphovascular invasion wasnot detected There was also no evidence of lymph nodemetastasis She did not receive adjuvant therapy and herCA19“ level decreased to within almost normal limitsTable Tumor recurrence was not detected at the month followup after admission The consent of the patient for publication was recorded according to the Ethics Committee of Nagoya University and the principlesof the Declaration of HelsinkiDiscussionThis is the first case of FDCS in the uterine cervix Afterreported surgery and chemotherapy we reached a finaldiagnosis Our experience demonstrates the difficultiesassociated with accurately diagnosing FDCS due to alack of familiarity with the pathologies of rare tumorsincluding sarcoma Although FDCS in the gynecologicalsystem is markedly rarer than other carcinomas it needsto be considered as a differential diagnosis because of itspotentiallyappropriatetreatmentprognosiswithoutfatalFDCS has been reported in approximately casesworldwide with an age range of to years and amalefemale ratio of The primary lesion of FDCS isFig Surgical specimen of the tumor The tumor with a necroticlesion arose from the posterior side of the cervix arrowheadsFig Magnetic resonance imaging of the patient at the initialpresentation A solid tumor developing from the posterior of theuterine cervix was confirmed arrowheadsposterior of the uterine cervix Fig and — and — mm spaceoccupying lesions were also detectedaround the rectus which were suspected to be lymphnode metastases Chestabdominal computed tomography CT showed no enlarged lymph nodes or distantmetastasis except for the tumors already detected Positron emission tomographyCT revealed the accumulation offluorodeoxyglucose maximum standardizeduptake value at the cervical tumor only Serumtumor marker levels were as follows carcinoembryonicantigen ngml cancer antigen125 UmLcancer antigen199 Uml squamous cell carcinomaantigen ngml soluble interleukin2 receptor Uml The other results of the blood examination including renal function liver enzymes and electrolyteswere within normal limitsBiopsy of the cervical tumor was performed and ahistopathological examination indicated that the tumorhad atypically spindle ovoid and polygonal cells with aneosinophilic hyalinerich cytoplasm with whorled andcordlike forms Nuclei were oval to round and had mildatypia Inflammatory cells were scattered to various degrees around tumor cells The results of immunohistochemical staining were as follows cytokeratin AE1AE3positive epithelial membrane antigen EMA negativeCAM52 negative cytokeratinMNF116 positive S100negative vimentin positive actin negative desminnegative cluster of differentiation CD negative CD8negative CD10 negative CD5 negative CD20 negativeCD79a negative PgR PgR inhibin negative thyroid transcription factor1 negative EpsteinBarr virusencoded RNA in situ hybridization negativeSince the tumor was suspected to be undifferentiatedcarcinoma of the uterine cervix the patient receivedchemotherapy with four cycles of paclitaxel mgm2 0cNakamura BMC Women's Health Page of Fig The histopathology of the cervical tumor The tumor had atypically spindle ovoid and polygonal cells with an eosinophilic hyalinerichcytoplasm with whorled and cordlike forms Nuclei were oval to round and had mild atypia a Tumor cells were positive for FDC bmostly in the lymph nodes in the neck axilla and mediastinum The remainder of FDCS originate from extralymphatic lesions such as the liver mesenterium stomach smallintestine pharynx tonsils retroperitoneumand ovary [“] however FDCS in the uterine cervixhas not yet been reported in the English literatureDue to the wide range of primary lesions there is noknown specific initial symptom of FDCS some patientsmay exhibit symptoms associated with an increase intumor volume such as lymph node swelling [] Sincethere are no specific findings in blood examinations orimaging studies difficulties are associated with makingan accurate diagnosis prior to the initiation of treatmentThe diagnosis of FDCS is pathologically confirmed usingbiopsy or resected specimens however due to the rarityof this tumor it is important to conduct appropriate immunohistochemistry in consideration of FDCS from thefindings of hematoxylin and eosin staining The tumorcells of FDCS are spindle to epithelioid in shape with thecoexistence of multinucleated cells Single nuclear inflammatory cells infiltrate and form bundles flowers andswirls structures Some giant cells and ReedSternbergcells are also detected in these tumors To reach a definitive diagnosis of FDCS FDC markers such as CD21CD35 KiM4p and CNA42 are used to distinguish itfrom other tumors that may have a mesenchymal structure such as undifferentiated carcinoma meningiomaand paraganglioma [ ] Alternatively FDCS may befound within lesions of Castleman™s disease particularlyits hyalinevascular type [] It has been reported thatFDCS occurred after the excision of a lesion of hyalinevascular type Castleman™s disease and occupied most ofthe lesions It is also important to distinguish FDCSfrom inflammatory pseudotumorlike FDCS associatedwith EpsteinBarr virus This tumor is often found in theabdominal ans particularly in the liver and spleenand has the characteristics of positive FDCS markersand the detection of EBV by in situ hybridization It progresses more slowly than FDCS and longterm survivalhas been reported even after recurrence []The treatment for cervical cancer principally involvessurgeryradiation and chemotherapy New surgicaltechniques such as laparoscopic radical hysterectomyand sentinel lymph node biopsy have also become available [ ] On the other hand a basic therapeuticstrategy for FDCS is prioritized to guarantee completesurgical tumor resection because of the low responserate to established chemotherapy and radiotherapy [“] Saygin examined patients with FDCS andreported 2year survival rates of and forearlylocally advanced and distant metastasis diseaserespectively Patients who underwent complete tumorresection had a better prognosis than those with unresectable localized tumors Furthermore no prognosticdifference was observed between the surgery group andpostoperative radiotherapy groups A large tumor sizelarger than cm and lymphoplasmacytic cell invasionhave been identified as poor prognostic factors [] InTable Level of tumor markers in each followup periodTumor marker Normal rangeCA125 UmL AdmissionCA199 UmL CEA UmL SCC ngmL At surgery m after admissionAt final followup m after admissionsIL2R UmL Abbreviation CA cancer antigen CEA carcinoembryonic antigen SCC squamous cell carcinoma antigen sIL2R soluble interleukin2 receptor 0cNakamura BMC Women's Health Page of the present case as the tumor remained localized andwas completely resected the patient could follow favorable clinical course without any tumor recurrenceConclusionFDC sarcoma is a rare tumor that may develop in theuterine cervix FDCS needs to be considered when confirming a mesenchymal cervical tumor and appropriateimmunohistochemistry needs to be performed for bothan accurate diagnosis and selection of a therapeuticstrategyAbbreviationsFDC Follicular dendritic cell sarcoma FDCS Follicular dendritic cellCT Computed tomography CD Cluster of differentiationAcknowledgmentsThe authors thank Dr Sakata and Dr Hirata for supporting the managementof the case with patience and knowledgeAuthors™ contributionsTN and MY developed the study concept interpreted the data and wrotethe manuscript ST and HK participated in designing the study and plannedthe investigations FK drafted the study design and supervised all of thework All authors have approved the final manuscriptShia J Chen W Tang LH Carlson DL Qin J Guillem JG Extranodalfollicular dendritic cell sarcoma clinical pathologic and histogeneticcharacteristics of an underrecognized disease entity Virchows Arch “Ruco LP Gearing AJ Pigott R Pomponi D Burgio VL Cafolla A et alExpression of ICAM1 VCAM1 and ELAM1 in angiofollicular lymph nodehyperplasia Castleman's disease evidence for dysplasia of folliculardendritic reticulum cells Histopathology “ Maeda K Matsuda M Suzuki H Saitoh HA Immunohistochemicalrecognition of human follicular dendritic cells FDCs in routinely processedparaffin sections J Histochem Cytochem “Arber D Kamel O van de Rijn M Davis RE Medeiros LJ Jaffe ES et alFrequent presence of the EpsteinBarr virus in inflammatory PseudotumorHum Pathol “ Casarin J Bogani G Papadia A Ditto A Pinelli C Garzon S et alPreoperative Conization and risk of recurrence in patients undergoinglaparoscopic radical hysterectomy for early stage cervical cancer amulticenter study J Minim Invasive Gynecol “ Rossetti D Vitale SG Tropea A Biondi A Lagan  AS New procedures for theidentification of sentinel lymph node shaping the horizon of futureManagement in Early Stage Uterine Cervical Cancer Updat Surg “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsFundingNo funding was obtained for this studyAvailability of data and materialsNot applicableEthics approval and consent to participateNot requiredConsent for publicationWritten consent to publish this information was obtained from studyparticipantsCompeting interestsAll authors declare that there are no competing interestsAuthor details1Department of Obstetrics and Gynecology Anjo Kosei Hospital Anjo AichiJapan 2Department of Obstetrics and Gynecology Nagoya UniversityGraduate School of Medicine Tsurumacho Showaku Nagoya AichiJapanReceived April Accepted August References Monda L Warnke R Rosai J A primary lymph node malignancy withfeatures suggestive of dendritic reticulum cell differentiation A report of cases Am J Pathol “Tew JG Kosco MH Burton GF Szakal AK Follicular dendritic cells asaccessory cells Immunol Rev “Saygin C Uzunaslan D Ozguroglu M Senocak M Tuzuner N Dendritic cellsarcoma a pooled analysis including cases with presentation of ourcase series Crit Rev Oncol Hematol “Pileri SA Grogan TM Harris NL Banks P Campo E Chan JK Tumoursof histiocytes and accessory dendritic cells an immunohistochemicalapproach to classification from the international lymphoma study groupbased on cases Histopathology “Chan JK Fletcher CD Nayler SJ Cooper K Follicular dendritic cell sarcomaClinicopathologic analysis of cases suggesting a malignant potentialhigher than currently recognized Cancer “ 0c"

Thyroid_Cancer

"rebound effect after stopping treatment with denosumab may be associated with rapid loss ofthe gains in bone mineral density achieved with treatment high levels of bone remodeling markers the occurrenceof vertebral fractures and even hypercalcemiaCase presentation A 64yearold osteoporotic Caucasian woman suffered from a fracture of her second lumbarvertebra in From January she was treated with denosumab for years with good densitometry resultsfor her hip and lumbar areas and no fractures over the last years of treatment Ten months after the treatmentwith denosumab was stopped a cascade of vertebral fractures including some in unusual locations third thoracicvertebra and multiple rib fractures in a context of hypercalcemia suggested possible malignancy A completeevaluation including systemic biological and biopsy analyses ruled out this hypothesis The hypercalcemia wasassociated with normal plasma phosphate and vitamin D concentrations and a high parathyroid hormone levelwith an abnormal fixation of the lower lobe of the thyroid on sestamethoxyisobutylisonitrile scintigraphyHistological analysis of the excised parathyroid tissue revealed hyperplasia The associated thyroidectomy goiterled to the discovery of a thyroid papillary microcarcinomaConclusions We consider the consequences of this rebound effect not only in terms of the major loss of bonedensity return to basal values within years and the multiple disabling fracture episodes but also in terms of thehypercalcemia observed in association with apparently autonomous tertiary hyperparathyroidism Several cases ofspontaneous reversion have been reported in children but the intervention in our patient precluded anyassessment of the possible natural course The discovery of an associated thyroid neoplasm appears to befortuitous Better understanding of the various presentations of the rebound effect after stopping treatment withdenosumab would improve diagnostic management of misleading forms as in this case Bisphosphonates couldpartially prevent this rebound effectKeywords Osteoporosis Denosumab rebound Fracture Hypercalcemia Hyperparathyroidism Correspondence yvesmaugarschunantesfrRheumatology Department Nantes University Hospital place AlexisRicordeau Nantes Cedex France The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMaugars Journal of Medical Case Reports Page of IntroductionSome warning signs need to be explored and are wellknown aftertreatment with denosumab A majorrebound effect after stopping denosumab can be responsible for rapid bone loss with vertebral crushes [“]Some other manifestations have been described such ashypercalcemia in both children [“] and adults []hyperparathyroidism [] and vertebral osteonecrosis[] A suspected increase in the number of cases ofprimary neoplasia has been reported in a recent metaanalysis [] We discuss a new observation with allthese manifestations together which poses diagnosisproblems and several explorations to eliminate a neoplasia This could have been avoided with better knowledge of these rebound manifestationsCase presentationA 64yearold Caucasian woman suffered from a firstvertebral fracture in the second lumbar vertebra L2 in following a fall from her bicycle She did not obtainany treatment Dual Xray absorptiometry DXA relumbar Tscore of ˆ’ standardvealed osteoporosisdeviation SD Our patient™s characteristics during follow up are summarized in Table Her phosphorus andcalcium levels were normal plasma calcium concentration mmoll normal range to mmollparathyroid hormone PTH concentration was normal ngl normal range to ngl and vitamin D levelwas low ngml normal range to ngl Shewas included in the FREEDOM protocol comparingdenosumab mg subcutaneously every monthsplus mg of calcium and IU of vitamin D dailywith placebo for the treatment of postmenopausal osteoporosis in January The unblinding of the trial years later showed that she had been randomized to thedenosumab group Several vertebral fractures occurredTable Patient™s characteristics during follow upduring this 3year period fifth thoracic vertebra T5eighth thoracic vertebra T8 and an aggravation of theL2 fracture She continued to participate in the extension protocol in mode for years and then withdrew of her own volition with a finalinjection ofdenosumab in July there were no new vertebralfractures during this entire period DXA in September demonstrated increased bone mineral densityBMD of in her lumbar region Tscore ˆ’ SD and of in her total left hip Tscore ˆ’ SD She was a former tobacco smoker and her medicalhistory included osteoarthritis of the knee a hiatus hernia hypertensionand colonicpolyps Calcaemia monitoring revealed a return to normal values until January nmoll normal range to mmoll Checkups while our patient wasstill on denosumab yielded values of nmoll in January and in September Fig amlodipineallergyIn May our patient complained of acute intensespinal pain that resisted standard painkillers and required treatment with opiates An evaluation was carriedout in hospital in June Spinal Xrays revealed fractures of the fourth thoracic vertebra T4 wedge grade T5 biconcave grade T8 wedge grade ninththoracic vertebra T9 crush grade tenth thoracic vertebra T10 wedge grade 11th thoracic vertebra T11crush grade first lumbar vertebra L1 biconcave grade L2 wedge grade and third lumbar vertebra L3 biconcave grade Fig Bone scintigraphy revealedhypersignals in all these vertebrae except L2 and T5 andin several ribs Magnetic resonance imaging MRI identified vertebra T4 in hypersignal on a T2weighted sequence and hyposignal on a T1weighted sequence withno signs of infiltration or suspected lysis Fig T9 T10T11 L1 and L3 also showed hypersignal and T5 T8 andL2 were older vertebral fractures with no bone marrowFracturesL2Tscore lumbartotalhip BMD SDˆ’ˆ’CalcaemiammollClinicalpresentationBicycle fallNo painFollow up of thepatient January FREEDOM 3yearevaluationJanuary FREEDOM extensionSeptember T5 T8 and aggravationof L2ˆ’ˆ’No painNo new fractureˆ’ˆ’TreatmentsInitiation denosumab mg semiannuallycalcium g daily vitamin D IU dailyPursuit denosumab mg semiannuallycalcium g daily vitamin D IU dailyAll treatments stoppedNoneZoledronate two infusions of mg annuallyvitamin D IU quarterlyHospitalizationJune Acute intensedorsal painNew evaluationSeptember Chronic dorsaland lumbar painT4 T9 T10 T11 L1 L3ˆ’ˆ’No new fractureˆ’ˆ’ˆ’ˆ’New evaluationAugust BMD bone mineral density L1 first lumbar vertebra L2 second lumbar vertebra L3 third lumbar vertebra SD standard deviation T4 fourth thoracic vertebra T5fifth thoracic vertebra T8 eighth thoracic vertebra T9 ninth thoracic vertebra T10 tenth thoracic vertebra T11 11th thoracic vertebraChronic lumbarpainTreatments stoppedNo new fracture 0cMaugars Journal of Medical Case Reports Page of Fig Calcemia bone mineral density and fracture events for a patient treated with denosumab for years who experienced a rebound effectwhen treatment was stopped with a combination of hypercalcemia related to hyperparathyroidism multiple fractures and rapid bone lossedema Lumbar and dorsal pain remained severe throughout this period of exploration justifying bed rest Biologicaltests revealed hypercalcemia with plasma concentrations of mmoll for calcium normal range to mmoll Fig and mmoll for phosphate normal range to mmoll hypercalciuria mmol24 hoursnormal range to mmol24 hours a 25OH vitaminD3 concentration of ngml normal range to ngla PTH concentration of pgml normal range to ngl a Creactive protein CRP concentration of mglnormal values mgl normal protein electrophoresiswith no Bence Jones proteinuria and a plasma creatinineconcentration of μmollrange to μmoll Blood formula and plasma concentrationsthyroidstimulating hormone TSH parathyroidofhormonerelated peptideand 25OH2D were normal Carboxyterminal collagen crosslinklevels were very high μgl normal valuesCTX μgl but were difficult to interpret in the context ofvertebral fracture DXA performed year after the last injection of denosumab revealed BMD losses of in our patient™s lumbar region and in her total hipPTHrpnormalcortisolThe association of fractures that are unusual for osteoporosis T4 acute and persistent back pain other rib fractures and hypercalcemia were suggestive of a potentialneoplasia which led to systemic explorations vertebralbiopsy and hyperparathyroidectomy and thyroidectomyknown goiter at the ultrasound exploration A thoracicabdominalpelvic computed tomography CT scan showedonly a heterogeneous multinodular goiter Sestamethoxyisobutylisonitrile MIBI scintigraphy revealed a small areaof fixation of the posterior lower right thyroid lobe and alower lobe nodule displaying clear uptake Fineneedleaspiration results were negative A biopsy of the T4 wascarried out under CT control and produced normal resultsWith hindsight a gassy image of the upper facet of the T4was suggestive of necrosis A parathyroid neoplasia couldhave been evoked too Surgery was performed at the end ofJuly to remove the right upper parathyroid gland × × mm weight g and histological analysissuggested nodular hyperplasia Associated total thyroidectomy led to the detection of a dystrophic goiter withmacrovesicular nodules and a mm isthmic papillarymicrocarcinoma with no associated adenopathy 0cMaugars Journal of Medical Case Reports Page of Fig Lumbar and thoracic Xray in January months before denosumab was stopped and in June months after denosumabwas stopped three old vertebral fractures L2 in and T5 T8 L2 aggravation during the period 20052008stars and new fractures T4 T9T10 T11 L1 L3arrows with denosumab rebound Stars are the old fractures and arrows the new oneHer pain was initially acute but of the mechanical typewith a generally favorable outcome Her calcaemia normalized the day after surgery mmoll with a plasma PTHconcentration of ngl normal range to ngl Anew bone densitometry evaluation was carried out in October at which time bone losses of for the lumbarregion Tscore ˆ’ SD and for the total left hipTscore ˆ’ SD were recorded Fig She continued tocomplain of disabling spinal pain Her phosphorus andcalcium evaluation results remained normal as did her vitamin D levels with the continuation of substitution treatment Given the considerable decrease in BMD she wasplaced on risedronate in September but this wasbadly tolerated She was then placed on zoledronate mgHer calcaemia remained stable at mmoll normalrange to mmoll After two infusions October and October a new DXA in August showed stabilization of the lumbar BMD and a significant loss in the total hip BMD ˆ’ Her plasma calcium levels remained normal mmoll and she did nothave any new vertebral or peripheral fracturesDiscussion and conclusionsThis patient who was included in the initial FREEDOMprotocol [] had benefited from denosumab treatmentwith no new vertebral fractures in the last years oftreatment and an increase in BMD to values exceedingˆ’ SD with no secondary effects However monthsafter stopping the treatment a cascade of vertebral fractures some in unusual locations T4 although this canbe possible when there are multiple lower vertebral fractures and multiple rib fracturesin a context of 0cMaugars Journal of Medical Case Reports Page of Fig Bone scintigraphy computed tomography scan and magnetic resonance imaging in June Magnetic resonance imaging and bonescintigraphy confirmed that there were six recent vertebral fractures fourth thoracic vertebra ninth thoracic vertebra tenth thoracic vertebra11th thoracic vertebra first lumbar vertebra and third lumbar vertebra small white arrows There were some costal fractures on bonescintigraphy sixth posterior on the right side and laterally tenth 11th and 12th on the left side black arrows A computed tomography scan alsoshowed old vertebral fractures fifth thoracic vertebra eighth thoracic vertebra and second lumbar vertebra white starssuggestedpossible malignancy Ahypercalcemiacomplete evaluation with systemic biological and biopsy T4 analyses ruled out this hypothesis Histological analysis ofthe tissue removed during theparathyroid intervention revealed hyperplasia but no adenoma The hypothesis of coincidental hyperparathyroidism had to be considered Before the treatment withdenosumab our patient™s calcaemia and PTH levels werenormal Based on retrospective analyses of calcaemia results we concluded that the increase in calcaemia became abnormal after years of denosumab treatmentHyperparathyroidism could have appeared during thedenosumab treatment phase with no obvious link between the two occurrences however there were no clearincreases in calcaemia during the first years and hypercalcemia was markedly aggravated by stopping denosumab The link between hyperparathyroidism anddenosumab was the subject of a recent publication []However the hyperparathyroidism described occurredrapidly after a single injection of denosumab with a fold increase in PTH levels at week normalization at months and normal calcaemia throughout [] This caseresolved within a year In our case calcaemia was highafter years of treatment No PTH determinations werecarried out in parallel during this period It is therefore difficult to determine the date of onset of the hyperparathyroidism as the denosumab treatment may have masked thehypercalcemia Similarly as our patient underwent surgery it is impossible to know what spontaneous course itwould have takenTwo similar cases of hypercalcemia during the rebound effect after stopping treatment with denosumabhave been reported one with low PTH levels and aspontaneously favorable outcome over several months[] and the other after a high dosage of denosumab mg quarterly [] The excessive bone remodelingobserved in the absence of associated fractures may havebeen due to major bone reabsorption potentially accounting for the hypercalcemia as in immobilizationrelated osteoporosis The high hypercalcemia observed isconsistent with this hypothesis Alendronate was administered and the patient™s plasma calcium concentrationsreturned to normal values within months In contrastour patient had a PTH concentration that was well 0cMaugars Journal of Medical Case Reports Page of nothighandanddisplayedcontrolledrapidnormalization within hours of calcaemia after theintervention Normal phosphate midupper calcaemiaand normal creatinine were not in favor of tertiaryhyperparathyroidism However the hyperparathyroidismdescribed occurred rapidly after a single injection ofdenosumab with a 22fold increase in intact PTHlevels at “ weeks and normal calcaemiaiPTHthroughout in a case report [] This dramatic increasein iPTH resolved spontaneously within a year Given thesurgical intervention carried out on our patient we cannot determine what the natural course of this hypercalcemia might have been in the same way that half thecases of hyperparathyroidism in kidney transplant recipients resolve within year for example [] The surgerywas carried out because we were concerned that our patient might be suffering from a parathyroid carcinomaThis rebound effect has been reported after stoppingdenosumab administered at an oncological dosage mg monthly for months [] Seven nonmalignantvertebral crushes were observed months after the lastinjection of denosumab At lower dosages denosumab mg halfyearly for years patients with breastcancer receiving aromatase inhibitors developed vertebral crushes after stopping denosumab [] The riskof vertebral fracture was higher if the treatments werelonger and if the patients had preexisting osteoporosistumorssometimesEight other cases of hypercalcemia have been reportedin children Denosumab was administered in these casesfor giantcellfibrous dysplasia brittle bonedisease and juvenile Paget™s disease [“] The hypercalcemia occurred early to months after the denosumab injection and wassevere plasmacalcium concentrations of up to mmoll but it occurred in a context of high doses and bone remodelingnot comparable with the contextin adults and therebound effect The hypercalcemia regressed over a fewmonths either spontaneously or on zoledronate Reactional bone hyperreabsorption was again suggestedThis hyperreabsorption seems to be related to therelease of receptor activator of nuclear factor kappaBligand RANKL with high crosslinked carboxyterminaltelopeptide of type collagen CTX1 and low Dickkopf1DKK1 and sclerostin [] Osteocytes are known to be theprincipal source of RANKL [] We have suggested thatosteocytes may undergo apoptosis during this reboundeffect when the treatment with denosumab is stoppedpotentially accounting for the necrosis and strong hyperreabsorption reported in certain patients presenting thisrebound effect []The discovery of a thyroid papillary microcarcinomaappears to have been fortuitous in this case Other studies have shown that denosumab may not only decreasethe frequency of bone tumor events but even have adirect or indirect antitumoral effect [] However ametaanalysis comparing denosumab and zoledronateand including four randomized trials patientsfound a significantly higher risk of primary neoplasiawith a cumulative annual incidence of on denosumab versus on zoledronate [] No particular cancer type profile was identified but the number of caseswas small n The cumulative doses in our patientamounted to approximately g of denosumab corresponding to months at the dose of mgmonth prescribed to prevent secondary bone tumor complicationsIt is not possible to draw any firm conclusions concerningour case as goiter is itself a risk factor for thyroid cancerwith a poorly defined incidence of between and forcancer in patients undergoing surgery for goiter []In conclusionit is important to be aware of thisrebound effect with strong bone hyperreabsorption incertain patients after stopping treatment with denosumab Several explorations to eliminate a neoplasm couldbe avoided with knowledge of these cases It can takeseveral different forms a simple loss of the BMD gainedon the treatment vertebral fractures or transient hypercalcemia which in this context may raise concerns of orsimulate malignant bone diseases or hyperparathyroidism as in our case The rebound after stopping treatment with denosumab makes it necessary to checkcalcaemia and CTX early before the risk of further vertebral fractures DXA will be carried out at the end ofthe denosumab sequence but an early new comparativeDXA would be less sensitive than CTX dosage We canpropose CTX and calcaemia months after the last injection of denosumab as a reference and then or months later A marked increase will require preventionThe most appropriate therapeutic approach remains unclear The effects of gradually decreasing the dose ofdenosumab have not yet been reported Bisphosphonatesseem to be only partially effective according to publishedpreliminary results []AbbreviationsBMD Bone mineral density CRP Creactive protein CT Computedtomography CTX Carboxyterminal collagen crosslink CTX1 Crosslinkedcarboxyterminal telopeptide of type collagen DKK1 Dickkopf1 DXA DualXray absorptiometry iPTH Intact parathyroid hormone L1 First lumbarvertebra L2 Second lumbar vertebra L3 Third lumbar vertebraMRI Magnetic resonance imaging PTH Parathyroid hormonePTHrp Parathyroid hormonerelated peptide RANKL Receptor activator ofnuclear factor kappaB ligand SD Standard deviation T4 Fourth thoracicvertebra T5 Fifth thoracic vertebra T8 Eighth thoracic vertebra T9 Ninththoracic vertebra T10 Tenth thoracic vertebra T11 11th thoracic vertebraTSH Thyroidstimulating hormone MIBI MethoxyisobutylisonitrileAcknowledgementsNAAuthors™ contributionsAll authors contributed to the work contribution to the observation YMCDL the discussion JMB BLG PG JG and writing the manuscript YMCDL All authors read and approved the final manuscript 0cMaugars Journal of Medical Case Reports Page of function Curr Drug Saf “ httpsdoi102174 Maugars Y Bart G Guillot P Multiple vertebral osteonecrosesKümmell's Disease after years on denosumab is osteocyte apoptosis toblame Calcif Tissue Int “ Chen F Pu F Safety of denosumab versus zoledronic acid in patients withbone metastases a metaanalysis of randomized controlled trials Oncol ResTreat “ Papapoulos S Lippuner K Roux C The effect of or years ofdenosumab treatment in postmenopausal women with osteoporosisresults from the FREEDOM Extension study Osteoporos Int “ Nakamura Y Kamimura M Ikegami S Changes in serum vitamin D andPTH values using denosumab with or without bisphosphonate pretreatment in osteoporotic patients a shortterm study BMC Endocr Disord“Torres A Lorenzo V Salido E Calcium metabolism and skeletal problemsafter transplantation J Am Soc Nephrol “ GonzalezRodriguez E AubryRozier B Stoll D Zaman K Lamy O Sixtyspontaneous vertebral fractures after denosumab discontinuation in women with earlystage breast cancer under aromatase inhibitorsBreast Cancer Res Treat “ httpsdoi101007s10549019054588Tyan A Patel SP Block S Hughes T McCowen KC Rebound VertebralFractures in a Patient with Lung Cancer After OncologyDose DenosumabDiscontinuation A Cautionary Tale Mayo Clin Proc Innov Qual Outcomes“Fassio A Adami G Benini C Vantaggiato E Saag KG Giollo A Lippolis IViapiana O Idolazzi L Orsolini G Rossini M Gatti D Changes in Dkk1sclerostin and RANKL serum levels following discontinuation of longtermdenosumab treatment in postmenopausal women Bone “ Bonewald LF The amazing osteocyte J Bone Miner Res “ de Groot AF AppelmanDijkstra NM van der Burg SH Kroep JR The antitumor effect of RANKL inhibition in malignant solid tumors A systematicreview Cancer Treat Rev “Sahbaz NA Tutal F Aksakal N Cancer frequency in retrosternal goiterAm Surg “Lamy O Stoll D AubryRozier B Rodriguez EG Stopping Denosumab CurrOsteoporos Rep “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsFundingNone for this workAvailability of data and materialsAll original data are available corresponding authorEthics approval and consent to participateInclusion in the FREEDOM protocol signedConsent for publicationWritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images A copy of the writtenconsent is available for review by the EditorinChief of this journalCompeting interestsWe do not have any competing interestsReceived July Accepted May ReferencesAnastasilakis AD Polyzos SA Makras P AubryRozier B Kaouri S Lamy OClinical features of patients with reboundassociated vertebral fracturesafter denosumab discontinuation systematic review and additional cases JBone Miner Res “Dupont J Laurent MR Dedeyne L Luyten FP Gielen E Dejaeger MReboundassociated vertebral fractures after stopping denosumab Reportof four cases Joint Bone Spine “ httpsdoi101016jjbspin201907010Fernández Fernández E Benavent Núñez D Bonilla Hernán G Monjo HenryI García Carazo S Bernad Pineda M Balsa Criado A Aguado AP MultipleVertebral Fractures Following Discontinuation of Denosumab TreatmentTen Clinical Cases Report Reumatol Clin httpsdoi101016jreuma201811002 [Epub ahead of print]Florez H Ramírez J Monegal A Guañabens N Peris P Spontaneousvertebral fractures after denosumab discontinuation A case collection andreview of the literature Semin Arthritis Rheum “Popp AW Varathan N Buffat H Senn C Perrelet R Lippuner K Bone mineraldensity changes after year of denosumab discontinuation inpostmenopausal women with longterm denosumab treatment forosteoporosis Calcif Tissue Int “TriptoShkolnik L Rouach V Marcus Y RotmanPikielny P Benbassat CVered I Vertebral fractures following denosumab discontinuation inpatients with prolonged exposure to bisphosphonates Calcif Tissue Int“Koldkjær Sølling AS Harsløf T Kaal A Rejnmark L Langdahl BHypercalcemia after discontinuation of longterm denosumab treatmentOsteoporos Int “Kurucu N Akyuz C Ergen FB Denosumab treatment in aneurysmalbone cyst Evaluation of nine cases Pediatr Blood Cancer httpsdoi101002pbc26926 Epub Dec PubMed PMID Uday S Gaston CL Rogers L Osteonecrosis of the jaw and reboundhypercalcemia in young people treated with denosumab for giant celltumor of bone J Clin Endocrinol Metab “Setsu N Kobayashi E Asano N Severe hypercalcemia followingdenosumab treatment in a juvenile patient J Bone Miner Metab “ Gossai N Hilgers MV Polgreen LE Greengard EG Critical hypercalcemiafollowing discontinuation of denosumab therapy for metastatic giant celltumor of bone Pediatr Blood Cancer “ Boyce AM Chong WH Yao J Denosumab treatment for fibrousdysplasia J Bone Miner Res “Trejo P Rauch F Ward L Hypercalcemia and hypercalciuria duringdenosumab treatment in children with osteogenesis imperfecta type VI JMusculoskelet Neuronal Interact “ Roux S Massicotte MH Huot Daneault A BrazeauLamontagne L DufresneJ Acute hypercalcemia and excessive bone resorption following antiRANKLwithdrawal Case report and brief literature review Bone “ Mazokopakis EE Denosumabinduced normocalcemic hyperparathyroidismin in a woman with postmenopausal osteoporosis and normal renal 0c"

Thyroid_Cancer

pulmonary disease COPD is due to structural changes and narrowing of small airways and parenchymaldestruction loss of the alveolar attachment as a result of pulmonary emphysema which all lead to airflow limitation Extracorporeal shock waves ESW increase cell proliferation and diï¬erentiation of connective tissue fibroblasts To date no studiesare available on ESW treatment of human bronchial fibroblasts and epithelial cells from COPD and control subjects We obtainedprimary bronchial fibroblasts from bronchial biopsies of patients with mildmoderate COPD and control smokers with normallung function 16HBE cells were also studied Cells were treated with a piezoelectric shock wave generator at low energy mJmm2 pulses After treatment viability was evaluated and cells were recultured and followed up for and h Cellgrowth WST1 test was assessed and proliferation markers were analyzed by qRTPCR in cell lysates and by ELISA tests in cellsupernatants and cell lysates After ESW treatment we observed a significant increase of cell proliferation in all cell types CKitCD117 mRNA was significantly increased in 16HBE cells at h Protein levels were significantly increased for cKit CD117 at h in 16HBE p and at h in COPDfibroblasts p � for PCNA at h in 16HBE p � for y1 CD90 at and h in CSfibroblasts p � and p � for TGF1 at h in CSfibroblasts p � for procollagen1 at h inCOPDfibroblasts p � and for NFκBp65 at and h in 16HBE p � and p � In the peripheral lung tissueof a representative COPD patient alveolar type II epithelial cells TTF1 coexpressing cKit CD117 and PCNA were occasionally observed ese data show an increase of cell proliferation induced by a low dosage of extracorporeal shock waves in16HBE cells and primary bronchial fibroblasts of COPD and control smoking subjects Backgrounde progressive chronic airflow limitation in chronic obstructive pulmonary disease COPD is due to two majorpathological processes i remodeling and narrowing ofsmall airways and ii destruction of the lung parenchymawith loss of the alveolar attachments as a result of pulmonaryemphysema [] Chronic ‚ammation in the lung plays a 0cCanadian Respiratory Journaltherapycentral role in both the small airway remodeling and thepulmonary emphysema [“] Lung volume reductionsurgery and lung transplantation while possible in endstageCOPD are restricted to just a few selected patients []httpwwwgoldcopdcom RegenerativeforCOPD includes mesenchymal stromal cell MSC or tissueengineering therapies But while bone marrow MSC oradipose tissue MSC treatments showed promising results inmice with induced emphysema [] clinical trials performedin COPD patients have been discouraging [ ] ere are alarge number of animal studies in which lung regenerationhas been successfully stimulated For instance in a rat modelof elastaseinduced emphysema administration of alltransRA ATRA stimulated alveolar regeneration [] keratinocyte growth factor KGF FGF7 administered afterpneumonectomy augmented alveolarization [] administration of HGF stimulated alveolar regeneration enhancedlung vascularization and improved exercise tolerance andgas exchange [] intratracheal administration of bFGF torats and dogs with elastaseinduced emphysema improvedalveolar dimensions and lung microvessel density [] andVEGF administration enhanced postpneumonectomy alveolar growth in mice [] But again the attempts tostimulate lung regeneration in COPD patients with emphysema with orally administered ATRA yielded no differences in computed tomography CT lung function orquality of life scores between treatment groups [ ] andRARc selective agonist administration also showed nodiï¬erences in CT scores or lung function in treated vsnontreated COPD patients [ ] However the therapeutic potential of regenerative pharmacology is still at thebeginning of its development And many authors haveshown that the human lung also in adulthood retains asignificant regenerative potential from the large to the smallairways and in terminal and respiratory bronchioles [] andthat tissue regeneration is achieved in two ways by proliferation of common diï¬erentiated cells andor by deployment of specialized stemprogenitor cells [ ]Extracorporeal shock wave therapy ESWT is applied inmany musculoskeletal diseases and in regenerative medicinebased on its capability to induce neoangiogenesis osteogenesis regeneration and remodeling through stem cellstimulation [] ESW in combination with tenogenicmedium improved the diï¬erentiation of human adiposederived stem cells hASCs into tenoblastlike cells []ESW combined with osteogenic medium increased the osteogenic diï¬erentiation of treated hASCs [] while stemcell diï¬erentiation into myofibroblasts was partially reducedby ESW treatment [] But to our knowledge no data areavailable on ESW treatment of primary bronchial fibroblastsof patients with COPD and control healthy smokers orbronchial epithelial cells 16HBEMarkers of cell proliferation include CD117 cKit orSCFR a receptor tyrosine kinase protein that binds to stemcell factor SCF expressed on hematopoietic stem cells Itcan also be expressed by mast cells melanocytes in the skininterstitial cells of Cajal in the digestive and urogenital tract[] cardiac pericytes [] amniotic fluid stem cells []stemprogenitor cells in conducting airway epithelium ofporcine lung [] and dendritic cells in the lung []Another marker of cell proliferation is proliferating cellnuclear antigen PCNA It is expressed in the nuclei of cellsand is involved in DNA replication DNA repair andchromatin remodeling [ ] In the lung of COPD patients alveolar type II epithelial cells and endothelial cells[] and small airway bronchiolar epithelium [] expressdecreased PCNA levels compared with related nonCOPDcontrol groups A third marker of cell proliferation is CD90y1thymocyte diï¬erentiation antigen1 a glycophosphatidylinositol cell surface protein expressed by thymocytes CD34 cells mesenchymal stem cells endothelialcells and cardiac fibroblasts It is also considered a marker ofmultipotent mesenchymal stem cells when expressed inassociation with other markers CD29 CD44 CD73CD105 [ ]We aimed in this study to analyze the proliferative eï¬ectof shock waves when applied as an external challenge toprimary bronchial fibroblasts of COPD patients and controlsmokers and to immortalized bronchial epithelial cells16HBE To this end we investigated cell markers expression related to this proliferative stimulus Methods Ethics Statement Collection and processing of bronchialbiopsies at the Institute of Veruno NO and collection andprocessing of the peripheral lung tissues at the UniversityHospital of Orbassano during lung resection for a solitaryperipheral neoplasm were approved by the ethics andtechnical committees ofthe Istituti Clinici ScientificiMaugeri CTS p102 and San Luigi Hospital OrbassanoTO CE N Italy the study complied withthe Declaration of Helsinki and written informed consentwas obtained from each participant Cell Culture and Treatments We used the SV40 large Tantigentransformed 16HBE cellline which retains thediï¬erentiated morphology and function of normal humanbronchial epithelial cells NHBE [] and primary humanbronchial fibroblasts obtained from bronchial biopsies ofpatients with COPD n � and control smoking subjectsn � with normal lung function Primary bronchial fibroblasts were obtained from bronchial biopsies obtainedfor diï¬erent protocol studies [] Bronchial biopsies weretreated with type II collagenase min at °C InvitrogenGIBCA and cultured in DMEM until confluentprimary fibroblasts were obtained 16HBE cells and primarybronchial fibroblasts were maintained in Dulbecco™s modified minimum essential medium DMEM supplementedwith vv fetal bovine serum FBS IUmL penicillin μgmL streptomycin 1x nonessential amino acids mMsodium pyruvate and mM glutamine °C CO2 []When cells were “ confluent the complete mediumwas replaced with DMEM with FBS for starvation time h e shockwave generator utilized for the in vitroexperiments was a piezoelectric device Piezoson Richard Wolf Knittlingen Germany designed for clinical 0cCanadian Respiratory Journaluse in orthopedics and traumatology Aliquots of mL ofcell suspension adjusted to × cellsmL were placed in mm polypropylene tubes completely filled with culturemedium e shock wave unit was kept in contact with thecellcontaining tube by means of a waterfilled cushionCommon ultrasound gel was used as a contact mediumbetween the cushion and tube ESW treatment was as follows energy flux density EFD � mJmm2 pulsesfrequency � shockss is EFD is a mediumhigh energywe already used for previous in vitro diï¬erentiation studiesin tendons [] After treatment cell viability was evaluatedby trypan blue exclusion and primary fibroblasts werepassaged in DMEM complete for hours 16HBEcells were cultured for and h because of their lowerresistance to starvation T0 corresponds to hours post ESWtreatment for all experiments reported Nontreated fibroblasts or 16HBE cells were used as controls Cell growth wasevaluated by the colorimetric test WST1 All experimentswere performed in quadruplicate ie four independentexperiments for each type of treatment ESW or noESWand each time exposure Extraction and Quantification of RNA and qRTPCRfrom Primary Bronchial Fibroblasts and 16HBE Total RNAfrom treated and nontreated cells was purified and isolatedusing an RNAspin Mini RNA Isolation kit GE HealthcareLife Sciences Pittsburgh USA following the manufacturer™sinstructions Total RNA was resuspended in μL nucleasefree water RNA concentration was determined using aUVvisible spectrophotometer λ260280 nm EppendorfBioPhotometer plus and stored at ˆ’°CQiagenQT00000728e expression of genes of interest was measured usingSYBR Green Qiagen UK for qPCR in a Corbett RotorGene Corbett Cambridge UK system Onestep realtime PCR was carried out by amplifying mRNA using theQuantiFast„¢ SYBR Green RTPCR kitITaccording to the manufacturer™s instructions and the genespecific primers Qiagen IT We detected the expression ofcKit or SCFR CD117 Cat QT01844549 Qiagen PCNACat QT00024633 y1 CD90Cat QT00023569TGF1CatProcollagenIQT01005725 and NFκBp65 Cat QT01007370 Weperformed independent experiments and quantitative PCRmeasurements in quadruplicate for each type of treatmentESW or noESW and each time exposure Briefly the PCRreaction mix prepared in a total volume of μL was run onthe Rotor Gene Q Qiagen IT and the following PCR runprotocol was used °C for min reverse transcription°C for min PCR initial activation step amplificationcycles of °C for s denaturation and °C for scombined annealingextension followed by melting curveanalysis to ensure the specificity of PCR amplificationGlyceraldehyde phosphate dehydrogenase GAPDHQT01192646 Qiagen was used as the reference gene forevery target gene per sample and the data were normalizedagainst the respective GAPDH signaling Cycle thresholdCT values were determined using the Rotor Gene Qsoftware RotorGene Q Series Software eCatexpression levels of all genes studied were normalized toGAPDH levels in each sample to determine the expressionbetween treated and nontreated cells using the ˆ’ΔCt method[] for primary bronchial fibroblasts and the ˆ’ΔΔCt for16HBE cells [] ELISA Tests in the Supernatants or Cell Lysates of ESWTreated and Nontreated Cells Protein extraction andquantification in the supernatants or cell lysates of ESWtreated and nontreated cells were performed as reported inTable Suppliers Cat Numbers dilution conditions anddetection limits of the ELISA kits used are also reported eELISA kits WST1 cell proliferation kit and MPERmammalian protein extraction kit were used according tothe manufacturer™s instructions Table CKit CD117PCNA and NFκBp65 were quantified in cell lysates CD90TGF1 and procollagen1 were quantified in the cellsupernatants Immunohistochemistry of the Lung Parenchyma of Patients with COPD Samples were frozen in liquid nitrogenprecooled is tane after embedding in OCT and used forcryostat sectioning and immunostaining of some cellproliferationrelated molecules Single immunostainings offrozen sections were performed with mouse anti“thyroidtranscription factor1 TTF1 sc53136 Santa Cruz rabbitanticKit CD117 ARG51826 ARGBIO and rabbit antiPCNA PAS27214 ermo Fisher primary antibodiesAntibody binding was demonstrated with secondary antibodies antimouse Vector BA and antirabbitVector BA followed by ABC kit AP AK5000VECTASTAIN and FastRed Substrate red color Doublestainings were performed using also ABC kit Elite PK6100VECTASTAIN and diaminobenzidine substrate browncolor for identification of TTF1 positive alveolar type IIepithelial cells [] coexpressing cKit CD117 or PCNAantigens Slides were included in each staining run usinghuman tonsil nasal polyp or breast cancer as positivecontrols For the negative control slides normal nonspecificmouse or rabbit immunoglobulins Santa Cruz Biotechnology Santa Cruz CA USA were used at the same proteinconcentration as the primary antibodiesmean± standardthe unpaired ttest Probability values of p were Statistical Analysis Group data were expressed asorinterquartile range IQR for morphologichistologic dataDiï¬erences between treatment groups were analyzed usingor mediandeviationrangeconsidered significant Data analysis was performed usingthe Stat View SE Graphics program Abacus Concepts IncBerkeley CA USA Results ESW Eï¬ects on Cell Proliferation ESW treatment at adosage of mJmm2 pulses frequency � shockssof primary bronchial fibroblasts from COPD patients n � 0cCanadian Respiratory JournalPackyearsExsmokercurrent smokerTable Clinical characteristics of chronic obstructive pulmonary disease COPD patients and control smokers who provided bronchialfibroblasts for œin vitro experimentsSubjectsCOPD1COPD2COPD3± Mean± SD± Mean± SDIndividual and mean± standard deviation SD data M male F female FEV1 forced expiratory volume in s BD bronchodilator FVC forced vitalAge years MFMMM”MMM”± ± ± ± ± ± ± CurrentCurrentCurrentFEV1 postBDFEV1 preBDCurrentCurrentNDNDND”FEV1FVCCS1CS2CS3Ex””capacity ND not determined Patients were classified according to the Global Initiative for Chronic Obstructive Lung disease httpgoldcopdorg levels ofseverity for COPD For COPD patients FEV1FVC are postbronchodilator values ANOVA test FEV1 p � FEV1FVC p � No significantdiï¬erences were observed for age p � and packyears p � smokedTable List of ELISA tests cell proliferation and protein extraction kits used For ELISA tests dilution of the supernatants or cell lysatesamples used and detection limits are also reportedTargetcKit or SCFR CD117PCNAy1 CD90TGF1Procollagen1NFκBp65WST1 cell proliferationMPER mammalian protein extraction ermo Scientific ngmL “ ngmL ngmL “ ngmL pgmL “ pgmL pgmL “ pgmL pgmL “ pgmL17pgmL “ pgmLCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpSEA121 HuSEA591MiSEB404 HuSEA124 HuSEA957 HuKHO0371KA1384Dilution PBS PBSDetection limit range diluent buï¬erInvitrogenAbnovaNo dilNo dilNo dilSupplierCat ashowed a significantly increased proliferation index at and h after treatment compared with nontreated bronchial fibroblasts Figure 1a ESWtreated primary bronchial fibroblasts from control smokers with normal lungfunction n � also showed a significant increase of theproliferation index at and h aftertreatmentFigure 1b Treated bronchial epithelial cells 16HBEshowed significantly increased proliferation index values at and h after treatment when compared with nontreated16HBE cells Figure 1c ESW Eï¬ects on mRNA and Protein Levels of Cell Proliferation and Cell Remodeling Markers Primary bronchialfibroblasts from COPD patients n � control smokersn � and human bronchial epithelial cells 16HBE werestimulated with extracorporeal shock waves at a dosage of mJmm2 pulses and compared with paired nonstimulated primary bronchial fibroblasts and 16HBE cellsCKit mRNA was significantly increased in ESWtreated16HBE cells at h p � and decreased in CSfibroblasts at h p � compared with nontreated cellsFigures 2b and 2c Furthermore a tendency to increased cKit mRNA levels was observed after ESW treatment for COPDfibroblasts Figure 2a CKit protein wassignificantly increased in the cell lysates at h after ESWtreatment in primary bronchial fibroblasts of COPD patientsp � Figure 2d and in 16HBE cells p at h after ESW treatment Figure 2f No significantchanges were observed for cKit protein in ESWtreatedprimary bronchial fibroblasts from control smokers CSbronchialfibroblastswith normal lung function Figure 2e PCNA mRNAlevels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared with nontreatedcells Figures 3a“3c PCNA protein in the cell lysatesshowed a tendency to be increased in primary bronchialfibroblasts of CS p � at h after ESW treatmentFigure 3e and a significant increase was observed at hT0 in 16HBE cells p � after ESW treatmentFigure 3f No significant changes were observed inprimaryof COPD patientsFigure 3d y1 CD90 mRNA levels were not significantly diï¬erent in ESWtreated fibroblasts and 16HBE cellscompared with nontreated cells Figures 4a“4c y1CD90 protein in the cell supernatants was significantlyincreased in primary bronchial fibroblasts of CS at hp � after ESW treatment Figure 4e No significant changes were observed in primary bronchial fibroblastsof COPD patients or in 16HBE cells Figures 4d and 4fTGF1 mRNA levels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared withnontreated cells Figures 5a“5c TGF1 protein in thecell supernatants was significantly increased in primarybronchial fibroblasts of CS at h p � after ESWtreatment Figure 5e No significant changes were observed in primary bronchial fibroblasts of COPD patients orin 16HBE cells Figures 5d and 5f Procollagen1 mRNAlevels were not significantly diï¬erent in ESWtreated fibroblasts and 16HBE cells compared with nontreated cellsFigures 6a“6c Procollagen1 protein in the cellsupernatants wasincreased in primarysignificantly 0cCanadian Respiratory JournalabIncreased cell proliferation was observed in all cellular types studied after challenge with ESW Ttestˆ—ˆ—p andˆ—ˆ—ˆ—p Figure WST1 test for evaluation of cell proliferation after extracorporeal shock wave ESW stimulation of primary bronchial fibroblastsof COPD patients n � a primary bronchial fibroblasts of control smokers n � b and bronchial epithelial cells 16HBE ccbronchial fibroblasts of COPD patients at h p � after ESW treatment Figure 6d No significant changeswere observed in primary bronchial fibroblasts of CS or in16HBE cells Figures 6e and 6f NFκBp65 mRNAlevels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared with nontreatedcells Figures 7a“7c NFκBp65 protein in the cell lysates was decreased in primary bronchial fibroblasts ofCOPD patients at h p � after ESW treatmentFigure 7d and increased in 16HBE cells at h p � and h p � after ESW treatment Figure 7f Nosignificant changes were observed in primary bronchial fibroblasts of CS Figure 7e Immunohistochemistry in the Lung Parenchyma of COPDPatients of Alveolar Type II Epithelial Cells Expressing cKitand PCNA In the lung parenchyma of COPD patientsalveolar type II epithelial cells were identified by the use ofanti“thyroid transcription factor1 TTF1antibodyImmunopositivity for cKit CD117 and PCNA was alsooccasionally observed in alveolar septa Figure Doublestaining used for identification of TTF1 cells coexpressingcKitFigures 9a and 9b and PCNAFigures 9c and 9d showed that alveolar type II epithelial cells coexpressing cKit and PCNA were present eventhough rarely observedCD117 Discussionis study shows that extracorporeal shock waves induce cellproliferation of bronchial epithelial cells 16HBE and primary bronchial fibroblasts of COPD patients and controlsmokers As far as markers of cell proliferation are concerned cKit CD117 was increased in bronchial epitheliumat both mRNA and protein levels h after ESW treatmentand it was also increased in primary bronchial fibroblasts at h after ESW challenge Other markers indicative of cellproliferation were also increased PCNA protein increased inCOPDWST1NO ESWESWT24T48T72T0012345ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—CST0T24T48T72NO ESWESWWST1012345ˆ—ˆ—ˆ—ˆ—ˆ—16HBET0T24T48NO ESWESWWST1012345ˆ—ˆ—ˆ—ˆ—ˆ—ˆ— 0cCanadian Respiratory JournaladbecfFigure CKit CD117 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBEcKit increased at mRNA c and protein f levels In primary bronchial fibroblasts of COPD patients cKit increased at protein level d Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Proliferating cell nuclear antigen PCNA mRNA a b c and protein d e f expression after ESW treatment in primary bronchialfibroblasts of COPD patients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchialepithelium 16HBE PCNA increased at protein f level Ttest was used for comparative purposes and p values are reported in the graphsT0T24T48T7201234102030COPD CNCOPD ESWcKit CD117 “ˆ† Ct0180021103960767CS CNCS ESWcKit CD117 “ˆ† CtT0T24T48T7202461020304050002016HBE CN16HBE ESWcKit CD117 “ˆ†ˆ† CtT0T24T480246204060800435041800320010020030006839038680037305624COPD CNCOPD ESWcKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESW000500100015002000250006727038680877507636cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW16HBE CN16HBE ESW020406080P 000010791501364cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT0T24T48T72PCNA “ˆ† Ct0005101520COPD CNCOPD ESWT0T24T48T7205101520PCNA “ˆ† CtCS CNCS ESWT0T24T48000510152025PCNA “ˆ†ˆ† Ct16HBE CN16HBE ESW01450027520139305288COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00102030PCNA ngmL00002004006008010000512084270367304489PCNA ngmLCS CNCS ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW0123004620190109820PCNA ngmL16HBE CN16HBE ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SW 0cCanadian Respiratory JournalacebdfFigure y1 CD90 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts ofcontrol smokers y1 increased at protein level at and h e Ttest was used for comparative purposes and p values are reported in thegraphsbronchial epithelial cells at h after ESW challenge y1CD90 protein increased in CS“primary bronchial fibroblasts at and h after ESW treatment molecules morerelated to remodeling such as TGF1 protein were increased in CS“primary bronchial fibroblasts at h afterESW treatment and procollagen1 protein increased at hfollowed by a decrease at h in COPD“primary bronchialfibroblasts after ESW treatment A marker of ‚ammationtranscription factor NFκBp65 protein was decreased inCOPD“primary bronchial fibroblasts at h after ESWtreatment but it was increased in CS“primary bronchialfibroblasts and in bronchial epithelial cells after ESWtreatment Markers of cell proliferation such as cKit andPCNA were observed in the peripherallung of COPDT0T24T48T72COPD CNCOPD ESWThy1 CD90 “ˆ† Ct0005101520CS CNCS ESWThy1 CD90 “ˆ† CtT0T24T48T72012316HBE CN16HBE ESWThy1 CD90 “ˆ† ˆ†CtT0T24T4801020304009523087570853209221COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00200040006000800010000Thy1 CD90 pgmLCS CNCS ESW01500003150239300410T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000200004000060000Thy1 CD90 pgmL16HBE CN16HBE ESW035960811001447T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW010203040Thy1 CD90 pgmL 0cCanadian Respiratory JournaladbecfFigure TGF1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPD patients ad primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts of controlsmokers TGF1 increased at protein level at h e Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Procollagen1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts ofCOPD patients procollagen1 increased at protein level d at h T0 followed by a decrease at h panel d Ttest was used forcomparative purposes and p values are reported in the graphsT0T24T48T72TGF 1 “ˆ† Ct0005101520COPD CNCOPD ESWT0T24T48T72TGF 1 “ˆ† Ct00051015CS CNCS ESWT0T24T48TGF 1 “ˆ† Ct0005101516HBE CN16HBE ESW07196046450373903445T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESWTGF 1 pgmL005010015004487044930863500385T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESWTGF 1 pgmL0005001000150004757089490102101199T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW16HBE CN16HBE ESWTGF 1 pgmL010203040T0T24T48T72Procollanen1 “ˆ† Ct000510152025COPD CNCOPD ESWT0T24T48T72Procollagen1 “ˆ† Ct000510152025CS CNCS ESWT0T24T48Procollagen1 “ˆ†ˆ† Ct00051015202516HBE CN16HBE ESW00220057350024202359T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESW00100020003000Procollagen1 pgmL00541053750944605958T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000100002000030000Procollagen1 pgmLCS CNCS ESW010340898407490T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW050100150200Procollagen1 pgmL16HBE CN16HBE ESW 0cCanadian Respiratory JournaladbecfFigure NFκBp65 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPD patientsa d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBE NFκBp65 increased at protein panel f level at and h of exposure In primary bronchial fibroblasts of COPD patients NFκBp65 decreased atprotein level d at h In primary bronchial fibroblasts of control smokers NFκBp65 increased at protein level e at h Ttest wasused for comparative purposes and p values are reported in the graphspatients and both these markers were occasionally coexpressed by alveolar epithelial type II cells TTF1 in thesepatientsExtracorporeal shock wave therapy is applied in regenerative medicine since it is capable of inducing neoangiogenesis osteogenesis and remodeling through stemcell stimulation [] On the other hand while regenerativetherapy applied to mice with induced emphysema has shownpromising results [] clinical trials performed in COPDpatients were discouraging [ ] Since the human lung alsoin adulthood maintains a significant regenerative potential[“] due to proliferation of diï¬erentiated of stemprogenitor cells andor by their stimulation [ ] we hereinvestigated the proliferative action of ESW at low dosage inbronchial epithelial cells and in primary bronchial fibroblasts of control smokers CS and patients with COPD Ourdata show that all the cell types studied significantly increased their proliferation index WST1 test after ESWtreatment in agreement with data previously obtained formuscle cells or tendon fibroblasts [] Interestingly thecKit CD117 receptor tyrosine kinase protein and mRNAwere increased in 16HBE cells and cKit protein also increased in primary bronchial fibroblasts of COPD patientsafter ESW stimulation It is not clear however if this cellresponse represents an intermediate dediï¬erentiation step ora simple proproliferative stimulus for stimulated 16HBEcells and COPD“primary bronchial fibroblasts Since weexposed welldiï¬erentiated cells we believe that this transitory increment may be interpreted as a proproliferativestimulus induced by ESW exposureIn bronchial epithelial cells 16HBE proliferating cellnuclear antigen PCNA considered a marker of cellproliferation was increased after ESW stimulation confirming again the proproliferative role of ESW exposurefor these lung structure cells is finding in view of thedecreased PCNA levels reported in the lung of COPDpatients [ ] compared with control subjects is particularly relevant since ESW stimulation may contrastthese lower PCNA levels characterizing the damaged lungof these patientse increased y1 CD90 protein level shown afterESW exposure in CS“primary bronchial fibroblasts was notobserved in ESWtreated COPD“primary bronchial fibroblasts or in 16HBE treated cells PCNA protein alsotended to be higher in CS“primary bronchial fibroblastsafter ESW treatment but not in COPD“primary bronchialfibroblasts ese diï¬erences in the response to ESWchallenge of COPD“ and CS“primary bronchial fibroblastsmay in part be due to the reduced proliferation capacity ofthese cells derived from COPD lungs as previously reported [ ] In our welldiï¬erentiated ESWexposedfibroblasts we interpret the increment of y1 protein NFκBp65 “ˆ† Ct012345T24T48T72T0COPD CNCOPD ESW NFκBp65 “ˆ† Ct01234T24T48T72T0CS CNCS ESW NFκBp65 “ˆ†ˆ† Ct000510152025T24T48T016HBE CN16HBE ESW00805026820020907045NFκBp65 pgmL0050000100000150000T72 CNT72 SWT24 SWT48 CNT48 SWT0 SWT24 CNT0 CNCOPD CNCOPD ESWNFκBp65 pgmL036510427702233003830020000400006000080000100000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CNT72 SWT72 CNCS CNCS ESWNFκBp65 pgmL0015500002062865000100001500002004006008001000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CN16HBE CN16HBE ESW 0cCanadian Respiratory JournalFigure Photomicrographs showing thyroid transcription factor1 TTF1 expression panels a b cKit CD117 c d and proliferatingcell nuclear antigen PCNA e f in the peripheral lung tissue of a representative patient with chronic obstructive pulmonary diseaseCOPD Arrows indicate positively stained cells mainly located in the alveolar septa Bars � micronsafter ESW treatment”like that of cKit”as a proproliferative stimulus induced by the treatmentWe found increased levels of secreted TGF1 inCS“primary bronchial fibroblasts h after ESW stimulation TGF signaling pathways are involved in the regulationof many cell functions and in the maintenance of cellularhomeostasis [] We recently reported a decrease of TGF1and TGF3 in bronchiolar epithelium and alveolar macrophages of COPD patients compared with CS [] and thisdecrease may favor the increase of autoimmunity responsesin these patients [] We speculate that the inductionthrough ESW challenge of an increase of TGF in bronchialfibroblasts may play a role in the TGF repositioning andgain in homeostatic function of this important protein in thelungs of COPD patientsTGF induced extracellular matrix and procollagen1production has been reported in pulmonary fibroblasts[] even though it was also reported that the increase ofprofibrotic markersincluding procollagen1 in humanlung fibroblasts may be NLRP3 ‚ammasome dependentand TGF independent [] and associated with increased‚ammation ofthe lung [] We here observed aTTF a0Lung COPDCD a0Lung COPDPCNA a0Lung COPDabcdef 0cCanadian Respiratory JournalFigure Photomicrographs showing alveolar type II epithelial cells TTF1 cells red color coexpressing cKit CD117 brown color ab and PCNA brown color c d in the peripheral lung tissue of a representative patient with COPD Positive doublestained cells can berecognized in the alveolar septa even though their presence was only rarely observed Arrows indicate positively stained cells located in thealveolar septa Bars � micronstransitory increase of procollagen1 protein in COPD“primary bronchial fibroblasts at h after ESW

Thyroid_Cancer

Rapid repair of human disease‘specific single‘nucleotide variants by One‘SHOT genome editingYuji Yokouchi12 Shinichi Suzuki2 Noriko Ohtsuki12 Kei Yamamoto12 Satomi Noguchi12 Yumi Soejima3 Mizuki Goto34 Ken Ishioka5 Izumi Nakamura12 Satoru Suzuki6 Seiichi Takenoshita7 takumi era123Many human diseases ranging from cancer to hereditary disorders are caused by single‘nucleotide mutations in critical genes Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases However current procedures for repairing deleterious single‘nucleotide mutations are not straightforward requiring multiple steps and taking several months to complete In the current study we aimed to repair pathogenic allele‘specific single‘nucleotide mutations using a single round of genome editing Using high‘fidelity site‘specific nuclease AsCas12aCpf1 we attempted to repair pathogenic single‘nucleotide variants SNVs in disease‘specific induced pluripotent stem cells As a result we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single‘nucleotide marker followed by absolute markerless scarless repair of the RET SNV with no detected off‘target effects The markerless method was then confirmed in human type VII collagen‘encoding gene COL7A1 Thus using this One‘SHOT method we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one resulting in allele‘specific repair that can be completed within weeks with or without a single‘nucleotide marker Our findings suggest that One‘SHOT can be used to repair other types of mutations with potential beyond human medicineThe human genome contains extensive variation including an estimated — singlenucleotide variants SNVs that determine how we look and function as well as our specific disease tendencies1“ Some SNVs are pathogenic and either directly or indirectly cause hereditary disorders4“ such as multiple endocrine neoplasia type 2B MEN2B7 and dystrophic epidermolysis bullosa DEB8 MEN2B is an autosomal dominant syndrome characterised by thyroid adrenal gland and neuronal tumours and skeletal abnormalities The majority of MEN2B cases result from a single aminoacid substitution Met918Thr in the RET protooncogene which is caused by a pathogenic SNV RET c2753TC at the second base of the codon7 DEB is an inherited disease characterised by severe recurrent skin ulcers and blistering It is caused by genetic mutations in the human type VII collagenencoding gene COL7A1 the product of which is an anchoring fibril connecting the epidermis to the dermis8 To model diseases such as these in a0vitro diseasespecific induced pluripotent stem cells iPSCs carrying pathogenic SNVs or other genetic mutations can be obtained from patients9“ Repairing these iPSCs to generate isogenic revertant cells is a promising strategy for genome repair and s up new avenues for drug 1Pluripotent Stem Cell Research Unit in Department of Thyroid and Endocrinology School of Medicine Fukushima Medical University Hikarigaoka Fukushima Japan 2Department of Thyroid and Endocrinology School of Medicine Fukushima Medical University Fukushima Japan 3Department of Cell Modulation Institute of Molecular Embryology and Genetics IMEG Kumamoto University Kumamoto Japan 4Department of Dermatology Faculty of Medicine Oita University Yufu Japan 5Department of Microbiology School of Medicine Fukushima Medical University Fukushima Japan 6Office of Thyroid Ultrasound Examination Promotion Radiation Medical Science Centre for the Fukushima Health Management Survey Fukushima Medical University Fukushima Japan 7Fukushima Medical University Fukushima Japan email yokouchyfmuacjpScientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0c discovery1314 However the repair process remains problematic and a precise and convenient genome editing procedure has not yet been developedArtificial genome repair andor modification generally starts from a targetspecific doublestrand break generated by sitespecific nucleases1516 Doublestrand breaks are then repaired by a cell™s own genome repair machineries1516 However most of the break sites are incorrectly repaired by nonhomologous end joining and can result in gene knockout through the generation of nonspecific insertions or deletions indels1718 If a repair template carrying a repair base is coadministrated however the cleavage sites can be accurately repaired via homologydirected repair HDR1516 Sitespecific nucleases such as transcription activatorlike effector nucleases or the Streptococcus pyogenes Sp Cas9 nuclease are typically used as genome editing tools for human iPSCs19“SpCas9 is a type IIA endonuclease in the class clustered regularly interspaced short palindromic repeats CRISPRCas system that has been repurposed as a programmable sitespecific nuclease for genome engineering22“ Indeed SpCas9 has become a popular genome editing tool for genetically modifying human pluripotent stem cells19 Despite its efficient cleavage activity wildtype SpCas9 has a low DNA repair rate using HDR following plasmidbased administration It recuts the repaired site because the guide RNA has a “2base mismatch tolerance during sequence recognition leading to incorrect repair by nonhomologous end joining1718 To prevent recutting a blocking mutation must be introduced into the seed sequence of the guide RNA or into the protospaceradjacent motif PAM26“However for œscarless genome editing repairs with wildtype SpCas92749 each method has its obvious strengths and weaknesses For example CORRECT which includes excellent tricks to prevent recutting of the edited target by the editing tool can be performed even if the target recognition ability of the genome editing tool is insufficient however the need for two consecutive editing steps27 In comparison MhAX has high genome editing efficiency but cannot achieve completely scarless editing because singlebase markers are required Further as with the CORRECT method MhAX editing requires two consecutive edits49 increasing cost and time requirementsAnother recentlyidentified bacterial programmable sitespecific nuclease CRISPRCas12aCpf1 is a type VA endonuclease belonging to the class CRISPRCas system32 Among identified Cas12a enzymes those from Acidaminococcus sp BV3L6 As and Lachnospiraceae bacterium ND2006 show strong cleavage activity in mammalian cells32 These Cas12a endonucleases have unique features that complement Cas9 and expand the genome editing range First Cas12a recognises a Trich PAM upstream of the protospacer whereas Cas9 recognises a Grich PAM downstream of the protospacer32 Second two PAMinteracting variants have been generated that expand the Cas12a target range3334 Third Cas12amediated cleavage generates a staggered cut on the PAMdistal region of the target sequence as opposed to the PAMproximal blunt ends generated by Cas932 Finally and perhaps most importantly the guide or CRISPR RNA crRNA exhibits highfidelity target recognition meaning that Cas12a can precisely distinguish the target sequence at a singlebase resolution3536 Consequently the resulting offtarget effects are kept to a background level These features suggest that Cas12a might be suitable for precise diseasespecific iPSC repair because its recut activity is lowCas12a has already been used to knock out pathogenic genes in cancer cells37 generate insertions or twonucleotide substitutions in iPSCs38 and to induce exonskipping in diseasespecific iPSCs39 Thus we investigated whether Cas12a could be used to carry out allelespecific singlenucleotide repair of iPSCs carrying the pathogenic SNVs found in MEN2B and DEB patients in a single round of genome editing To accomplish this we used an AsCas12a PAM variant and singlenucleotide mismatch detection polymerase chain reaction SNMDPCR analysis in diseasespecific iPSCs to develop a precise convenient genomeediting procedure we have called OneSHOT One allelespecific single HDR and singlestranded oligodeoxynucleotide ssODN transient drug selection with SNMDPCR screening OneSHOT provides scarless singlenucleotide substitution of a pathogenic SNV in diseasespecific iPSCs within a0weeks The final modification rate is within a practical range for handpicking cloning Our findings suggest that this simple low cost procedure could be used for genome editing in a single step drastically reducing the time currently needed for scarless SNV repairResultsPrinciples of One‘SHOT repair of single‘nucleotide mutations AsCas12a is a highfidelity RNAguided sitespecific nuclease that binds to the target genomic DNA site via a 20nt guide sequence in the crRNA allowing it to discriminate the target sequence at the single nucleotide level Fig a0 Following the addition of a crRNA designed for a specific target sequence containing a singlenucleotide mutation AsCas12a selectively binds to the target sequence on the mutant allele and induces a doublestrand break leaving the wildtype sequence on the alternative allele unaffected Fig a0 In the presence of a ssODN wildtype sequence template the mutant nucleotide in the target sequence can be œrepaired to the wildtype sequence via the cellular HDR machinery To mark the repaired allele we labelled the ssODN with a singlenucleotide marker in the vicinity of the mutant nucleotide This label allowed us to easily identify the generepaired clones by allelespecific amplification40“42SNMDPCR detection of the singlenucleotide marker Fig a0 A complete outline of the OneSHOT workflow for SNV repair is provided in the Supplementary Information and in Supplementary Fig a0S1Allele‘specific single‘nucleotide substitution in MEN2B‘specific iPSCs Before carrying out allelespecific singlenucleotide repair of the pathogenic RET mutation we assessed whether the OneSHOT approach could be used to accomplish allelespecific singlenucleotide substitution of the wildtype alleleWe established FB414 human iPSCs from a patient with MEN2B using a Sendai viral vector protocol43 We then confirmed that the FB414 cells exhibited an embryonic stem celllike morphology and expressed pluripotent gene markers indicating that they were authentic iPSCs Supplementary Fig a0S2 and X7 To identify Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0cguide seqcrRNAHigh Fidelity SiteSpecific Nuclease SSN AsCas12aCpf1target alleleDSBTYCValleleTYCVHDRTYCVTYCVby SNMDPCR ntssODN w markerFigure a0 OneSHOT principles AsCas12a pale yellow and crRNA orange and grey lines selectively bind to a target sequence containing a pathogenic SNV red triangle on the target allele Binding leads to a doublestrand break in the target sequence on the target allele left but not in the corresponding wildtype sequence containing the wildtype nucleotide blue triangle on the alternative nontarget wildtype allele right When the ssODN repair template bluegreen line with the wildtype nucleotide blue triangle and a singlenucleotide marker a silent mutation for SNMDPCR screening green triangle is cotransfected with AsCas12a into the cells the target site on the pathogenic allele is repaired using the template by the endogenous HDR machinery In this case the intended geneedited clones are easily identified by positive screening for the singlenucleotide marker because the repaired expathogenic allele now carries the singlenucleotide markerpossible target sites for AsCas12a around the SNV of interest we first searched for PAMs recognised by wildtype AsCas12a or the RR and RVR variants which recognise TYCV and TATV PAMs respectively3334 We identified two PAM sites for the RR variant TYCV Y CT V ACG TTCC located 12bp upstream of the target nucleotide on the sense strand and TTCA located 7bp upstream of the target nucleotide on the antisense strand Fig a02a magenta lines Based on this information we designed two pairs of crRNAs crRNA_RET1 and crRNA_RET1 a0m and crRNA_RET2 and crRNA_RET2 a0m which contain guide sequences that specifically recognise wildtype and mutant target sequences respectively Fig a02aTo test the cleavage activity and targetrecognition specificity of AsCas12a_RR using these crRNAs we performed a T7E1 assay using 409B2 human iPSCs carrying the wildtype RET sequence in the target site Fig a02a middle The crRNAs for the wildtype sequence crRNA_RET1 and crRNA_RET2 each exhibited significant cleavage activity towards the wildtype target sequence Fig a02b P and P respectively By contrast the crRNAs for the mutant sequence crRNA_RET1 a0m and crRNA_RET2 a0m showed extremely weak activity Fig a02b P for both A more accurate ICE analysis showed no significant activity of the crRNAs on the WT allele Supplementary Fig a0X2a These results indicate that the crRNAs for the mutant sequence do not have significant if any activity on the WT alleleHowever the observed cleavage activity of AsCas12a_RR in conjunction with crRNA_RET1 was significantly higher than that with crRNA_RET2 Fig a02b P Supplementary Fig a0X2a Puromycin treatment further promoted the cleavage activity of AsCas12a_RR with crRNA_RET1 Fig a02b P To test the applicability of the method to carry out allelespecific singlenucleotide substitution in human iPSCs we attempted to replace the wildtype nucleotide RET c2753T at the Met918 site in the wildtype allele in FB414 MEN2BiPSCs Fig a02c Following electroporation of the pY211puro vector which expresses AsCas12a_RR and crRNA_RET1 Fig a02c blue line and a ssODN modification template ssODN_RET_M918T_I913silentC carrying both a variant nucleotide at Met918 and a singlenucleotide marker at Ile913 Fig a02c red C and light green C respectively into FB414 cells we conducted SNMDPCR screening Overall clones were positive for the substitution Fig a02c d and GE1 in Table a0 Direct sequencing of the target sequence revealed that clones contained the wildtype allelespecific introduction of the mutant nucleotide at the target site T C substitution resulting in the Met918Thr substitution Fig a02e red arrow along with the singlenucleotide marker T C substitution leading to a silent mutation at Ile913 Fig a02e blue arrow The HDR efficiency was Table a0We then searched for offtarget sequences corresponding to the target sequence using the web tool CHOPCHOP v244 and detected no indels in either of the predicted two offtarget sites by Sanger sequencing Table a0 GE1 and by AmpliSeq Supplementary Table a0X4 These results indicated that the OneSHOT method could be used to replace a single nucleotide in an allelespecific manner while minimising offtarget effects As in the preliminary experiment direct sequencing analysis around the target sites revealed no duplication events in the unintended geneedited clones suggesting that most of the intended geneedited clones had clonally proliferated Supplementary Fig a0S3 GE1Scientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0c–¸Figure a0 Singlenucleotide substitution of the RET wildtype sequence in MEN2B iPSCs a Human RET locus containing the MEN2B mutation and crRNA of AsCas12a_RR for the mutation Top exon of the RET locus Middle the wildtype WT allele sequence Blue letters indicate the wildtype nucleotide at Met918 underlined Bottom the mutant allele sequence Red letters indicate the single missense mutation caused by a TC substitution producing a Met918Thr substitution underlined Coloured lines indicate the guide sequence template for the crRNA The pink line indicates the AsCas12a_RR PAM Coloured dashed lines indicate the sites cleaved by AsCas12a_RR with the corresponding crRNA b T7E1 assay using human wildtype iPSCs 409B2 electroporated with AsCas12a_RR and the different crRNAs crRNA_RET1 crRNA_RET1 a0m crRNA_RET2 or crRNA_RET2 a0m targeting exon Left the cropped gel images Arrowheads indicate cleaved bands The fulllength gels are presented in Supplementary Figure a0S7 Right statistical analysis of the cleavage activity and specificity of AsCas12a_RR with the crRNAs following selection with different concentrations of puromycin c HDRmediated editing for generating artificial homozygous MEN2B using AsCas12a_RR with crRNA_RET1 selectively targeting RET_Met918 in the wildtype allele d SNMDPCR analysis of the first round of screening The cropped gel image is shown here The arrowhead indicates positive PCR amplicon a0bp The fulllength gel is presented in Supplementary Figure a0S8 e Sequencing of the original and modified MEN2B iPSCs FB414 Top original sequence of RET exon with a T C substitution in the MEN2B mutant allele Bottom the modified RET sequence The T C substitution resulting in a homozygous Met Thr substitution Red and blue arrows indicate the positions of the pathogenic SNV and the singlenucleotide marker respectively Underlining indicates the codons affected by the editing A more detailed explanation is provided in the œExtended Figure Legends in the Supplementary InformationAllele‘specific single‘nucleotide repair of a pathogenic RET variant To repair the pathogenic SNV RET c2753TC in the mutant allele in FB414 cells we first tested the cleavage activity and target recognition specificity of AsCas12a_RR using crRNA_RET1 a0m and crRNA_RET2 a0m Fig a02a in a homozygous MEN2B iPSC line with mutations in RET exon in both alleles GE19 genotype RETMet918ThrMet918Thr RETIle913 silentC Fig a02e bottom The T7E1 assay confirmed that the MEN2B target sequence was selectively cleaved by AsCas12a_RR with either crRNA_RET1 a0m or crRNA_RET2 a0m but not with crRNA_RET1 or crRNA_RET2 Fig a03a The ICE analysis revealed that only the AsCas12a_RR with crRNA_RET1 a0m exhibited strong cleavage activity on the target sequence Supplementary Fig a0X2b therefore we selected the crRNA_RET1 a0m for use in subsequent experimentsWe then carried out OneSHOT repair in FB414 cells using AsCas12a_RR with crRNA1m and a ssODN repair template containing a repair nucleotide at Met918 and a singlenucleotide marker at Ile913 Fig a03b T in blue and C in light green respectively Subsequent SNMDPCR screening showed that clones were positive Fig a03c GE2 in Table a0 while direct sequencing confirmed that of the positive clones contained the introduced wildtype nucleotide at the target site C T substitution leading to a Thr918Met substitution repair Fig a03d red arrow These clones also contained the singlenucleotide marker T C substitution leading to a silent mutation at Ile913 Fig a03d blue arrow The overall HDR efficiency was Table a0 GE2 and we detected no offtarget effects by Sanger sequencing Table a0 GE2 and by AmpliSeq Supplementary Table a0X4 As in the preliminary experiment direct sequencing analysis around the target sites revealed no duplication events in the unintended geneedited clones suggesting that most of the intended geneedited clones had clonally proliferated Supplementary Fig a0S3 GE2Allele‘specific single nucleotide repair of a pathogenic variants in RET and COL7A1 without a single‘nucleotide marker We next investigated whether the OneSHOT method could be used to repair the pathogenic SNV in RET without including the singlenucleotide marker which would achieve true scarless repair We therefore performed OneSHOT repair in the FB414 cells using AsCas12a_RR crRNA_RET1 a0m and the ssODN repair template with only a wildtype nucleotide at Met918 In the subsequent SNMDPCR screening for the pathogenic SNV no amplicons were obtained from repaired clones because the pathogenic SNV was lost from the mutant allele Fig a04a Overall we identified negative clones by SNMDPCR screening for the pathogenic SNV and direct sequencing revealed that carried only the wildtype nucleotide at Met918 Fig a04cd and GE4 in Table a0 In this experiment the overall HDR efficiency was Table a0 GE4 and no indels were detected in the two predicted offtarget sites by Sanger sequencing Table a0 GE4 and by AmpliSeq Supplementary Table a0X4We next attempted to perform scarless repair of a pathogenic SNV in iPSCs derived from a patient with DEB to confirm the applicability of the approach for other hereditary diseases We generated iPSCs from a patient with DEB autosomal recessive compound mutation COL7A1pGly2138Ter COL7A1c3591del13insGG and aimed to substitute the pathogenic SNV c6412G T pGly2138Ter in exon Supplementary Fig a0S4ab Scarless OneSHOT using AsCas12a_RR with crRNA_COL7A11 a0m plus the repair template scarlessly repaired the pathogenic SNV in the mutant allele Supplementary Fig a0S4cde and GE5 in Table a0 with a substitution rate of No indels were detected in the seven predicted offtarget sites Supplementary Table a0S2 Supplementary Table a0X4 Unlike the scarless OneSHOT for RET_Met918Thr in FB414 cells Fig a0 GE4 in Table a0 identical sequences within the target site were observed among the unintended geneedited clones suggesting that these clones were likely duplicated Supplementary Fig a0S3 GE5Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0cabcRET locus on Chr 10q11exon M918PAM crRNA_ RET1 bpWT allelecrRNA_RET2 PAMPAM M918TcrRNA_RET1mMutant allelecrRNA_RET2m PAM M NCpuro crRNA_RET 1m 2m T7E1 assay in WT iPSC 409B2 nsns xedni ledniNCcr2mpuro cr2mpuro cr2mpuro cr2puro cr2puro cr1puro cr1puro cr1mpuro cr1mpuro cr2puro cr1puro cr1mpuro crRNApuro treatmentRETI913 C T PAMRET M918 C TcrRNA_RET1WT allele sequencessODN_RET_M918T_I913silentCModified WT allele sequence Mutant allele sequencede1KM M1KIle913 A T T A T TCMet918 A TC G A C GIleIleMetThrThrScientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0cGene editing CellGE1GE2GE3GE4GE5FB414FB414FB414FB414B1173Genotype phenotypeOriginal †’ DestinationRETM918T †’ RETM918TM918T I913_silentCMEN2Ba †’ MEN2B homo with SN MarkerRETM918T †’ RET I913_silentCMEN2Ba †’ MEN2B revertant with SN MarkerRETM918T †’ RET I920_silentCMEN2Ba †’ MEN2B revertant with SN markerRETM918T †’ RETMEN2Ba †’ MEN2B scarless revertantCOL7A1G2138X del13 ins GG †’ COL7A1 del13 ins GGDEBb †’ DEB scarless revertantNo of total picked clones TCNo of 1st screening passed clones SNMD PCRNo of 2nd screening passed clonessequencing1stTC 2nd1st 2ndTC Table OneSHOT and scarless OneSHOT gene editing GE experiments After electroporation of the AsCas12a_RR expression vector and the ssODN template into the cells the crude DNA samples from the singlecell derived colonies that expanded on the master plates were subjected to SNMDPCR in the first screening round For positive screening colonies with amplifiable “200bp fragments from the SNMDPCR primer pair were the intendedclone candidates GE13 For negative screening colonies lacking PCR amplification were the intendedclone candidates GE4 and In the second screening round we directly read the sequences around the target site of the DNA fragments amplified by Tks Gflex DNA polymerase in each sample silentC a silent mutation generated by replacement with a cytidine for SN marker a Multiple endocrine neoplasia type 2B B Dystrophic epidermolysis bullosa Positive screening results Negative screening resultsSampleSiteGenomic location No of mis matchesOriginalRET exon target1chr10 GE1GE1GE2GE2GE3GE3GE4GE4Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget chr15 chr4 chr15 chr4 chr15 chr4 chr15 chr4 Sequencea including mismatchesTTCC AGT TAA ATG GAT GGC AAT TGTTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG Indel ratio b Table Offtarget effects of AsCas12a_RR in gene editing experiments “ GE1GE4 After amplifying the offtarget candidates predicted by CHOPCHOP v2 from the intended geneedited iPSC clones we directly read the sequences around the candidate sites after Sanger sequencing with specific primers a Underline indicates the PAM of the AsCas12a_RR variant Lower letters indicate mismatched bases in the offtarget candidates as compared with the original target sequence b Number of indel clones relative to the number of analysed clonesDiscussionMany hereditary human diseases are caused by singlenucleotide mutations These singlebase alterations have the potential to drastically alter protein structure and function Although most singlenucleotide mutations are completely harmless silent repair of pathogenic SNVs would significantly improve the quality of life and life expectancy of patients with hereditary diseases Thus in the present study we investigated whether we could achieve scarless repair of pathogenic SNVs in pluripotent stem cells from patients with two different types of hereditary disease MEN2B and DEB More importantly we aimed to carry out the repairs in a single stepUsing the OneSHOT approach developed in this study we successfully repaired a RET gene SNV in MEN2B iPSCs with the addition of a singlenucleotide selective marker in a single step We then confirmed that the same technique could be used to carry out scarless repair in MEN2B and DEBspecific iPSCs without the need for the singlenucleotide marker Scarless repair where no trace of gene editing is left around the target sequence is the goal of any gene editing technique because it safely repairs mutations in noncoding genomic regions without any secondary effects In contrast the inclusion of marker sequences during gene editing can have downstream effects Such secondary effects include the introduction of noncoding SNVs to cryptic splice sites Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0ccausing abnormal RNA splicing4546 and mutations that introduce a premature termination codon resulting in unstable mRNA46 Noncoding mutations affecting regulatory elements can also interfere with gene regulation through loss of function resulting in reduced gene expression or gain of function resulting in gene mis or overexpression4748 Therefore scarless repair is crucial for maintaining genome integrity and preventing unknown secondary effects in the target geneSeveral other methods of pathogenic SNV repair have been developed including CORRECT2627 and MhAX49 However all currently available methods have inherent obstacles to achieving scarless SNV repair in a fast and errorfree manner To overcome some of these obstacles we used the AsCas12a nuclease which has highfidelity targetrecognition3536 circumventing the need for a blocking base to inhibit recutting as is required in other methods2627 We also performed SNMDPCRbased negative screening for the pathogenic SNV which easily detects candidate clones containing the intended alteration As a result of these modifications we achieved absolute scarless editing of the RET and COL7A1 SNVs see Fig a0 Supplementary Fig a0S4 and GE4 and GE5 in Table a0 Another advantage of the AsCas12a nuclease was the ability to carry out SNV repair in a single step because only one round of HDR is required for gene editing Fig a0 The OneSHOT method was used to repair the SNVs in RET and COL7A1 within a 3week period with sufficient efficiency for handpicking In contrast other methods can take up to “ a0months to generate the intended geneedited clone because two rounds of HDRMMEJ may be required262749 However similar to our approach the CORRECT method can achieve scarless singlenucleotide substitution thus ensuring high sequence fidelity around the target site in geneedited cells Fig a0 and Supplementary Fig a0S4 Conversely MhAX leaves a silent single nucleotide mutation around the target site for use in screening49 Another difference is that the dsDNA template in MhAX can be randomly integrated into the genome outside of the target site by nonhomologous end joining50 whereas the ssODN templates used for OneSHOTscarlessOneSHOT and CORRECT approaches are not randomly integrated51 Thus the OneSHOT method developed for SNV repair in the current study appears to have several advantages over currently available methods see Supplementary Table a0S3In the CORRECT procedure the cuttomutation distance the distance between the CRISPRSpCas9 cleavage site and the blocking mutation is a crucial factor for HDR efficiency and zygosity determination2627 We therefore searched for more appropriate sites for the singlenucleotide markers by first comparing the efficacies of three singlenucleotide markers set in different positions around the target site using a PCRrestriction fragment length polymorphism RFLP assay52 We found that two of the markers showed similar HDRspecific cleavage activity while no cleavage activity was detected for the third marker Supplementary Information and Supplementary Fig a0S5 suggesting that Ile920 could be used as an alternative singlenucleotide marker Testing of HDR efficiency in FB414 cells following OneSHOT repair using the alternative marker again confirmed that the singlenucleotide substitutions in the geneedited clones were effectively detected by positive screening using SNMDPCR for a singlenucleotide marker Supplementary Information and Supplementary Fig a0S5 We do note however that the efficiency of identification might depend on the position of the singlenucleotide marker and the primers used for SNMDPCRDespite our success in repairing the pathogenic SNVs in a single step the study has several limitations The OneSHOT method only requires one PCR run thereby reducing the time and cost compared with standard PCR“RFLP screeningbased methods which require up to three steps5253 However we found that falsepositive clones are included in the population after the first SNMDPCR screen Supplementary Fig a0X8 Therefore we are currently designing a simple way to discriminate false clones from authentic clones using a PCRbased procedure We also noted that the geneedited cell lines generated by OneSHOT are not always clonal This situation arises because high cell densities occur in the culture during puromycin selection a0days and in the recovery culture “ a0days prior to clonal expansion However assessment of our data suggests that a 1day recovery culture and sufficient singlecell suspension at the reseeding stage can prevent duplication and ensure clonal establishment of the geneedited cells Using the current protocol we estimate that the HDR substitution rate is “ While this is sufficient to permit a handpicking cloning protocol it is lower than that achieved by Cas12a in fertilised eggs from model animals3954 We hoped to improve this rate by combining OneSHOT with other procedures based on alternative principles such as introducing a blocking base into the repair template2627 andor using HDRNHEJ modification compounds3863“ We have examined whether the modification compounds can promote HDR however the compounds examined in this study had no HDRpromoting effects in our experimental system Supplementary Fig a0X4It is important to emphasise though that the procedure depends on highfidelity target recognition by the sitespecific nuclease Thus the only enzymes appropriate for the OneSHOT procedure include highfidelity variants of engineered SpCas955“ or naturally highfidelity Cas9 orthologues59“ Finally while we confirmed the expression of pluripotency markers in the geneedited clones data not shown we next aim to carry out functional analyses to confirm the differentiation potential of the repaired cells Therefore further work is needed to finetune the protocol and to confirm differentiation potential and functionality of the proteins in the corrected cell populationsTo increase the reliability of the OneSHOT method it is important to show the robustness of OneSHOT and the fidelity of the repair In order to demonstrate these issues we performed targeted NGSbased deep AmpliSeq analysis of the target sequence With regard to repair fidelity the AmpliSeq analysis showed that accurate singlenucleotide substitutions were achieved by HDR that were faithful to the ssODN template and occurred at sufficient frequency Supplementary Fig a0X3ac “ These results suggest that the method has good repair fidelity With regard to the robustne

Thyroid_Cancer

Polyphenol induced improvements in glucose metabolism are associated with bile acid signaling to intestinal farnesoid X receptorKevin M Tveter1 Jose A Villa Rodriguez Alrick J Cabales1 Li Zhang2 Fiona G Bawagan1 Rocio M Duran1 Diana E Roopchand To cite Tveter a0KM Villa Rodriguez a0JA Cabales a0AJ et a0al Polyphenol induced improvements in glucose metabolism are associated with bile acid signaling to intestinal farnesoid X receptor BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 –º Additional material is published online only To view please visit the journal online http dx bmjdrc KMT and JAV R contributed equallyReceived March Revised June Accepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Food Science Rutgers The State University of New Jersey New Brunswick New Jersey USA2Key Laboratory of Genomic and Precision Medicine Beijing Institute of Genomics Chinese Academy of Sciences Beijing Branch Beijing ChinaCorrespondence toDr Diana E Roopchand roopchand sebs rutgers eduIntroduction Bile acid BA biotransformation by gut bacteria impacts BA profile and signaling to nuclear receptors such as the farnesoid X receptor FXR regulating glucose metabolism Altered BA FXR signaling was therefore investigated as a potential mechanism linking polyphenol induced gut bacterial changes and improved glucose metabolismResearch design and methods Diabetic dbdb were fed low fat diet LFD or LFD supplemented with a proanthocyanidin rich extract of grape polyphenols LFD GP for weeks Metabolic phenotypes serum BAs gut microbiota composition and gene expression markers relevant to gut barrier and glucose metabolism were assessed Gut anoids were used to investigate effects of individual BAs on ileal FXR activityResults Compared with LFD fed controls GP supplemented dbdb mice showed improved glucose metabolism decreased relative abundance of gut bacteria associated with production of secondary BAs SBAs and depleted serum levels of SBAs taurohyodeoxycholic acid THDCA ωmuricholic acid ωMCA and tauroωmuricholic acid TωMCA Serum levels of primary BAs PBAs increased consistent with higher gene expression of PBA synthesis enzyme Cyp7a1 GP induced BA changes associated with FXR inhibition as evidenced by reduced expression of FXR responsive genes Shp Fgf15 and Fabp6 in ileum tissue as well as hepatic Shp which negatively regulates PBA synthesis GP treatment did not affect expression of hepatic Fxr or expression of Abcb11 Slc51b and Obp2a genes controlling BA transport Ceramide biosynthesis genes Smpd3 Sptlc2 and Cers4 were decreased in liver and intestine suggesting lower tissue ceramides levels may contribute to improved glucose metabolism THDCA ωMCA and TωMCA behaved as FXR agonists in ileal anoid experiments therefore their depletion in serum of GP supplemented dbdb and wild type WT mice was consistent with FXR inhibitionConclusion These data suggest that by altering the gut microbiota GPs modify BA FXR signaling pathways to promote glucoregulationINTRODUCTIONDietary polyphenols in plant based foods contribute to improved glycemic control in Significance of this studyWhat is already known about this subject –º Dietary polyphenols such as proanthocyanidins alter the gut microbial community and are associated with improved metabolic resilience in humans and mice –º Bile acids BAs signal to farnesoid X receptor FXR a nuclear transcription factor that regulates hepatic BA synthesis and glucose metabolismWhat are the new findings –º Grape polyphenol GP supplementation decreased abundances of gut bacterial taxa associated with secondary BA SBA production concomitant with depletion of SBAs and increased primary BAs PBAs in murine serum –º GP supplementation suppressed expression of FXR regulated genes Fgf15 Fabp6 and Shp an inhibitor of PBA synthesis which was consistent with increased serum PBAs –º GP induced FXR inhibition was associated with decreased expression of genes required for biosynthesis of ceramides which impair glucose homeostasis –º The SBAs depleted in GP treated mice were revealed as FXR agonists in ileal gut anoidsHow might these results change the focus of research or clinical practice –º This study highlights BA FXR signaling pathways as an important mechanism for further investigation in human intervention studies of dietary polyphenols and metabolic healthmice and humans1“ Improved glucose metabolism in mice supplemented with berryfruit extracts was related to a proanthocyanidin PAC induced bloom of Akkermansia muciniphila1 a microbe shown to attenuate symptoms of metabolic syndrome MetS and type2 diabetes T2D in obese mice and humans5 We hypothesized that metabolic improvements also result from changes in BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismhost derived bacterial metabolites regulating host energy metabolismBile acids BAs are signaling molecules linking the gut microbiota to host energy metabolism Primary BAs PBAs synthesized in the liver are conjugated with taurine mice or glycine humans7 Bacterial derived bile salt hydrolases deconjugate taurine or glycine from the sterol core of PBAs followed by bacterial transformations such as αdehydroxylation dehydrogenation and epimerization which generate secondary BAs SBAs7 PBAs and SBAs signal to key regulators of energy metabolism such as nuclear transcription factor farnesoid X receptor FXR and Takeda G protein coupled receptor TGR57 Fxrˆ’ˆ’ mice were protected from high fat diet HFD induced obesity and had altered BA and gut microbiota higher Bacteroidetes less Firmicutes profiles compared with wild type WT mice8 Transfer of cecal microbiota from HFD fed Fxrˆ’ˆ’ mice to germ free WT mice resulted in less adiposity and improved glucose metabolism compared with mice that received microbiota from HFD fed WT mice suggesting the altered gut microbial and BA profiles in FXR deficient mice contributed to metabolic improvements8 Studies using tissue specific FXR knockout mice showed that intestinal FXR activity was required to mediate HFD induced metabolic dysfunctions9 Pharmacological inhibition of intestinal FXR using tempol antibiotics metformin or glycinemuricholic acid Gly MCA led to gut microbial remodeling and improved glucose and lipid homeostasis9“In addition to the A muciniphila bloom mice fed HFD supplemented with PAC rich grape polyphenols GPs showed other profound bacterial community changes raising the possibility that altered BA profile and signaling could contribute to observed improvement in glucose homeostasis1 The present study provides compelling evidence in support of this hypothesisRESEARCH DESIGN AND METHODSDietsA complex of grape polyphenols and soy protein isolate GP SPI was prepared as previously described1 Nutritional profiles for SPI and GP SPI are provided in online supplementary table Isocaloric ingredient matched diets Research Diets New Brunswick New Jersey USA used in this study were previously described4 Mice were fed low fat diet LFD containing SPI LFD formulated with GP SPI delivering GP LFD GP HFD containing SPI or HFD formulated with GP SPI delivering GP HFD GP Online supplementary table provides diet formulation detailsAnimalsFour week old dbdb mice B6BKSD LeprdbJ stock no Jackson Laboratory Bar Harbor Maine USA were single housed on a hour lightdark cycle to hours light with ad libitum access to LFD and water in a controlled temperature room °C±°C for week for acclimation Mice were randomized to receive LFD n7 or LFD GP n7 for days Metabolic phenotyping included food intake body weight body composition EchoMRI in1 system Echo Medical Systems Houston Texas USA and oral glucose tolerance tests as previously described1 On day mice were euthanized by CO2 inhalation followed by cardiac puncture and collection of tissues as previously described1 Fecal and cecal samples were collected for microbial community profiling Wild type C57BL6J mice n10 aged weeks were acclimated on LFD for week and randomly divided into two groups and fed HFD or HFD GP n5 per group for weeks after which mice were euthanized by CO2 inhalationTissue gene expression analysisRNA was extracted from ileum jejunum colon and liver “ mg with RNeasy Plus Universal Mini Kit QIAGEN followed by RNA cleanup Machery Nagel RNA purification kit RNA µg was reverse transcribed to cDNA and qPCR was performed using TaqMan primers online supplementary table as previously described4 Data were analyzed using 2ΔCT method using hydroxymethylbilane synthase HMBS as housekeeping geneqPCR of A muciniphila in fecal and cecal samples was performed as previously described1 Briefly gDNA extracted from fecalcecal samples was diluted to ngµL for quantification of A muciniphila abundance relative to total bacteria and archaea by qPCR using A muciniphila AM1 AM2 and universal primer U341F U515R sets1 16S rRNA gene sequencingGenomic DNA was extracted from fecal and cecal samples collected from dbdb mice Illumina protocols were used to prepare V4 amplicons of the 16S rRNA gene for sequencing on a MiSeq system resulting in × reads Denoising and clustering were conducted using DADA2 algorithm to differentiate sequences into amplicon sequence variants ASVs for downstream analysis using QIIME Details are available in online supplementary materialsSerum biochemistrySerum for BA analysis was collected by cardiac puncture BAs were analyzed on a Water™s Alliance e2695 HPLC system Waters Milford Massachusetts USA coupled to a Water™s Acquity QDA mass spectrometer equipped with an electrospray interphase ESI Waters Milford Massachusetts USA and quantified by external calibration curves using pure standards Details are provided in online supplementary materials Serum leptin polypeptide YY PYY interleukin6 IL6 and insulin were determined using a MILLIPLEX MAP Mouse Metabolic Hormone Magnetic Bead kit Millipore with a MagPix instrument Luminex as previously described4anoid experimentsIntestinal crypts were isolated from WT C57BL6J mouse ileum according to established methods16 Crypts were counted and added to Matrigel five crypts per µL BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cCorning growth factor reduced and µL was added per well in well plates and allowed to polymerize °C for “ min followed by addition of mL of complete growth medium CGM see online supplementary materials CGM without Y27632 dihydrochloride monohydrate was replaced every “ days anoids were passaged every “ days ratio Mature anoids days postpassage were treated in triplicate n3 wells with methanol vehicle chenodeoxycholic acid CDCA µM alone or CDCA plus another BA µM in CGM for hours RNA was extracted for qPCR analysis using TaqMan primers as described above4Statistical analysesAnalyses were conducted using Prism GraphPad Software La Jolla California USA Significant differences two groups were assessed with a two tailed unpaired Student™s t test with Welch correction for unequal variance when needed or by one way or two way analysis of variance groups followed by Sidak™s or Tukey™s multiple comparison test Statistical analysis of alpha and beta diversity metrics was calculated using QIIME details in online supplementary materials ADONIS and permutation analysis were conducted using R Studio V342 R Studio Software Boston Massachusetts USA and Python RESULTSGPs improve glucose metabolism in dbdb mice independent of obesityLeptin receptor deficient dbdb mice develop obesity gut barrier dysfunction and hyperglycemia independent of HFD feeding17 Compared with LFD fed controls dbdb mice fed LFD containing PAC rich GPs LFD GP for weeks showed significantly improved oral glucose tolerance figure 1A4 Area under the curve remained stable over time for the LFD GP group but increased in the LFD group figure 1A Mice fed LFD GP initially exhibited a transient decrease in food intake presumably due to taste but at later time points LFD and LFD GP groups consumed similar amounts of food ± and ± gdaymouse respectively p005 online supplementary figure 1A The LFD GP group consumed ± mg of GPs per day Both groups had similar body weight gain body composition and liver weights online supplementary figure 1B“DGPs promote a bloom in A muciniphila without improving markers of metabolic endotoxemiaCompared with controls GP supplemented dbdb mice had decreased αdiversity as evidenced by richness Shannon index and Faith™s phylogenetic diversity index figure 2A Principal coordinate analysis showed that GPs significantly altered fecal community structure within days online supplementary figure As previously observed1 GPs promoted increased cecal mass online supplementary figure Metabolism1E a phenotype common to antibiotic treated and germ free mice and consistent with reported antibacterial properties of PACs18 GP supplemented mice had an increased relative abundance of phylum Verrucomicrobia at the expense of Firmicutes in fecal days “ and ceca samples day figure 2BC online supplementary figure 3AB Ceca of GP supplemented mice had higher relative abundance of Bacteroidetes 25LFD although except for day fecal Bacteroidetes remained similar between groups figure 2B online supplementary figure 3C GP supplementation did not consistently alter levels of Proteobacteria or Actinobacteria figure 2B online supplementary figure 3DE Consistent with increased Verrucomicrobia quantitative qPCR analyses confirmed that GPs promoted a bloom in Akkermansia muciniphila figure 2C at the expense of other taxa figure 2DReduced abundance of A muciniphila and metabolic endotoxemia characterized by gut dysbiosis compromised gut barrier integrity lipopolysaccharide LPS leakage and intestinal inflammation was associated with impaired glucose metabolism in obese mice and humans5 Oral administration of A muciniphila in obese diabetic mice and humans resulted in improved glucose homoeostasis and attenuated metabolic endotoxemia through improved gut barrier integrity5 GP induced improvement in glucose metabolism in HFD fed mice has therefore been considered a consequence of the A muciniphila bloom leading to reduced inflammation and increased gut barrier integrity1 Reduced metabolic endotoxemia could not however explain the improved glucose tolerance in GP supplemented dbdb mice despite increased A muciniphila in feces day and cecum figure 2C Relative to control GP supplementation did not change intestinal gene expression of markers of inflammation Tnf Il6 iNOS gut barrier integrity Tjp1 Ocln Muc2 peripheral lipid deposition Fiaf or glucose transport Glut2 online supplementary figure 4A“C GP supplemented dbdb mice had less Muc3 expression in jejunum and ileum online supplementary figure 4AB suggesting lower mucus secretion There were no differences in serum insulin IL6 or glucoregulatory hormones PYY and leptin online supplementary table These data suggested that other mechanisms were driving improved glucose metabolismGPs reduced gut bacterial taxa associated with production of SBAsAlthough total bacterial and archaeal abundance was not significantly different between LFD and LFD GP groups figure 2E GPs induced profound gut microbial changes online supplementary figure that would be expected to alter BA diversity abundance and signaling Targeted liquid chromatography mass spectrometry LC MS analysis revealed that GP supplemented dbdb mice had higher serum concentrations of BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure GPs improve glycemic control in obese dbdb mice A Oral glucose tolerance tests were performed after mice consumed LFD squares n7 or LFD GP closed squares n7 for and weeks Blood glucose concentrations mgdL expressed as mean±SD were measured at the indicated time points “ min following administration of glucose gkg Between group difference was determined by unpaired two tailed t test with Welch™s correction p01 p001 B Scatter plot of blood glucose AUC determined for individual mice at weeks and where mean AUC±SD are shown as horizontal and vertical bars Difference was determined using two way ANOVA followed by Tukey™s intragroup posthoc test different letters indicate significant difference p005 or by Sidak™s intergroup posthoc test p001 ANOVA analysis of variance AUC area under the curve GP grape polyphenol LFD low fat dietPBAs driven by increased cholic acid CA and taurocholic acid TCA figure 3A and C online supplementary figure 6A Concentrations of muricholic acid MCA tauroMCA TMCA tauroαMCA TαMCA taurochenodeoxycholic acid TCDCA were similar between groups αMCA was not detected in LFD GP group figure 3A online supplementary figure 6A Increased PBA pool correlated with an overall reduction in SBAs figure 3C where ωMCA TωMCA and taurohyodeoxycholic acid THDCA were undetectable in dbdb mice fed LFD GP although deoxycholic acid DCA was increased figure 3A Total serum BAs were similar between groups figure 3BThe GP induced depletion of SBAs was unrelated to the leptin receptor mutation in dbdb mice Compared with HFD fed controls wild type C57BL6J mice fed HFD supplemented with GP HFD GP for weeks also showed serum depletion of SBAs THDCA ωMCA and TωMCA reduced overall levels of serum SBAs and no difference in concentration of total serum BAs online supplementary figures 6B and Unlike dbdb mice GP supplemented WT mice had decreased tauro deoxycholic acid TDCA and no significant difference in CA or overall PBA pool although TCA concentration was increased online supplementary figures 6B and In dbdb mice multiple correlation analyses were performed to associate GP induced changes in serum BAs figure to changes in fecalcecal gut bacteria online supplementary figure The GP induced increase of CA TCA PBAs and DCA SBA was positively and significantly associated with increased abundance of Akkermansia Blautia Clostridium ASV59 and S247 figure 3EF Blautia and Clostridium possess BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Supplementation of GPs remodeled gut microbial composition and diversity A Microbial αdiversity metrics of fecal samples collected from mice after consuming LFD squares n7 or LFD GP closed squares n7 for indicated number of days “ and of cecal Cec samples collected at endpoint day Difference was determined by two way ANOVA followed by Sidak™s test B Per cent relative abundance of main bacterial phyla based on Naïve Bayes taxonomic classifier non rarified data Phyla present at relative abundance were classified as ˜Other™ Percentage of C A muciniphila DNA and D nonA muciniphila DNA relative to total bacterial and archaeal DNA in fecal and cecal samples where relative abundance was determined by qPCR using primers specific for A muciniphila AM1AM2 and universal V4 primers 515F806R E Total 16S bacterial and archaeal gene countsng of gDNA extracted from fecal or cecal samples Group mean±SD at each time point are illustrated by horizontal and vertical lines Difference between diet groups over time panels C“E was determined using two way ANOVA followed by Sidak™s posthoc test intergroup comparison or Tukey™s posthoc test intragroup comparison Different letters a b and c indicate significant difference within diet groups p005 while the same letter indicates no difference Between group differences panels A C D E p005 p001 p0001 p00001 ANOVA analysis of variance ASV amplicon sequence variants AUC area under the curve GP grape polyphenol LFD low fat diet PD phylogenetic diversity7αdehydroxylating activity therefore taxa within these genera may be responsible for DCA production via dehydroxylation of CA21 GP treated mice had lower levels of taxa within the Clostridiales order ASV50 and Ruminococcaceae and Lachnospiraceae families ie ASV56 and and Clostridium genus ASV74 online supplementary figure which are reported to possess 7αdehydroxylating activity required for conversion of PBAs to SBAs21“ In agreement with evidence from mice and humans the GP induced decrease in these bacterial taxa encoding 7αdehydroxylation activity was highly correlated to the GP associated depletion of SBAs ωMCA and TωMCA figure 3EF21 Finally positive and significant associations were found between reduced αMCA TωMCA andor ωMCA and reductions in taxa belonging to RF39 Anaeroplasma Ruminococcus Butyricicoccus Dorea Dehalobacterium Christensenellaceae Lactococcus Streptococcus and Oscillospira figure 3EFGPassociated BA changes promote inhibition of FXR signaling and upregulation of classical BA synthesis pathwayTo investigate the consequences of GP induced serum BA changes gene expression of FXR TGR5 and their downstream targets were analyzed in tissues GP supplementation did not change ileal Fxr gene expression however FXR transcriptional activity was decreased as expression of its target genes fibroblast growth factor Fgf15 small heterodimer partner Shp and ileal BA binding protein I BABP gene Fabp6 were suppressed in ileum figure 4A Intestinal FXR signaling negatively regulates hepatic PBA synthesis through interaction of Fgf15 with hepatic fibroblast growth factor receptor 4Klotho receptor complex or by regulating BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure GPs increased PBAs and reduced SBAs in serum in association with depletion of bacterial ASVs related to SBA production A LC MS and pure standards were used to determine the mean concentration of individual PBAs and SBAs mean±SD in serum samples n7 samples collected from individual mice fed LFD white bars or LFD GP purple bars B Total serum BA concentration mean±SD was determined based on sum of individual PBA and SBA concentrations shown in panel A quantified for each mouse fed LFD n7 or LFD GP n7 C Serum PBA and SBA concentrations mean±SD in LFD versus LFD GP diet groups were calculated by summing the individual PBAs or SBAs shown in panel A D Using data from panels B and C pie charts illustrate pooled PBAs green and pooled SBA gray as a percentage of total serum BA concentration quantified for LFD and LFD GP groups For panels A“C significant difference was determined using unpaired two tailed t test followed by Welch™s correction p005 p001 Heatmap representation of the Spearman™s r correlation coefficient between serum BA profile and significantly changed bacterial ASVs at genera or family level of taxonomy in GP treated mice relative to control diet group from E day27 fecal samples or F day29 cecal samples Shades of red indicate serum BA and bacterial taxa are positively correlated to while shades of blue indicate a negative correlation to ˆ’ Significant positive or negative correlations are shown p005 p001 p0001 ASV amplicon sequence variant CA cholic acid DCA deoxycholic acid GP grape polyphenol LC MS liquid chromatography mass spectrometry LFD low fat diet MCA muricholic acid PBA primary bile acid SBA secondary bile acid TCA taurocholic acid TCDCA taurochenodeoxycholic acid TDCA tauro deoxycholic acid THCDA taurohyodeoxycholic acid TαMCA tauroαMCA TMCA tauroMCA TωMCA tauroωMCA TUDCA tauro ursodeoxycholic acidBMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Expression of genes involved in FXR signaling ceramide synthesis and glucose metabolism in response to GP supplementation Scatter plot of relative mRNA levels of indicated genes expressed in A ileum and B liver tissues collected from individual mice fed LFD squares or LFD GP closed squares Group mean±SD n7 samples group is illustrated by horizontal and vertical lines Data represent qPCR of technical duplicates analyzed by ˆ’ΔCT method Between group difference was determined by unpaired two tailed t test with or without Welch™s correction for unequal variance p005 p001 p0001 FXR farnesoid X receptor GP grape polyphenol LFD low fat dietthe hepatic expression and gene repressive function of Shp via liver receptor homologue1 and hepatocyte nuclear factor4α26“In agreement with reduced ileal Fgf15 hepatic Shp expression was significantly decreased figure 4B Fgf15 and Shp negatively regulate BA synthesis therefore their reduced expression was consistent with hepatic upregulation of cytochrome P450 family subfamily A member Cyp7a1 the rate limiting enzyme in the classical BA synthesis pathway7 and a trending increase in downstream enzyme Cyp8b1 p006 figure 4B Gene expressions of Cyp27a1 and Cyp7b1 online BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismsupplementary figure 4E involved in the alternative BA synthesis pathway were unaffected by GP treatment To determine if reduced hepatic Shp expression altered hepatic FXR activity we examined FXR target genes Abcb11 encoding bile salt export pump Bsep Slc51b encoding anic solute transporter Ost30 and Obp2a encoding lipocalin Lpn13 an acute phase protein32 Compared with control GP treatment did not affect hepatic expression of Fxr Abcb11 Slc51b or Obp2a figure 4B These data suggest that GP mediated effects on FXR activity are restricted to intestine resulting in suppression of ileal Fgf15 and Shp transcription and increased Cyp7a1 activity and PBA synthesis figure FXR activity regulates Tgr5 expression therefore we measured Tgr5 mRNA levels and downstream targets33 On activation by BAs TGR5 signals the release of incretin glucagon like peptide1 Glp1 to promote euglycemia33 Glp1 is produced when proglucagon protein Gcg is cleaved by prohormone convertase PC13 encoded by Pcsk134 GP supplemented mice showed reduced ileal expression of Tgr5 and no changes in Gcg or Pcsk1 figure 4A GPs did not change expression of Fxr Fgf15 or Tgr5 in colon tissue GPs induced an increase in colonic Gcg however Pcsk1 was unchanged indicating Glp1 levels were unaffected online supplementary figure 4D These data further support the idea that GPs inhibit ileal FXRGPmediated FXR inhibition is associated with downregulation of ceramide synthesis genes and improved markers of hepatic energy metabolismTissue accumulation of ceramides is linked to insulin resistance and diabetes which can be ameliorated by pharmacological or genetic inhibition of ceramide biosynthesis35 FXR activity positively upregulates genes required for ceramide synthesis in ileum which leads to impaired glucose metabolism and hepatic steatosis in mouse models of MetST2D10 Synthesis and accumulation of ceramides in liver contributes to hepatic insulin resistance steatohepatitis and metabolic disease35 We found that GP induced FXR inhibition was associated with lower expression of de novo ceramide synthesis genes specifically sphingomyelin phosphodiesterase Smpd3 in ileum figure 4A and serine palmitoyltransferase long chain base subunit Sptlc2 and ceramide synthase Cers4 in liver figure 4B Consistent with lower expression of ceramide biosynthesis genes GP supplemented mice showed improvements in markers of hepatic energy metabolism evidenced by lower hepatic expression of carbohydrate response element binding protein Chrebp a transcription factor that activates key enzymes of de novo lipogenesis38 glucose phosphatase G6Pase which catalyzes the final step in hepatic glucose production and Idh3a a subunit of the IDH3 isocitrate dehydrogenase heterotetramer complex that regulates fatty acid metabolism and whose inhibition is associated with hepatic glycogen synthesis figure 4B39 GP supplemented mice had reduced expression of Lbp encoding LPS binding protein suggesting decreased liver inflammation and insulin resistance40 Hepatic gene expression of gluconeogenesis enzyme phosphoenolpyruvate carboxykinase Pck1 and CEBP homologous protein Chop which are normally upregulated during hepatic endoplasmic reticulum stress41 were similar between groups online supplementary figure 4ESBAs depleted in GPsupplemented mice are FXR agonists that promote expression of ceramide synthesis genesThe majority of secreted BAs are reabsorbed in ileum and returned to the liver via the portal vein7 Cecum colon and feces have similar BA profiles while the serum BA profile is most closely related to that of ileum and portal vein as a minor fraction of reabsorbed BAs enter systemic circulation25 We sought to investigate how individual BAs altered by GP supplementation might affect ileal FXR signaling Gut anoids were cultured from ileal crypts isolated from WT mice figure 5A and treated with individual PBAs or SBAs that were differentially detected in serum of GP supplemented mice figure 5B To validate the system anoids were treated with CDCA a potent FXR agonist or CDCA in combination with the FXR antagonist TMCA As expected CDCA increased the expression of Fxr Fgf15 and Shp compared with vehicle treated anoids and this effect was attenuated by addition of TMCA figure 5B When anoids were treated with individual BA alone ie µM in the absence of CDCA activator FXR activity was unaffected as Fxr Fgf15 and Shp mRNA expression remained similar to untreated anoids online supplementary figure We therefore investigated individual BAs in the presence of FXR activator CDCA as previously reported11 to reveal agonistic or antagonistic effects on FXR signaling anoid cultures treated with CDCA in combination with ωMCA TωMCA THDCA or DCA increased CDCA induced expression of Fxr Fgf15 and Shp indicating that these SBAs are FXR agonists figure 5B In agreement with FXR agonistic activity anoids treated with CDCA in combination with TωMCA resulted in increased expression of Smpd3 Cers4 and Sptlc2 ceramide synthesis genes Cotreatment with CDCA and ωMCA THDCA or DCA only upregulated Sptlc2 figure 5B CA a PBA reported to be a weak FXR agonist and detected at higher concentration in GP supplemented dbdb but not WT mice increased CDCA induced activation of Fxr Fgf15 and Shp and increased expression of Cers4 and Sptlc243 TCA a PBA detected at higher concentration in GP supplemented dbdb and WT mice attenuated CDCA induced gene expression of Fxr and Fgf15 but not Shp and reduced CDCA induced gene expression of Cers4 figure 5B These anoid data suggest that GPs led to the depletion of FXR activators ωMCA TωMCA and THDCA and increase of an FXR antagonist TCA Consistent with in vivo data the net effect BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Ileal anoids treated with BAs revealed agonistic or antagonistic effects on FXR and ceramide pathway genes A Ileal crypts were isolated and cultured in Matrigel medium detailed in methods Spheroids representative day photo matured into anoids representative day photo B Scatter plot of relative mRNA levels of indicated genes expressed in anoids after hours of treatment with vehicle methanol black diamonds µM CDCA closed red circles a combination of µM CDCA and known FXR inhibitor TMCA µM a combination of µM CDCA and indicated PBA µM red circles a combination of µM CDCA and indicated SBA µM blue squares Data shown were combined from two independent experiments and for each experiment three wells containing mature anoids were treated with indicated BAs Data represent qPCR of technical duplicates analyzed by ˆ’ΔCT method Group mean±SD n6 wells total per treatment group is illustrated by horizont

Thyroid_Cancer

inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the p65 and actin cytoskeleton regulatory pathwaysNaze G Avci1 Sadaf Ebrahimzadeh‘Pustchi1 Yasemin M Akay1 Yoshua Esquenazi2 Nitin Tandon Jay‘Jiguang Zhu Metin Akay1Glioblastoma GBM is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy GBM tumors show nuclear factor‘κB activity that has been associated with tumor formation growth and increased resistance to therapy We investigated the effect of inhibitor BAY ‘ with Temozolomide TMZ on the signaling pathways in GBM pathogenesis GBM cells and patient‘derived GBM cells cultured in 3D microwells were co‘treated with BAY ‘ and TMZ or BAY ‘ and TMZ alone and combined experiments of cell proliferation apoptosis wound healing assay as well as reverse‘phase protein arrays western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells The results revealed that the co‘treatment significantly altered cell proliferation by decreasing GBM viability suppressed pathway and enhanced apoptosis Moreover it was found that the co‘treatment of BAY ‘ and TMZ significantly contributed to a decrease in the migration pattern of patient‘derived GBM cells by modulating actin cytoskeleton pathway These findings suggest that in addition to TMZ treatment can be used as a potential target to increase the treatment™s outcomes The drug combination strategy which is significantly improved by inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatmentGlioblastoma multiforme GBM is the most malignant primary brain tumor in the central nervous system Current standard of care therapy includes surgery followed by radiotherapy and concomitant and adjuvant chemotherapy with the alkylating agent Temozolomide TMZ which provides survival benefits for patients with GBM1 However even with the advances in surgical resection combined with TMZ therapy and irradiation the prognosis for newly diagnosed GBM patients remains poor In fact due to its rapid proliferation increased invasion and migration capacity and chemoresistance to the alkylating agents a0the median survival is only a0months with the ˜Stupp™ regimen radiation with daily TMZ — “ a0weeks followed by cyclic TMZ2 and 5year survival rate is less than which is the lowest longterm survival rate of malignant brain tumors3“ TMZ methylates DNA at the O6 positions of guanine and DNA repair enzyme O6methylguanine methyltransferase MGMT removes alkyl groups from O6 position of guanine in DNA making cells resistant to TMZ6 Therefore new therapies are necessary to prevent cell proliferation and induce apoptosis for GBM patientsNuclear factorkappa B NFκB is a regulatory transcription factor of the Rel gene family including p50 cRel RelB or p65 subunits It is involved in the control of tumor cell proliferation migration immune response and apoptosis7“ Studies have shown that NFκB gene was involved in the regulation pathways of different cancer types such as thyroid cancer head and neck squamous cell carcinoma and colorectal cancer711“ Increased 1Department of Biomedical Engineering University of Houston Cullen Blvd Houston TX USA 2UTHealth Neurosurgery McGovern Medical School Memorial Hermann at Texas Medical Center The University of Texas Health Science Center at Houston Houston TX USA email makayuheduScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cactivation of NFκB has also been identified in GBM tumors where the expression of NFκB was much higher in GBM tissue compared with nonGBM tissue1415 NFκB also promotes chemoresistance to TMZ and regulates MGMT activity in GBM by promoting MGMT gene expression through NFκB binding sites within the MGMT promoter16 NFκB inhibitors such as parthenolide do not completely eradicate tumors therefore they are mostly used in combination with other drugs17 When used in combination with TMZ NFκB inhibitor parthenolide has been shown to activate mitochondrial apoptosis signaling in U87MG and U373 GBM cells which lead to cell death18 and had a combined effect on cell cytotoxicity in LN18 and T98G glioma cells19 NFκB inhibitor CBL0137 has been shown to bind DNA leading the functional inactivation of the Facilitates Chromatin Transcription FACT complex a chromatin remodeling complex regulating transcription replication and DNA repair2021 In a0vitro evaluation of the CBL0137 on FACT p53 and NFκB has been done using U87MG and A1207 GBM cells It was shown that CBL0137 induced loss of chromatinunbound FACT activated p53 and inhibited NFκB dependent transcription21 In a0vivo studies showed that CBL0137 was effective in increasing survival rates in TMZresistant orthotopic mouse models21 Moreover Wang et a0al indicated that NFκB inhibitor BAY suppresses the expression of MGMT and enhances the TMZinduced apoptosis in TMZ resistant U251 cells22 However there is still a lack of characterization of the precise pattern of NFκB activation in combination with TMZ in GBM cell populations that have been a0surgically resected from patientsIn vitro and in a0vivo identifications and validations of molecular targets of GBM are important as they can progress into clinical studies Studies reported that combining multiple gene targets may prevent tumor growth and improve the treatment strategy for GBM23“ Both Bay and TMZ exert antitumoral activities individually in different tumor types28“ Therefore in this study we aimed to analyze functionally the combined effect of Bay and TMZ in different GBM cells For this purpose first we used our 3D PEGDAbased hydrogel microwell platform31“ to provide reliable preclinical models that can recapitulate in a0vivo features of the GBM tumors We cultured GBM cells U87 and LN229 and patientderived GBM cells in 3D microwells for a more precise and personalized treatment approach We then treated GBM cells with Bay and TMZ in combination or alone Our results indicated that the cotreatment of Bay and TMZ significantly reduced cell viability in all three cell lines in correlation with a significant decrease in the spheroid size The levels of NFκB protein and its subunits p65 and p50 were also significantly decreased compared with the control and single drug applications Similar a0decreases in the cell viability and protein levels were observed in all three GBM cells Tumor biopsy samples could give more realistic information about how tumors respond to drugs when they are used for in a0vitro or in a0vivo studies35“ Therefore we decided to continue our experiments with only using our patientderived GBM cells We treated patientderived GBM cells with Bay and TMZ or alone and analyzed specific cellular proteins along with their posttranslational modifications via reversephase protein arrays RPPA to elucidate the mechanism of action of the proteins3839 We observed that several cell signaling pathways including cell metabolism proliferation apoptosis were significantly affected by the combination of the drugs which were consistent with the literature4041 Furthermore our RPPA data revealed that there was a significant change in the modulation of actin cytoskeleton and following experiments including western blot analysis for the expression of FAK protein and wound healing assay for cell migration patterns confirmed the RPPA results We observed a significant decrease in both actin fluorescence intensity and migration pattern in the a0cotreated patientderived GBM cells To the best of our knowledge the effect of cotreatment of Bay and TMZ has never been studied previously on the actin modulation of patientderived GBM cells These results suggested that Bay and TMZ induced alteration in the a0actin filament anization by reducing the level of focal adhesion protein which might implicate in cell apoptosis The effect of Bay with TMZ necessitates further exploration to better understand its mechanism of action in GBM and potential therapeutic tools for GBM treatmentResultsCo‘treatment of Bay ‘ and TMZ reduced viability of GBM cells We used our previously a0published data to select the most effective drug concentrations for this study42 We cultured LN229 U87 and patientderived cells in the microwells for a0days where they formed 3D spheroids and we added a0µM of Bay and a0µM of TMZ in combination or alone Then we cultured the spheroids for more days with or without drug Control group did not receive any treatment The cell viability assay was performed on day after drug administration The results showed that the a0cotreatment significantly reduced cell viability of GBM cells LN229 and U87 and patientderived GBM cells cultured in 3D PEGDA microwells respectively as shown in Fig a01ac When they were used alone TMZ reduced cell viability to and p and Bay reduced cell viability to and in LN229 U87 and patientderived GBM cells respectively compared to control groups Fig a01d However when they were used in combination the viability of the cells significantly decreased to and in LN229 U87 and patientderived GBM cells respectively compared to control groups p Fig a01d Tumor cells are generally less sensitive to drug treatments in 3D cultures than in 2D cultures4344 This could reflect reduced compound access or differences in the response to cell death To confirm that cotreatment was more effective compared to single drug use we quantified the size of the spheroids using ImageJ45 Our data showed that after a0days of drug treatment the spheroids™ sizes were significantly reduced in the cotreatment by and in LN229 Fig a01e U87 Fig a01f and patientderived GBM cells p Fig a01g respectively compared to control group p When we compared the spheroids™ sizes of the cotreatment with TMZ alone there was a reduction of and in LN229 U87 and patientderived GBM cells respectively p Finally the spheroids™ sizes of the cotreatment compared with Bay alone showed a decrease of and in LN229 U87 and patientderived GBM cells respectivelyScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Representative images of the GBM tumor cells cultured in the PEGDA microwells a“c LN229 U87 and patientderived GBM cells were cultured in the microwells for a0days respectively After day Bay and TMZ were applied either alone or in combination onto the cell spheroids Control group did not receive any treatment The cells were cultured with or without drugs additional more days The images were taken on Day Day and Day after the drug application to observe the disruption in the spheroids Dotted black lines represent the edge of the tumor spheroid Scale bars a0µm d Bar graph showing trypan blue staining for cell viability of LN229 U87 and patientderived GBM cells e“g Spheroid size quantification was done using ImageJ for LN229 U87 and Patientderived GBM cells respectively Twotailed ttest followed by Wilcoxon test were done GraphPad Prism v5 Data represent the mean ± SD of three biological replicates p and p Suppression of activity in GBM cells by co‘treatment of Bay ‘ and TMZ As a readout of NFkB activity after drug treatment we first quantitatively assessed the cytoplasmic activation of phosphorylated NFκB p65 subunit in both treated and untreated groups in all GBM cells NFκB pp65 subunit activity was observed in the control groups of all three GBM cells Fig a02a NFκB pp65 subunit activity decreased to and when TMZ applied alone and and when Bay was applied alone in LN229 U87 and patientderived cells respectively However the decrease in NFκB pp65 subunit was reduced to when LN229 U87 and patientderived cells respectively were cotreated p Fig a02a Bay specifically inhibits NFκB activation by blocking phosphorylation of IκBα46 In independent experiments we analyzed the abundance of phosphorylated NFκB p65 NFκB p50 and IκBα in all three GBM cells Qualitative and quantitative western blot analysis revealed that the exposure to Bay with TMZ significantly downregulated the abundance of NFκB p65 NFκB p50 and IκBα compared with control and Bay or TMZ alone Fig a02b Please note that loading controls were used for each experiment but only the representative loading control for p and tP65 and p and tP50 was presented Fig a02b The cell viability assay cells™ size and protein expressions in all three GBM cells revealed similar results without any dramatic change Therefore considering the importance of using patientderived tumor cells to elucidate the mechanism of drugs and respective signaling pathways35“ we further continued our experiments using patientderived GBM cellsApoptosis was promoted by co‘treatment of Bay ‘ and TMZ RPPA technology is designed for multiplexed antibodybased relative quantification where each array is tested with a validated antibody specific to a particular protein along with their particular posttranslational modifications47 In the attempt to elucidate the mechanism of action of Bay with TMZ by which NFκB subunits were modulated and to identify downstream signaling molecules we employed RPPA platform using our drug treated or untreated patientderived GBM cells RPPA results showed that many oncogenic pathways were altered by the drug treatments but more specifically by the cotreatment Fig a03a Decreased expression of NFκB was not only associScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 NF“kB activity in LN229 U87 and patientderived GBM cell lines a NF“kB p65 subunit activity in LN229 U87 and patientderived GBM cell lines respectively The cells cultured with or without drugs for a0days were collected from the microwells and subjected to ELISA Data represent the mean ± SD of three biological replicates p and p b Representative immunoblots LN229 U87 and patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels bottom panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p ated with changes in the a0NFκB pathway but also with apoptosis cell metabolism and proliferation which were confirmed by the analysis of downregulated RPPA proteins in Enrichr KEGG libraries4849 Fig a03c p One of the specific pathways given by RPPA was apoptosis Apoptosis is one of the important mechanisms that regulates cell death and suppress tumorigenesis Studies have demonstrated that Bcl2 family proteins can positively and negatively regulate apoptosis by regulating antiapoptotic protein Bcl2 and proapoptotic protein Bax4050 Our RPPA data using patientderived GBM cells showed that the fold change of Bcl2 relative to control was times higher in cotreated group TMZ alone Bay alone respectively Fig a03b In order to further confirm whether the expression of a0these proteins were downregulated by the cotreatment we performed western blot analysis Our results showed a similar decrease in Bcl2 protein expression in the cotreatment compared with the control and single drug a0treatment Fig a03d In contrast Bax protein fold change relative to control was times higher in cotreated group TMZ alone Bay respectively where we observed a significant increase after the cotreatment of Bay with TMZ compared with the control p Fig a03b Bcl2Bax ratio is a key indicator in susceptibility of the cells to apoptosis Western blot results confirmed the change in Bcl2Bax ratio in the cotreatment compared with the control group and single a0drug treatment Fig a03d Our RPPA data also showed a significant increase in the cleavedcaspase protein Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The effect of Bay and TMZ on signaling pathways in patientderived GBM cells a Heat map presentation of RPPA analysis showing the changes in the protein expression RPPA was performed on lysates treated with Bay and TMZ alone or in combination All relative protein level data points were normalized to the a0control group Red and green indicate up and down regulations respectively in the heat map The samples were run in duplicate n b Fold change of the a0selected proteins relative to the a0control group via RPPA Data represent the mean ± SD of two biological replicates p p Wilcoxon rank sum test c Analysis of downregulated RPPA proteins shows a a0significant activation in numerous Enrichr KEGG pathways The pathways were a0sorted by p value ranking d Representative immunoblot validation of significantly altered proteins involved in different KEGG pathways Patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p fold change relative to control times higher in the cotreatment compared with times higher in TMZ alone and times higher in Bay alone p Fig a03b To confirm if cotreatment triggered apoptosis correlated with caspase activation we performed western blot analysis with procaspase3 cas3 and cleavedcaspase3 Ccas3 We observed that Bay and TMZ induced apoptosis was associated with cas3 Fig a03d Please note that loading controls were used for each experiment but only the representative loading control for Bax cas3 and Ccas3 was presented Fig a03d Moreover another important mechanism of NFκB activation in GBM regulates through AKT phosphorylation of IκB Our RPPA data showed relative fold changes of in the cotreated group TMZ alone and Bay alone respectively p Fig a03b The western blot results also confirmed a significant decrease in the abundance of AKT pT308 Fig a03dTo further investigate whether cotreatment of Bay with TMZ can lead to glioma cell apoptosis and to confirm our RPPA and western blot results we performed apoptosis assay TUNEL The patientderived GBM cells were cotreated with Bay with TMZ or single drug treated and subjected to TUNEL assay to detect DNA damage Fig a04a The results indicated that TUNEL cells in the cotreatment were increased tenfold compared with control and and 24folds compared with TMZ alone and Bay alone respectively p Fig a04b Additionally in some TUNEL cells we observed a typical ring type chromatin aggregation underneath the nuclear membrane which suggested an early stage apoptosis51 Fig a04a red arrows There were also a few TUNEL cells that lacked the typical apoptotic ringlike nuclear structure indicating that they were either at a different stage of apoptosis or alternatively undergoing necrosis52 that we have not investigated furtherCo‘treatment of Bay ‘ with TMZ changed actin anization by inhibiting FAK phosphorylation and cell migration Actin filaments Factin are one of the main components of the cellular cytoskeleton which regulates actin dynamics and migration process in the cells The disruption of the actin cytoskeleton inhibits cell migration and adhesion53 Depolymerization or cleavage of actin lamins and other cytoskeletal proteins have been also found to be involved in cell apoptosis54“ To confirm the RPPA results showing changes in the actin modulation pathway and to understand the mechanism that regulates cytoskeletal Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Apoptosis assay TUNEL a Fluorescent images of TUNEL cells in patientderived GBM cells TUNEL assay was performed on cells treated with Bay and TMZ in combination or alone in the microwells Cells were collected from the microwells trypsinized and replated into 8well chamber slides TUNEL cells green with ringlike nuclear stain are indicated with red arrows Nuclei were counterstained with DAPI blue b Numbers of TUNEL cells are presented as mean ± SD of three biological replicates p and p X20 objective Scale bars a0µmanization we treated patientderived GBM cells co treated with Bay with TMZ or single drug treated 3D spheroids collected from the microwells were stained with phalloidin green and DAPI blue Staining cells with fluorescently conjugated phalloidin is considered the most reliable method of accurately labeling Factin in fixed cells57 In the control group intact cells formed finemeshed networks with a distinct Factin anization on both day Fig a05a upper panel and day Fig a05a bottom panel In single drug treated cells actin was still found to be polymerized to filaments as it can be seen by its interaction with phalloidin at both days and However the cells which were cotreated with Bay and TMZ lost their Factin anization and their shape compared with the control and the single drug treated groups at day Fig a05a bottom panel Changes in the a0actin distribution within the cells were quantified by measuring the staining intensity using Fiji Macro ImageJ as described previously5859 At day we observed a a0significant decrease in the fluorescence intensity of phalloidin when the cells were cotreated with Bay and TMZ compared with the a0control and single drug treated groups p Fig a05b To investigate the drug related Factin mechanism we examined the levels of FAK protein following cotreatment or single drug treatment As shown in Fig a05c cotreatment significantly decreased the level of phosphorylated FAK compared with both control and single drug applications p Furthermore we investigated cell migration patterns of the patientderived cells that were cotreated with Bay and TMZ or single drug treated We collected 3D spheroids from microwells after drug treatment and replated them in 24well plate to perform scratch wound healing assay We noted a significant increase in cell density in the scratch area in both control and Bay alone after and a0h of scratch formation p Fig a06a Although compared with the a0control cells both cotreatment and TMZ alone groups showed a decrease in the cell migration into the scratch area after a0h we observed that after a0h the migration rate of the cotreated cells was significantly slower than the cells that were treated with TMZ alone p Fig a06b These results indicated that the disanization of actin microfilaments was concomitant with the cell apoptosis after the a0cotreatment of Bay with TMZDiscussionDespite the increase in the median survival of GBM patients from to months4 the clinical efficacy of standard of care therapy including TMZ chemotherapy combined with surgery and radiotherapy is still limited Due to challenges in treating GBM significant attempts have been made to develop single or combined drug treatments60“ However given the cost long time frame and risks of failure associated with developing a new drug repurposing available drugs may be the most effective alternative therapeutic strategy Therefore it is important to evaluate potential drug combinations for GBM treatmentScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Changes in the actin cytoskeleton and migration pattern in patientderived GBM cells cotreated with Bay and TMZ or single drug treated in the microwells a Upper panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin green and DAPI blue Bottom panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin and DAPI Scale bars a0µm b Intensity of staining obtained with phalloidin was measured in each cell using ImageJ and displayed as boxplots with to confidence intervals A twoway ANOVA with Dunnett™s multiple comparisons test was performed to determine statistical relevance Three biological replicates n p p c Representative immunoblots show the levels of FAK pTyr397 and total FAK in patientderived GBM cell lysates cotreated with Bay and TMZ or single drug treated for a0days in the microwells The levels of the proteins were quantified using ImageJ right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p Due to the cell repellent property of PEGDA hydrogel tumor cells can form aggregates at the bottom of the microwells and selfassemble into spheroids in each well within a0days following cell seeding313363 Compared with 2D monolayer cell culture 3D spheroids have an important advantage their larger size Thus often drug effects can easily be monitored over time by measuring the size and shape of spheroids4344 Additionally using 3D in a0vitro tumor models can better recapitulate in a0vivo features of the tumors We used PEGDA hydrogelbased microwell platform313363 in order to culture different types of a0GBM cells commercially available GBM cell lines LN229 U87 and a0patientderived GBM cells However we investigated the effect of the drugs on the patientderived GBM cells more in detail since growing tumors from tumor biopsy samples could give very detailed information about how tumors respond to drugs35“ Considering the precious nature of the patient samples this platform which requires fewer cells compared with 2D monolayer cultures provides us with a robust tool to recapitulate in vivo features of GBM tumors and to test our drug combinationsNFκB is one of the major transcription factors associated with GBM and responsible for activating a series of cellular responses including cell proliferation survival invasion and apoptosis6465 Previous studies have shown that NFκB can activate Akt and promote cell survival and proliferation by downregulating the expression of phosphatase and tensin homolog deleted on chromosome ten1866 NFκB pathway can inhibit cell apoptosis by inhibiting a stressactivated protein kinase and a mitogenactivated protein kinase signaling pathway67 It can also be activated in response to treatment with cytotoxic drugs such as vinca alkaloids and topoisomerase inhibitors Several studies have demonstrated the activation of NFκB in GBM patientderived stemlike cells cultures96869 Moreover alkylating agents TMZ can activate NFκB through DNA damage pathway activation7071 The combination effect of Bay and TMZ have been showed in our previous study where we determined the most effective drug concentrations on GBM cells using our microfluidics platform42 Another study that investigated the combined effect of NFκB inhibitor BAY with TMZ showed that combined drug application induced TMZ resistant in U251 GBM cells22 However the characterization of the precise pattern of NFκB activation in different GBM cell populations from surgically resected tissues still remains elusive Therefore in this study we investigated the interaction of Bay with TMZ and their effects on the LN299 and U87 GBM cell lines as well as patientderived GBM cells in order to recapitulate NFκB activation as in a0vivo features of the GBM and its signaling pathways We applied a0µM of Bay and a0µM of TMZ3442 in combination or alone for all three GBM cell types First we observed a significant decrease in both cell viability and size of the spheroids in the cotreatment compared with control and single drug application Then we showed quantitatively and Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Cell migration of patientderived GBM cells by wound healing assay a Patientderived cells were cotreated with Bay and TMZ or single drug treated in the microwells trypsinized and replated in 24well plates After they reached to their confluency a scratch wound was formed with a 200μl tip and cells were incubated for the next a0h Images were taken 4x at a0h a0hr and a0hr Scale bars a0µm b The wound width was measured with ImageJ and the average wound width was shown Data represent the mean ± SD of three biological replicates p and p oneway ANOVA with Tukey™s post hoc testqualitatively the expression of NFκB in all three GBM cell types a0We noted a significant decrease in the cotreated group compared with control and single drug application Our western blot data also confirmed the decrease in the abundance of pP65 pP50 and pIKBa that Bay has been shown to inhibit its phosphorylation46 However in the cotreated group the decrease was significantly higher compared to both control and single drug application This data showed that cotreatment of Bay and TMZ has more effect on the inhibition of NFκB pathway than Bay or TMZ alone and suggests a a0decreased downstream transcription of oncogenic proteins72 Although there were slight differences in the NFκB expression patterns in three different GBM cell types a0we focused on the patientderived cells in the rest of the study due to their ability to better recapitulate the genomic similarities to primary disease7374Proteins that interact with each other activate multiple pathways which can result in apoptosis according to tissue type and pathological condition Glioblastoma tumors express high levels of antiapoptotic BCL2 family proteins such as Bcl2 and BclxL which may cause glioblastoma cells to resist apoptosis75 The proapoptotic members of Bcl2 family such as Bax and Bak are necessary for their proapoptotic effect Interactions and the ratio between antiapoptotic Bcl2 and proapoptotic Bax are decisive factors in the induction of apoptosis7677 Active NFκB can prevent cells from apoptosis by stimulating the expression of genes and promoting cell proliferation Although patientderived GBM samples have been shown to be highly resistant to apoptosis77 our data revealed changes in the expression of various members of Bcl2 family and NFκB signaling pathway after cotreatment of Bay and TMZ Our RPPA results outlined distinct molecular profiles in which apoptotic P53 signaling and NFκB signaling pathways were significantly affected after the a0cotreatment These results supported that the inhibition of NFκB expression could inhibit the expression of Bcl2 and promote the expression of Bax thus promote apoptosis Our data also suggested the possible interaction between Bcl2 and p53 in Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Proposed schematic of the a0signaling pathways involved in Bay and TMZmediated inhibition in GBM patientderived cells The effect of combined therapy of Bay and TMZ was achieved through the inhibition of SrcFAKVinculin which regulate the cytoskeleton anization through MAPKs JNK and PI3KAKT signaling pathways Exposure to both Bay and TMZ also leads to receptormediated activation of Bax but not Bcl2 in the subsequent inhibition of the downstream NFκB transcription factor Inhibition of NFκB in turn causes cell deathregulating cell survival and death7778 The activation of extrinsic and intrinsic molecular pathways can lead to the proteolytic activation caspases The extrinsic pathway is triggered by proapoptotic ligands that activate cell surface death receptors and procaspase8 which in turn leads to the cleavage of caspase3 and apoptosis79 Our results determined that the a0cotreatment significantly inhibited the expression of caspase3 while the expression of cleaved caspase3 was increased Additionally TUNEL assay which detects DNA strand breaks which could occur as an event in the apoptosis showed a dramatic increase in the TUNEL cells after the cotreatment compared with the a0control and single drug application Altogether these results suggested that the inhibition of cell proliferation Bcl2 and caspase3 by a0the cotreatment of Bay and TMZ may occur through the NFκB mediated apoptosis and they might be tightly coupled8081The literature provides evidence that supports crosstalk between PI3KAktmTOR signaling pathway and NFκB which is downstream of Akt NFκB activation in GBM regulates through AKT phosphorylation of IκB resulting in an activated NFκB that translocates to nucleus8283 Our data showed that when Bay was used with TMZ there was a decrease in the abundance of PI3Kp110 AktpS473 AktpT308 and mTORpS2448 This preliminary data is important to suppo

Thyroid_Cancer

Acute myeloid leukemia AML is a complex hematological disease characterized by genetic and clinical heterogeneity The identification and understanding of chromosomal abnormalities are important for the diagnosis and management of AML patients Compared with recurrent chromosomal translocations in AML t816p112p133 can be found in any age group but is very rare and typically associated with poor prognosisMethods Conventional cytogenetic studies were performed among AML patients recorded in our oncology database over the last years Fluorescence in situ hybridization FISH was carried out to detect the translocation fusion Array comparative genome hybridization aCGH was carried out to further characterize the duplication of chromosomesResults We identified three AML patients with t816p112p133 by chromosome analysis Two of the three patients who harbored an additional 1q duplication were detected by FISH and aCGH aCGH characterized a Mb and Mb gain in chromosome at band q321q44 separately in these two patients One patient achieved complete remission CR but relapsed months later The other patient never experienced CR and died years after diagnosisConclusion A 1q duplication was detected in two of three AML patients with t816p112p133 suggesting that 1q duplication can be a recurrent event in AML patients with t816 In concert with the findings of previous studies on similar patients our work suggests that 1q duplication may also be an unfavorable prognostic factor of the diseaseKeywords 1q duplication Acute myeloid leukemia t816p112p133 Prognostic factorBackgroundAcute myeloid leukemia AML is a common disease characterized by immature myeloid cell proliferation and bone marrow failure which can be subdivided into “ pathogenetically different subtypes [] Over the past two decades the incidence has increased by [ ] Furthermore AML has poor longterm survival with a Correspondence lzhang202003163com Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning People™s Republic of ChinaFull list of author information is available at the end of the high relapse rate [] Therefore AML represents a substantial health problem that requires strict monitoring and innovative treatment strategies The development of newer effective treatment strategies is necessary for AML patientsTo date the detection of cytogenetic abnormalities has been regarded as a critical prognostic tool for AML treatment [] Hence it is urgently necessary to identify chromosomal rearrangements in AML patients and provide the whole spectrum of cytogenetic abnormalities for AML [] According to the World Health anization classification system updated in AML with recurrent genetic abnormalities including t821q22q22 The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLiu a0et a0al Mol Cytogenet Page of t1517q24q21 t1517PMLRARA t11q23MLL inv16p131q22 and t1616p131q22 has been identified [ ] Nonrandom chromosomal abnormalities such as deletions and translocations have been detected in approximately of all adult AML patients Moreover chromosomal abnormalities have been recognized as genetic events that can cause and promote this disease [] Certain cytogenetic abnormalities including t821q22q22 t1517q24q21 and inv16p131q22 are associated with longer remission and survival while alterations of chromosomes 11q23 and complex karyotypes are associated with poor response to therapy and shorter overall survival [] Chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBMYH11 and t11q23MLL are usually found in AML patients [ ] However AML with t816p112p133KAT6ACREBBP is a very rare AML subtype and can be found in any age group from infancy to the eighth decade of life with a female predominance [“] A majority of adult patients with t816p112p133 are therapy related [“] and pediatric patients tend to be de novo [] There are approximately cases reported in the literature [“] and the first t816p112p133 in an infant was described in [] Some AML patients with t816 p112p133 have a bleeding tendency and disseminated intravascular coagulopathy which are overlapping clinical features that mimic acute promyelocytic leukemia APL [] Unlike APL AML with t816p112p133 has an unfavorable treatment response and outcome [ ] As a sole chromosomal anomaly t816p112p133 is found in more than of reported cases and one or more additional chromosomal anomalies can be seen in the remaining cases [] The most common secondary chromosomal anomalies are total or partial trisomy and monosomy or deletion of the long or short arm of chromosome [“ ] Comparatively the gain of 1q in variable sizes has also been frequently noticed in patients with t816p112p133 in these large studies [“ ]Recurrent cytogenetic abnormality t816p112p133 is seldom associated with AML and the 1q duplication in AML patients with t816p112p133 has never been discussed In the present study a total of de novo or treatmentrelated AML patients were collected from our laboratory oncology database Among them three patients were detected with t816p112p133KAT6ACREBBP and two of these three showed an additional copy of partial chromosome 1qMethodsPatientsThis study was approved by the Institutional Review Board IRB of Oklahoma University IRB Number A total of AML patient samples were studied cytogenetically from to at the Genetics Laboratory of Oklahoma University Health Sciences Center Bone marrow samples were obtained from three of the patients who had t816p112p133Conventional cytogenetic analysisShortterm cultures of unstimulated bone marrow samples were established and harvested according to standard laboratory protocols Karyotype analysis was performed using Giemsa and trypsin techniques for Gbanding The cytogenetic abnormalities were described according to the International System for Human Cytogenetic Nomenclature ISCN Fluorescence in a0situ hybridization analysisFluorescence in a0 situ hybridization FISH assays were performed according to the manufacturer™s instructions in combination with our established laboratory protocols A PMLRARA dualcolor dualfusion translocation probe Abbott Molecular Inc Des Plaines IL USA subtelomerespecific probes for chromosome parm and qarm and whole chromosome painting WCP probes for chromosomes and were purchased from Cytocell Ltd NY USA A spectrum greenlabeled probe mapping to the 8p1121 region and a spectrum orangelabeled probe mapping to the 16p133 region were created in house with the following BACPAC clones RP11642I24[chr8 4167633641856494hg19] and RP11589C21[chr8 4187370242036222hg19] RP11619A23[chr16 37200763914571hg19] and RP1195J11[chr16 38603744025510hg19] Children™s Hospital Oakland Research Institute Oakland CA USA The KAT6A gene located on 8p1121 and the CREBBP gene located on 16p133 were covered by the greenlabeled and redlabeled homebrewed probes respectively All probes were validated before use Chromosome spreads were counterstained with 46diamidino2phenylindole DAPI4 in antifade medium Vector Laboratories Inc CA USA Digital images carrying specific hybridization signals were captured and processed on CytoVision version Applied Spectral Imaging Carlsbad CA USAaCGH analysisGenomic DNA was extracted from each of the three patients™ bone marrow pellets according to the standard operating procedure using the phenol and chloroform method with a commercially available DNA extraction kit Puregene blood kit Qiagen Valencia CA or Nucleic Acid Isolation System QuickGene610L FUJIFILM Corporation Tokyo Japan Two aCGH platforms NimbleGen and Agilent were used in this study For the 0cLiu a0et a0al Mol Cytogenet Page of NimbleGen aCGH platform human reference genomic DNA was purchased from Promega Corporation Promega Corporation Madison WI USA The patient™s DNA and the reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming and then equal quantities of both labeled products were mixed and loaded onto a a0K oligonucleotide chip Roche NimbleGen Inc Madison WI USA to hybridize at a0 °C for a0 h in a MAUI hybridization system BioMicro Systems Salt Lake City UT according to the manufacturer™s protocols with minor modifications The slides were washed with washing buffers Roche NimbleGen Inc after hybridization and scanned using a Roche Scanner MS Microarray Scanner Roche NimbleGen Inc Images were analyzed using NimbleScan software version and SignalMap software version Roche NimbleGen Inc The genomic positions were determined using GRCh36hg18 UCSC Genome Browser For the Agilent aCGH platform human reference genomic DNA was purchased from Agilent Corporation Agilent Corporation Santa Clara CA USA The patient™s DNA and the purchased reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming Agilent Corporation Patient DNA labeled with Cy3 was combined with a normal control DNA sample labeled with Cy5 of the same sex and hybridized to an Agilent × a0K oligo microarray chip Agilent Technologies by incubating in an Agilent Microarray Hybridization Oven Agilent Technologies After a0h of hybridization at a0°C the slides were washed and scanned using the NimbleGen MS Microarray Scanner Roche NimbleGen Inc Agilent™s CytoGenomics software Agilent Technologies was applied for data analysis The genomic positions were determined using GRCh37hg19 UCSC Genome BrowserCase presentationCase An 82yearold male presented with anemia was referred to us for AML evaluation His subsequent lab results and hospital records were not available in our clinical databaseCase A 28yearold female presented with disseminated intravascular coagulopathy was referred to rule out APL Her complete blood examination and bone marrow aspirate smears were not available Flow cytometry revealed monocytic cells positive for CD4 CD11b partial CD13 bright CD14 partial CD15 CD33 bright and HLADR partial but negative for CD3 CD7 CD34 CD117 MPO and TdT consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient achieved hematological CR on day and cytogenetic CR on day after induction chemotherapy and then relapsed a0months laterCase A 69yearold female with a medical history of breast cancer after lumpectomy chemotherapy and radiation presenting with generalized weakness pancyt ia and fever was referred to us for disease progression evaluation A complete blood examination showed a white blood cell count of × 109L with blasts a hemoglobin count of a0 gL and a platelet count of × 109L Her bone marrow aspirate smear demonstrated over myeloblasts Flow cytometry revealed that of the blast cells expressed CD45 moderate CD34 dim CD38 HLADR CD13 CD15 and CD33 and were negative for CD117 consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient started consolidation chemotherapy but had spontaneous regression and died a0years after AML diagnosisResultsIn case routine chromosome analysis detected an abnormal karyotype with a translocation between the short arms of chromosomes and Fig a01a in of cells consistent with a diagnosis of AML with t816p112p133 The nomenclature of the cytogenetic findings in patient was t816p112p133[]46XY[] No other consistent karyotypic aberrations were detected Thus this male patient was excluded from subsequent FISH and aCGH analysesIn case chromosome analysis demonstrated the same chromosome rearrangement between and in all cells Besides of these cells showed an extra chromosome segment attached to chromosome Fig a01b The karyotypes in patient were described as 46XXt816p112p133 add14p112[]46XY[] Negative FISH t1517q24q21PMLRARA further ruled out a diagnosis of APL data not shown Metaphase FISH analysis confirmed the t816p112p133KAT6ACREBBP fusion and demonstrated a part of chromosome on chromosome Fig a02a and b In addition to characterizing the extrachromosomal material aCGH was carried out aCGH confirmed the FISH findings and detected a a0Mb gain from chromosome at bands q321q44 a0bp GRCh36hg18 USCS Genome Browser Fig a03aIn case t816p112p133 with a gain of a similar chromosome segment on the long arm of chromosome was detected in of cells by karyotyping analysis Fig a0 1c FISH confirmed the KAT6ACREBBP fusion and revealed additional chromosome material Fig a02c and d Loss of the end portion of the chromosome long arm was not found by FISH Fig a03e aCGH further detected a gain from chromosome at bands 1q321q44 results for 0cLiu a0et a0al Mol Cytogenet Page of a Patient b Patient Fig Representative abnormal karyotypes of three patients with t816p112p133 a Karyotype of patient showing 46XYt816p112p133 as the sole abnormality b and c Karyotypes of patients and showing 46XXt816p112p133 and an additional chromosome segment attached to the short arm of chromosome and the long arm of chromosome respectively Translocated derivatives and are indicated by black arrows and derivatives and are indicated by red arrows 0cLiu a0et a0al Mol Cytogenet Page of c Patient Fig continued a0 bp GRCh37hg19 UCSC Genome Browser Fig a0 3b The molecular size was a0MbDiscussionAML is one of the most common diseases characterized by the proliferation of blast cells in bone marrow or peripheral blood which accounts for approximately of adult leukemia cases As reported previously common chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBfrequently observed and numerous MYH11 are uncommon chromosomal aberrations also exist in AML [] The detection of these fusion transcripts is important for the diagnosis and progression monitoring of AML patients []t1517PMLRARA and In previous large studies approximately AML cases with t816p112p133 have been reported [“] Among them cases showed a gain by 1q of variable sizes [“ ] As an uncommon entity t816 accounts for “ of all cases of AML [“] In our study three patients with t816p112p133 were identified one man and two women The two women were both diagnosed with AML subtype M5 and showed an extra copy of 1q at the same bands q321q44 which were different from the nine reported cases above The clinical features and cytogenetic data of the cases of AML with t816p112p133 and 1q duplications are summarized in Table a0 To the best of our knowledge this is the first study of the delineation of 1q duplication by aCGH in AML patients with t816p112p133AML patients with this abnormality often show unique clinical and biological characteristics [] Compared with the current categories t1517 t821 inv16 and t11q23 in AML t816 is clustered closer to t11q23 and shares commonly expressed genes [] Xie et a0 al reported adult AML cases with t816p112p133 indicating that t816p112p133 commonly exhibits monoblastic or myelomonocytic differentiation and arises in patients with a history of cytotoxictreated cancer Patients with de novo AML with t816 or treatmentrelated AML with t816 without adverse prognostic factors have a good outcome [] Identifying adverse prognostic factors is of importance to the choice of therapy and evaluation of survival in AML patients with t816 0cLiu a0et a0al Mol Cytogenet Page of CREBBPKAT6A fusionKAT6A8p1121CREBBPKAT6A fusionKAT6ACREBBP fusionCREBBP16p133CREBBP16p133KAT6ACREBBP fusiona KAT6A8p1121c WCP14WCP1WCP14b WCP1WCP1TelVysion 3q WCP1WCP1WCP3WCP1d WCP3TelVysion 3p TelVysion 3p TelVysion 3q e Fig Metaphase FISH of patient a and c showing KAT6ACREBBP fusion signals WCP FISH indicating the extra chromosomal materials on chromosome and chromosome were both from chromosome b and d No loss of the end portion of the chromosome long arm was indicated eOver the past a0 years cytogenetic and molecular technologies have largely promoted the efficiency of the identification and characterization of this disease [] Compared with conventional cytogenetic analysis and FISH methods aCGH is an attractive method for the investigation of cancer genomes [] aCGH has higher resolution simplicity high reproducibility shorter turnaround time and precise mapping of aberrations Most importantly it avoids the need for cell culture and dividing cells [“] Furthermore aCGH chromosomal analysis facilitates rapid detection and duplication of cytogenetic abnormalities previously undetectable by conventional cytogenetics [] In our investigation we applied aCGH to characterize the additional chromosome materials in patients and and interestingly found that the two patients 0cLiu a0et a0al Mol Cytogenet Page of revealed the same extra copy of 1q at bands q321q44 Patients with 1q duplication have also demonstrated a wide range of multiple malformations such as intellectual disability macrocephaly large fontanels prominent foreheads broad flat nasal bridges higharched palates retrognathia lowset ears and cardiac defects [ ] More recent studies have shown that a 1q gain is also related to a portion of solid tumors For instance the gain of 1q is well known as a poor prognostic biomarker of Wilms tumor [] and it plays an important role in predicting poor clinical outcome in patients with thyroid carcinoma as well [] In addition patients with a 1q duplication showed worse survival and high risk in acute leukemia Burkitt lymphoma and myeloproliferative neoplasms [“] The outcomes of 1q duplication in the nine reported AML patients with t816p112p133 are summarized in Table a0 Seven patients™ data were available These seven patients two adult and five pediatric all received induction chemotherapy and six achieved CR At the time of last followup two adult patients and three of five pediatric patients had died Only two pediatric patients were alive We reported two adult patients here patient achieved CR but relapsed a0 months later and patient had spontaneous regression and died a0 years after diagnosis Taken together the findings suggest that 1q duplication might be associated with adverse outcomes in AML patients with t816p112p133 However the significance of the 1q duplication in AML with t816 needs to be further investigated Since such changes have been seldom reported the pathogenic effects of 1q duplication in AML patients with t816p112p133 require more studies to be delineatedConclusionThree patients were detected with t816p112p133 from an AML patient database Two female patients were identified with a 1q duplication by FISH and aCGH analyses Combining our investigation with the findings of published studies we conclude that 1q duplication is a recurrent finding in AML patients with t816 Our data also suggest that 1q duplication might be associated with unfavorable prognosis in these cases The understanding of cytogenetic data would contribute to the diagnosis and treatment evaluation of AMLFig aCGH results of patient and patient showing partial 1q gain duplicated 1q regions are indicated by red frames 0cLiu a0et a0al Mol Cytogenet Page of Table The previously reported AML cases with a0t816p112p133 and a01q duplicationSex Age years FAB type Karyotype1q BandsOutcome yearsLast stateCase Case FFHaferlach et al FM5M5M5aDiab et alM M4Diab et alDiab et alDiab et alDiab et alFFFFXie et alM Brown et alM Brown et alFM45M4M4M5M4M4M446XXt816p112p133 add14p112[]46XX[]46XXt816p112p133[]46idemadd3q27[]45XXt816p11p13der1013q10q10[]46XXder7t17q21q35t816p11p13[]46XX[]46XY1del1p22t816p11p1310der14t1014q112p112[]47XYdel1q11der1t18p11q112x2i5p10810der14t1014q112p112der16t8165XXt816p11p1318der21t121q12p13[]46XX[]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110q11p11[]46idemadd7p21[]46XX[46XYt816p11p13der14t114q31p11[]46XderXtX1q26q23t816p11p13der11t1111p11q1346XYder3t38q27q13del6p22t816p112p133del10q21q25add13p112del16p12del20p112del20q112q133[]46idemdel1p35p363del15q23add19p131[]46XYt816q27q13del12q21q241del13q21q3116der19t119q32p133mar[]46XYdel6p22t816p112p133[cp2]46XY[]47XderYtY1q12q21 6t816 p11p13[]47idemdel13q3q3 [checked with CAD data]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110 q11p11 []46idemadd7p21 []46XX []1q321q441q321q44CR after inductionRelapsed months laterspontaneous regression1q21NAPartial 1q gain CR for 1q121q111q311q23CR for CR for CR for NA1q32CR for monthsAliveDiedNADeadAliveDiedAliveNADead1q21No CRDied month after treatment1q11Early remission after course Relapsed at months and months after diagnosisDiedAML acute myeloid leukemia FAB French“American“Britishh M male F female NA not available CR complete remissionAbbreviationsAML Acute myeloid leukemia aCGH Array comparative genomic hybridization FISH Fluorescence in situ hybridization APL Acute promyelocytic leukemia WCP Whole chromosome painting CR Complete remissionAcknowledgementsNot applicableAuthors™ contributionsM Liu and YR gathered clinical information and drafted the manuscript YR YK and M Liu performed routine cytogenetic analysis and participated in the interpretation of the results M Li performed FISH analysis and participated in the interpretation of the results XL supervised the FISH analysis and helped draft the manuscript XW performed CGH array analysis and helped draft the manuscript LZ and SL conceived the study participated in its design and 0cLiu a0et a0al Mol Cytogenet Page of extensively reviewed and revised the manuscript All authors have read and approved the final manuscriptFundingThis study has received no funding supportAvailability of data and materialsAll data generated or analyzed during this study are included in this published Ethics approval and consent to participateThis study was approved by University of Oklahoma Institutional Review Board for the Protection of Human SubjectsConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning People™s Republic of China Department of Pediatrics University of Oklahoma Health Sciences Center Oklahoma City OK USA Department of Neurology The Second Hospital of Jilin University Jilin People™s Republic of China Received April Accepted August References Braess J Acute myeloid leukemia Dtsch Med Wochenschr “Luppi M Fabbiano F Visani G Martinelli G Venditti A Novel agents for acute myeloid leukemia Cancers Basel Cancer Research UK Acute myeloid leukaemia AML incidence statistics https wwwcance rrese archu khealt hprofe ssion alcance rstati stics stati stics bycance rtypeleuka emiaamlincid ence Accessed Aug Jung J Cho BS Kim HJ Han E Jang W Han K Lee JW Chung NG Cho B Kim M Kim Y Reclassification of acute myeloid leukemia according to the WHO classification Ann Lab Med “ Murphy T Yee KWL Cytarabine and daunorubicin for the treatment of acute myeloid leukemia Expert Opin Pharmacother “ Byrd JC Mrózek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B CALGB Blood “Lindsley RC Mar BG Mazzola E et al Acute myeloid leukemia ontogeny is defined by distinct somatic mutations Blood “ Vardiman JW Thiele J Arber DA et al The revision of the World Health anization WHO classification of myeloid neoplasms and acute leukemia rationale and important changes Blood “Saultz JN Garzon R Acute myeloid leukemia a concise review J Clin Med Döhner H Weisdorf DJ Bloomfield CD Acute myeloid leukemia N Engl J Med “ Strickland SA Shaver AC Byrne M et al Genotypic and clinical heterogeneity within NCCN favorablerisk acute myeloid leukemia Leuk Res “ Yang JJ Park TS Wan TSK Recurrent cytogenetic abnormalities in acute myeloid leukemia Methods Mol Biol “ Coenen EA Zwaan CM Reinhardt D et al Pediatric acute myeloid leukemia with t816p11p13 a distinct clinical and biological entity a collaborative study by the InternationalBerlinFrankfurtMunster AMLstudy group Blood “ Xie W Hu S Xu J Chen Z Medeiros LJ Tang G Acute myeloid leukemia with t816p112p133KAT6ACREBBP in adults Ann Hematol “ Haferlach T Kohlmann A Klein HU et al AML with translocation t816p11p13 demonstrates unique cytomorphological cytogenetic molecular and prognostic features Leukemia “ Gervais C Murati A Helias C et al Acute myeloid leukaemia with 8p11 MYST3 rearrangement An integrated cytologic cytogenetic and molecular study by the groupe francophone de cytogénétique hématologique Leukemia “ Diab A Zickl L AbdelWahab O et al Acute myeloid leukemia with translocation t816 presents with features which mimic acute promyelocytic leukemia and is associated with poor prognosis Leuk Res “ Schouten TJ Hustinx TW Scheres JM Holland R de Vaan GA Malignant histiocytosis Clinical and cytogenetic studies in a newborn and a child Cancer “ Brown T Swansbury J Taj MM Prognosis of patients with t816p11p13 acute myeloid leukemia Leuk Lymphoma “ Barrett R Morash B Roback D et al FISH identifies a KAT6ACREBBP fusion caused by a cryptic insertional t816 in a case of spontaneously remitting congenital acute myeloid leukemia with a normal karyotype Pediatr Blood Cancer Schumacher J Szankasi P Kelley TW Detection and quantification of acute myeloid leukemiaassociated fusion transcripts Methods Mol Biol “ DíazBeyá M Navarro A Ferrer G et al Acute myeloid leukemia with translocation 816p11p13 and MYST3CREBBP rearrangement harbors a distinctive microRNA signature targeting RET protooncogene Leukemia “ Veigaard C Nørgaard JM Kjeldsen E Genomic profiling in high hyperdiploid acute myeloid leukemia a retrospective study of cases Cancer Genet “ Yasar D Karadogan I Alanoglu G et al Array comparative genomic hybridization analysis of adult acute leukemia patients Cancer Genet Cytogenet “ van der Veken LT Buijs A Array CGH in human leukemia from somatics to genetics Cytogenet Genome Res “ Laskowska J Szczepanek J Styczyński J Tretyn A Array comparative genomic hybridization in pediatric acute leukemias Pediatr Hematol Oncol “ Mehrotra M Luthra R Ravandi F et al Identification of clinically important chromosomal aberrations in acute myeloid leukemia by arraybased comparative genomic hybridization Leuk Lymphoma “ Kulikowski LD Bellucco FTS Nogueira SI et al Pure duplication 1q41qter further delineation of trisomy 1q syndromes Am J Med Genet A 2008146A2663“ Nowaczyk MJM Bayani J Freeman V Watts J Squire J Xu J De novo 1q32q44 duplication and distal 1q trisomy syndrome Am J Med Genet A 2003120A229“ Cone EB Dalton SS Van Noord M Tracy ET Rice HE Routh JC Biomarkers for wilms tumor a systematic review J Urol “ Xu B Ghossein R Genomic landscape of poorly differentiated and anaplastic thyroid carcinoma Endocr Pathol “ Fournier A Florin A Lefebvre C Solly F Leroux D Callanan MB Genetics and epigenetics of 1q rearrangements in hematological malignancies Cytogenet Genome Res “ Lancman G Tremblay D Barley K et al The effect of novel therapies in highmolecularrisk multiple myeloma Clin Adv Hematol Oncol “ Bacher U Schnittger S Grüneisen A Haferlach T Kern W Haferlach C Inverted duplication dup1q32q21 as sole aberration in lymphoid and myeloid malignancies Cancer Genet Cytogenet “ Marcellino BK Hoffman R Tripodi J et al Advanced forms of MPNs are accompanied by chromosomal abnormalities that lead to dysregulation of TP53 Blood Adv “ Beach DF Barnoski BL Aviv H et al Duplication of chromosome [dup1q21q32] as the sole cytogenetic abnormality in a patient previously treated for AML Cancer Genet “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c'

Thyroid_Cancer

Cabozantinib is an oral multikinase inhibitor whose targets include vascular endothelial growth factor receptors MET and the TAM family of kinases TYRO3 AXL MER Cabozantinib is approved for patients with advanced hepatocellular carcinoma who have been previously treated with sorafenib based on improved overall survival and progressionfree survival relative to placebo in the phase III CELESTIAL study During CELESTIAL the most common adverse events AEs experienced by patients receiving cabozantinib included palmarplantar erythrodysesthesia fatigue gastrointestinalrelated events and hypertension These AEs can significantly impact treatment tolerability and patient quality of life However AEs can be effectively managed with supportive care and dose modifications During CELESTIAL more than half of the patients receiving cabozantinib required a dose reduction while the rate of treatment discontinuation due to AEs was low Here we review the safety profile of cabozantinib and provide guidance on the prevention and management of the more common AEs based on current evidence from the literature as well as our clinical experience We consider the specific challenges faced by clinicians in treating this patient population and discuss factors that may affect exposure and tolerability to cabozantinib IntroductionThere has been a marked increase in liver cancer deaths in recent years In there were a0 new cases of liver cancer worldwide and liver cancer accounted for almost deaths making it the sixth most prevalent cancer worldwide [] The most common primary malignancy of the liver is hepatocellular carcinoma HCC [] The frequency burden and etiology of HCC vary across geographic regions and populations but are linked to prevalence of predisposing chronic hepatic conditions such as Electronic supplementary material The online version of this s1152 contains supplementary material which is available to authorized usersKey Points Cabozantinib represents a treatment option for patients with advanced hepatocellular carcinoma who progress after sorafenibAdverse events associated with cabozantinib may be effectively managed with supportive care and dose modifications thereby allowing patients to continue treatment at the appropriate dose with minimum interruptionStudies of cabozantinib in the firstline setting are ongoing by understanding the safety profile of this drug clinicians will be able to balance efficacy with tolerability for each patient Gabriel Schwartz GabrielSchwartzucsfedu Gastrointestinal Medical Oncology Clinic University of a0California San Francisco Fourth St Fourth Floor San a0Francisco CA a0 USAIndiana University Health Simon Cancer Center Indianapolis IN USA Department of a0Medicine University of a0California San Francisco San a0Francisco CA USAIRCCS Istituto Clinico Humanitas Rozzano Milan Italy viral hepatitis and nonalcoholic fatty liver disease NAFLD or nonalcoholic steatohepatitis NASH which generally develop in the setting of cirrhosis [ ] In recent years the incidence of nonviral HCC has increased while the proportion of HCC cases related to viral hepatitis has declined [ ] Additional risk factors for HCC include alcohol consumption smoking obesity and diabetes [] As the epidemiology of these conditions has evolved so too has the etiology of HCC []Vol0123456789 0c G a0Schwartz et alFor patients with advanced HCC the vascular endothelial growth factor receptor VEGFR“targeting tyrosine kinase inhibitor TKI sorafenib has been a standard of care [] however the treatment landscape has been transformed in recent years with the introduction of newer TKIs immunotherapies and monoclonal antibody therapies [] This provides clinicians and patients with a variety of treatment options based on mechanism of action and safety profileCabozantinib is a multikinase inhibitor that targets VEGFR “ MET the TAM family of kinases TYRO3 AXL MER RET ROS1 KIT TRKB FLT3 and TIE2 [ ] several of which are implicated in tumor growth angiogenesis and immune regulation [] VEGFR MET and AXL have been implicated in the pathogenesis of HCC [“] A capsule formulation of cabozantinib was first approved in for treatment of progressive metastatic medullary thyroid carcinoma MTC [] The tablet formulation not bioequivalent or interchangeable with the capsule [] was subsequently approved for patients with advanced renal cell carcinoma RCC [ ] and more recently for patients with advanced HCC who have received prior sorafenib [ ] The approval in HCC was based on outcomes from the pivotal phase III CELESTIAL trial which showed significantly improved overall survival OS and progressionfree survival PFS with cabozantinib relative to placebo in patients who received prior sorafenib [] The safety profile of cabozantinib was manageable nearly all patients receiving cabozantinib experienced an adverse event AE but these were effectively managed with dose modification and supportive care measuresClinicians treating patients with advanced HCC can face significant challenges as many patients present with cirrhosis and comorbidities that can impact treatment tolerability Adequate assessment of liver function and management of comorbidities are therefore essential before and during HCC treatment [] Here we provide guidance on the management of AEs associated with cabozantinib in patients with advanced HCC We briefly review outcomes from CELESTIAL and focus on managing some of the more common AEs experienced by patients based on current evidence from the literature as well as our own clinical experience Cabozantinib in a0Hepatocellular Carcinoma CELESTIALIn the phase III CELESTIAL study patients with advanced HCC were randomized to treatment with cabozantinib a0mg daily or placebo [] Patients were required to have had prior treatment with sorafenib and could have received up to two prior systemic regimens for HCC Eastern Cooperative Oncology Group ECOG performance status PS of or and ChildPugh class A liver function see Electronic Supplementary Table a0 for definition were also required At the second planned interim analysis patients had been randomized The study met its primary endpoint with significantly improved OS with cabozantinib relative to placebo median OS was versus months hazard ratio confidence interval [CI] “ p a0 a0 Cabozantinib also improved PFS with a median of versus months hazard ratio CI “ p a0 a0 as well the objective response rate per Response Evaluation Criteria In Solid Tumors RECIST v11 vs a0 p a0 a0 Safety and a0TolerabilityAllcause AE rates were generally higher in the cabozantinib arm than in the placebo arm some of the more common AEs experienced by patients in the cabozantinib a0 versus placebo arms included diarrhea vs decreased appetite vs palmarplantar erythrodysesthesia PPE vs fatigue vs nausea vs hypertension vs vomiting vs asthenia vs and increased aspartate aminotransferase AST vs Fig a0 The most common grade AEs in the cabozantinib versus placebo arms were PPE vs hypertension vs increased AST vs fatigue vs and diarrhea vs Overall the safety profile of cabozantinib was consistent with those from the phase III studies in RCC and MTC with gastrointestinal GI events PPE fatigue and hypertension being the most common AEs experienced by patients across studies [ ]In addition to supportive care measures protocolspecified dose modification including dose interruption and reduction was utilized to manage AEs [] Eightyfour percent of patients in the cabozantinib arm had an AE that led to dose interruption and had a dose interruption due to a grade AE [] Sixtytwo percent of patients had at least one dose reduction due to an AE [] and dose reduced due to a grade AE [] Thirtythree percent of patients had a second dose reduction [] Median time to first and second dose reduction in the cabozantinib arm was a0days and a0days respectively PPE was the event that most commonly led to dose interruption and dose reduction followed by diarrhea and and fatigue and [] Although most patients receiving cabozantinib required a dose interruption the rate of discontinuation due to treatmentrelated AEs was relatively low in the cabozantinib arm vs in the placebo arm indicating that the majority of AEs were adequately managed with dose modification and supportive care In the cabozantinib group AEs that led to treatment discontinuation in ‰¥ a0 of patients were PPE fatigue decreased appetite diarrhea and nausea In a subgroup analysis of patients 0cAE Any grade [Grade Grade ]Fatigue [ ]Hypertension [ ]Increased AST [ ]Increased ALT [ ]Asthenia [ ]Nausea [ ]Vomiting [ ]Decrease appetite [ ]Weight loss [ ]Diarrhea [ ]Constipation [ ]Abdominal pain [ ]PPE [ ]Fig Incidence rates for select AEs experienced by patients with HCC receiving cabozantinib during the CELESTIAL trial [] AEs are color coded by system blue gastrointestinal purple skin and subcutaneous tissue green constitutional orange hepatic disorders red cardiovascularhematological disorders AE adverse event ALT alanine aminotransferase AST aspartate aminotransferase HCC hepatocellular carcinoma PPE palmarplantar erythrodysesthesiawho received sorafenib as the only prior treatment for HCC duration of prior sorafenib did not appear to impact the types or rates of grade AEs []Generally the more common AEs emerged in the first weeks of treatment Fig a0 However clinicians should be aware of infrequent or serious events that can occur in the later phases of treatment Hemorrhagic events of grade or higher were reported in of patients in the cabozantinib arm including five patients with a grade event Bleeding complications are associated with antiangiogenic therapies and may arise as a result of reduced vascular integrity [] Median time to onset of hemorrhagic events was a0weeks in CELESTIAL Other grade or higher rare but serious AEs in patients receiving cabozantinib included fistulas of patients GI perforations and arterial and venous or mixed thrombotic events [] Median time to first occurrence was approximately a0weeks for GI perforations weeks for venous and arterial thromboembolisms and weeks for fistulas [] Two patients in the cabozantinib arm had developed ChildPugh C ie decompensated cirrhosis by the week assessment []Reversible posterior leukoencephalopathy syndrome RPLS a syndrome of subcortical vasogenic edema diagnosed by magnetic resonance imaging has been reported with cabozantinib and other TKIs [ ] Although there were no RPLS events during CELESTIAL [] clinicians should be aware of the symptoms which include headaches seizures confusion changes to vision or altered mental function [ ] Osteonecrosis of the jaw ONJ whereby necrotic jaw bone becomes exposed is another rare but serious AE associated with TKIs including cabozantinib [“] although again there were no ONJ events reported during this study [] The use of antiresorptive drugs in patients with bone metastases is also associated with development of ONJ []A post hoc analysis estimated the incremental qualityadjusted lifeyears accrued with cabozantinib compared with placebo using the fivedimension fivelevel EuroQol questionnaire [] Cabozantinib treatment was associated with an initial decline in mean total qualityadjusted lifeyears during the first “ a0months relative to placebo followed by longterm improvement that was significantly greater than that observed with placebo p a0 a0Management of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c Fig Rates and timing of select AEs in patients with HCC receiving cabozantinib during the CELESTIAL trial The size of the circle is proportional to the AE rate AEs are color coded by system blue gastrointestinal purple skin and subcutaneous tissue green constitutional orange hepatic disorders red cardiovascularhematological disorders black generalother AE adverse event ATE arterial thrombotic event GI gastrointestinal GR grade HCC hepatocellular carcinoma PPE palmarplantar erythrodysesthesia VTE venous thrombotic eventMedian time to first dosereduction to mg weeksG a0Schwartz et alMedian time to seconddose reduction to mg weeksFistulas Hemorrhage GR ATEs VTE Wound complication GI perforations Hepatic encephalopathy Diarrhea PPE Hypertension Median time to first occurrence weeks Factors Affecting Tolerability of a0Cabozantinib Co‘morbiditiesHCC emerges primarily in older adults [] In addition to the underlying HCC etiology older adults with HCC are likely to have additional comorbidities such as cardiovascular or pulmonary disease [] and it is not uncommon for patients with HCC to have multiple comorbidities [] Liver cirrhosis with compromised liver function and decreased hepatic reserve is a major risk factor for HCC development Other HCCrelated comorbidities include hepatitis B virushepatitis C virus infection alcoholic liver disease NASH and diabetes [] In addition metabolic syndrome characterized by hyperlipidemia and hypertension is linked to development of NAFLD which may progress to NASH cirrhosis and finally HCC [] For patients with HCC assessment of liver function is a key step in treatment decisionmaking [] Patients with moderate or severe hepatic impairment are predominantly excluded from clinical trials in HCC therefore treatment of these patients is complicated by a lack of prospective clinical data as well as competing comorbidities []Although the number of patients with HCC and prior an transplant is limited these patients are generally excluded from clinical trials and treatment is complicated by the need for immunosuppression TKIs may be used to treat posttransplant HCC recurrence although supporting data are limited The use of TKIs in these patients is complex so treatment decisions should involve collaboration between the oncology and transplant medicine care teams The use of sorafenib in patients receiving mammalian target of rapamycin inhibitorbased immunosuppression has been associated with an increased risk of fatal bleeding [ ] Immunotherapies are associated with an increased risk of an rejection in posttransplant patients [] Cabozantinib Clearance and a0ExposureTKIs are associated with high interpatient variability in clearance and exposure which may affect both efficacy and tolerability This variability may be due to a variety of factors including genetic background drug“drug interactions drugfood interactions and renal or hepatic impairment [] As evidenced by exposureresponse modeling patients with low clearance of cabozantinib may have higher exposure and an increased risk of developing certain AEs [ ] Awareness of these nuances may help clinicians to mitigate their effects thereby balancing efficacy with tolerability Hepatic and a0Renal ImpairmentAccording to pharmacokinetic analyses of patients with HCC and other tumor types mild hepatic impairment is predicted to have a minimal effect on cabozantinib exposure [] therefore adjustment of the recommended 60mg starting dose is not necessary for patients with Child“Pugh A 0cliver function [ ] Data on the pharmacokinetics of cabozantinib in patients with moderate ChildPugh B or severe Child“Pugh C hepatic impairment are limited [] As per the US Food and Drug Administration FDA prescribing information the starting dose of cabozantinib should be reduced to mg in patients with moderate hepatic impairment while cabozantinib is not recommended for patients with severe hepatic impairment [] Note that the European Summary of Product Characteristics SmPC does not recommend dose adjustments for moderate hepatic impairment owing to limited data [] For patients with HCC increased exposure due to hepatic impairment should be considered if intolerable AEs develop and dose modification undertaken as recommended Fig a0 [ ] Cabozantinib should be used with caution in patients with mild or moderate renal impairment owing to the potential for increased exposure although no dose adjustments are necessary Cabozantinib is not recommended for use in patients with severe renal impairment owing to lack of data on safety and efficacy in this population [ ] Drug“Drug and a0Drug“Food InteractionsGiven the range of comorbidities that may exist in patients with advanced HCC it is important to review all concomitant medications for potential interactions prior to initiation of treatment with cabozantinib Certain medications and foods have been shown to modulate the pharmacokinetics of cabozantinib which may in turn impact exposure levels efficacy and risk of AEs Cabozantinib is metabolized in the liver primarily by the enzyme cytochrome P450 3A4 CYP3A4 [] therefore CYP3A4 inhibitors or inducers may impact exposure examples of CYP3A4 inducersinhibitors are shown in Electronic Supplementary Table a0 Strong CYP3A4 inhibitorsinducers should be avoided in patients receiving cabozantinib If concomitant administration of a strong CYP3A4 inhibitor is necessary then the cabozantinib Recommended dose at initiation mg Except for¢ Patients with moderate hepatic impairment or coadministration of a strong CYP3A4 inhibitor initiate cabozantinib at mg ¢ Patients with coadministration of a strong CYP3A4 inducer initiate cabozantinib at mg Safety assessmentNo AEsGrade Grade AE or ONJSupportive caresee Tables “DosemodificationImprovementtolerableelbarelotnIlitnu esod dloHgrade ‰¤Continue at tolerated doseReduce dose by mg and restart mg †’ mg mg †’ mg mg †’ mg mg †’ mg or discontinueImmediate Discontinuation¢ Severe hemorrhage¢ Development of GI perforation or unmanageable fistula¢ Serious thromboembolic event eg myocardial infarction cerebral infarction¢ Hypertensive crisis or severe hypertension despite optimal medical management¢ Nephrotic syndrome¢ Reversible posterior leukoencephalopathy syndromeFig Cabozantinib dosing algorithm [ ] AE adverse event CYP3A4 cytochrome P450 3A4 GI gastrointestinal ONJ osteonecrosis of the jawManagement of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c G a0Schwartz et aldose should be reduced by a0mg for example from to a0mg [] Conversely the cabozantinib dose should be increased by a0mg if strong CYP3A4 inducers need to be coadministered [ ]Cabozantinib should not be taken with any food as this may affect absorption [] The label recommends that cabozantinib be taken at least h before or at least h after eating [] Grapefruit and grapefruit juice are strong CYP3A4 inhibitors and should be avoided [ ]Cabozantinib may be used with caution in patients who are receiving concurrent antiarrhythmics or other QTprolonging agents [] This is based on a study of patients with MTC who received a daily 140mg capsule dose of cabozantinib recommended for this indication in which the mean deltadelta QT interval was increased by approximately “ a0ms with upper CIs not exceeding ms [] Such an increase is within the range considered to be acceptable for oncology drugs in this setting [] No patient in the aforementioned study or in CELESTIAL had a confirmed QTcF QT corrected using Fridericia™s method a0 ms [] which is considered clinically significant [] For patients receiving cabozantinib monitoring with periodic electrocardiogram and electrolyte measurements may be advisable particularly in patients with risk factors such as cardiac disease or a prior history of QT prolongation [] Concomitant use of proton pump inhibitors PPIs such as esomeprazole does not affect cabozantinib exposure levels [] However PPIs may cause hypomagnesemia which is linked to an increased risk of QT prolongation [] Therefore coadministration of PPIs and cabozantinib should be undertaken with caution following an individualized assessment of the patient™s baseline magnesium levels and concomitant medications that may also influence QT Pretreatment AssessmentsGiven the heterogeneity of the HCC patient population and the complexity associated with comorbidities and concomitant medications all patients should undergo a comprehensive assessment of medical history prior to initiation of treatment with cabozantinib Ideally the multidisciplinary care team should include an oncology pharmacist [] A œbrown bag medication review should be carried out prior to treatment initiation [] whereby the patient brings in all current medications including overthecounter medicines vitamins herbal remedies etc Therapeutic duplications should be eliminated for example concomitant PPIs and histamine H2 antagonists H2 blockers Switching and deprescribing should be considered where possible to minimize the risk of drugdrug interactions Adverse Event ManagementThe AE profile of cabozantinib is generally similar to that of other VEGFRtargeting TKIs with GIrelated AEs fatigue PPE and hypertension being the most common AEs [] Other AEs that occur less frequently can also have a significant impact on quality of life QoL and treatment adherence such as mucosal inflammation [] Hepatobiliary AEs such as elevated AST alanine aminotransferase ALT and bilirubin are particularly relevant in the context of advanced HCC and need to be carefully monitoredProphylactic and supportive care measures for the more common cabozantinibassociated AEs grade or tolerable grade are outlined in Tables a0 and discussed in the upcoming sections Symptom gradings are summarized in Electronic Supplementary Table a0 Dose interruption is recommended for management of intolerable grade AEs not resolved with supportive care measures or for any grade AEs Fig a0 [ ] Cabozantinib may be reinitiated at a reduced dose once the event resolves to grade ‰¤ a0 Palmar“Plantar ErythrodysesthesiaPPE is one of the more common events associated with anticancer therapies including VEGFRtargeting multikinase inhibitors [“] PPE is characterized by pain redness tingling and swelling of hands and feet [] Presentation may vary according to the etiologic agent PPE induced by TKIs is typically localized to pressurebearing areas in contrast to that caused by chemotherapy which has a more diffuse pattern It has been hypothesized that inhibition of multiple angiogenic pathways by TKIs may compromise repair of capillary microtrauma in areas exposed to mechanical stress such as the hands and feet [ ] Although not lifethreatening PPE can rapidly progress to a debilitating condition negatively impacting QoL [ ]Prophylaxis and prompt management of emerging symptoms may help to minimize the impact of PPE on QoL and adherence Table a0 Prophylactic measures predominantly involve skin care practices to remove hyperkeratotic areas and to minimize friction and damage prior to the start of treatment [ ] Recommendations include use of thick cotton gloves and socks padded insoles in shoes and avoidance of heat or friction on the hands and feet [ ] Patients with potentially predisposing comorbidities such as peripheral neuropathy [ ] as well as patients with persistent symptoms may benefit from involvement of a podiatrist andor dermatologist within their multidisciplinary care team [] Treatment strategies involve moisturization prevention of infection and analgesia [ ] Monitoring is crucial so that emerging symptoms can be proactively managed Patients should be assessed at baseline 0cTable Adverse event management strategies”palmar“plantar erythrodysesthesia PPE PPEProphylaxisProvide education on prophylactic skin care before starting treatment []Advise manicure and pedicure before and during treatment to remove hyperkeratotic areas [ ]Protect sensitive areas recommend sunscreen with SPF protection ‰¥ a0 thick cotton gloves and socks padded insoles and wellfitting shoes avoid heat sources and use cooling aids and avoid activities that may cause force or rubbing on the hands and feet eg heavy lifting dish washing [ ] delegate such tasks to caregiversAdvise on optimal hand cleaning avoid fragrancedfoaming soaps and hand sanitizers containing alcohol ensure hands are dried thoroughly after cleaning []Prophylactically administer keratolytic cream eg urea [ ]Monitor regularly in order to proactively manage skin toxicities evaluate at baseline monitor up to weekly for the first “ months and monthly thereafter [ ]Supportive careContinue prophylactic measures []Maintain moisture of skin using emollients [ ]Consider topical treatment with salicylic acid urea “ cream either alone or with tazarotene cream or fluorouracil cream andor clobetasol cream topical analgesics may be added for pain control [ ]Topical cortisone and clobetasol may also be used consider oral analgesics eg NSAIDs pregabalin cautious use of opioids [ ]Consult with a dermatologist to drain blisters and remove hyperkeratotic areas []To prevent infection of cracked skin soak in equal parts vinegar and water for min per day [] a0Antibiotics should be prescribed only if there is evidence of infection [] a0There is limited evidence for the use of pyridoxine vitamin B6 []NSAID nonsteroidal antiinflammatory drug SPF sun protection factorTable Adverse event management strategies”fatigue FatigueProphylaxisProvide patient education about fatigue management tools and available support []Establish baseline fatigue levels with a fatigue scale and remeasure regularly during patient visits []Ensure adequate fluid and nutritional intake []Advise behavioral modifications balancing rest with physical activity recommendations include relaxation massage yoga aerobic or resistance exercise programs and energy conservation strategies [“]Assess thyroid function prior to treatment and monitor during treatment [ ]Supportive careRule out alternative causes of fatigue eg anemia endocrine disorders such as hypothyroidism pain dehydration hypercalcemia or depressionanxiety [ ]Advise patient to increase activity consider referral to a physical therapist []Consider referral to nutritional counselor for nutritional therapy []Incorporate psychosocial measures including cognitive therapy social support biofeedback and sleep therapy []Incorporate management with psychostimulants eg methylphenidate [ ] or corticosteroids eg methylprednisolone []Owing to effects on CYP3A45 substrates including cabozantinib longterm use of modafinil should be avoided []CYP3A4 cytochrome P450 3A4 CYP2C19 cytochrome P450 2C19Management of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c G a0Schwartz et alTable Adverse event management strategies”gastrointestinal GastrointestinalDiarrheaProphylaxisInstruct patients to monitor food and fluid intake [] a0Recommended water intake per day from all beverages and food [] L oz for women L oz for men a0Advise patients to keep a stool diary and to promptly report diarrhea to their healthcare provider [ ]Advise patients to avoid foods that may cause GI events such as lactosecontaining foods caffeine highfat or highfiber food eg nuts seeds legumes and raw fruit and vegetables [ ]Implement dehydration prevention management through oral rehydration with electrolytes []Supportive careAdminister loperamide at the first sign of diarrhea [ ] a0 mg orally followed by mg every h until “ h after last bowel movement maximum of mg in h a0For chronic diarrhea “ mg twice daily titrated as needed a0Alternatives to loperamide include diphenoxylate and tincture of opium []Implement supportive dietary modifications continuous oral hydration correction of fluid and electrolytes small frequent meals avoid lactosecontaining food and drink [ ] a0The BRAT bananas rice applesauce toast diet may help to alleviate mild diarrhea []If there are signs of severe dehydration administer IV fluid replacement isotonic saline or balanced salt solution []Rule out nontreatmentrelated causes eg infectious diarrhea []Decreased appetiteProphylaxisAdvise patients to monitor their appetite and weight []Encourage patients to consume highprotein calorierich food fruit and vegetables nutritional supplements that they may snack on throughout the day [ ]Advise patients to preprepare and freeze nutritional preferred food []Supportive careTreat underlying nausea []Consider involving a dietitian who may recommend scheduled eating times []Recommend a highcalorie diet []Provide dietary education alongside dietary modifications andor nutritionalvitamin supplements []Use a pharmacologic agent to stimulate appetite such as a CB1 receptor agonist dronabinol [ ] systemic corticosteroid methylprednisolone [ ] progestin megestrol acetate [ ] or mirtazapine [ ]NauseavomitingProphylaxisAssess risk factors for nauseavomiting prior to treatment []Metoclopramide may be administered prophylactically []Advise patients to avoid foods that are overly sweet greasy fried or spicy []Supportive careAntiemetic agents such as dopamine receptor antagonists eg metoclopramide prochlorperazine or 5HT3 receptor agonists eg ondansetron are recommended for management of nausea or vomiting [ ] a0Certain NK1 receptor agonists eg aprepitant and netupitant and dexamethasone are inducers inhibitors andor substrates of CYP3A4 and thus could alter cabozantinib exposure [ ] however the potential for ondansetron to prolong the QT interval must also be considered [] There is moderate evidence for olanzapine an antipsychotic drug that blocks multiple neurotransmitters as an antiemetic in this setting [] 0cTable continuedMucosal inflammationstomatitisProphylaxisA comprehensive dental examination should be conducted prior to treatment to identify potential complications []Mitigation of potential risk factors [ ] a0Modification of illfitting dentures a0Appropriate care for preexisting dental problems such as caries ulcers etcRegular oral assessments should be conducted throughout treatment [ ]Educate patients on good oral hygiene and oral care protocols including written instructions [] a0The oral cavity should be washed using salinecontaining mouthwash up to four times daily and dentures should be regularly cleaned []Painful stimuli eg smoking alcohol hot fooddrink sharp or spicy food should be avoided [ ]Supportive careTreat pain with doxepin mouthwash or viscous lidocaine [ ]Lactobacillus lozenges may be used to reduce inflammation []Obtain bacterialviral culture if oral infection is suspected and treat infection as clinically indicated []5HT3 5hydroxytryptamine CB1 cannabinoid CYP3A4 cytochrome P450 3A4 GI gastrointestinal IV intravenous NK neurokininTable Adverse event management strategies”hypertension HypertensionProphylaxisMonitor BP before initiation of cabozantinib using a minimum of two standardized BP measurements alongside patient history physical assessment directed laboratory evaluation and an instrument test to determine cardiovascular risk factors [ ]Educate patients on BP selfmonitoring and advise they keep a BP log []BP should be well controlled prior to initiating cabozantinib ensure patients who have already been prescribed antihypertensive therapy are adherent and that therapy has been titrated to effective doses [ ]Check for potential drugdrug interactions of existing antihypertensive agents with cabozantinib Supplementary Table a0Consider effects of concomitant medications on BP eg antiinflammatory drugs can increase BP opiates can lower BP []Monitor BP during cabozantinib treatment weekly during first cycle every ‰¥ a0“ weeks thereafter []Supportive careAdd antihypertensive medications or increase dose of existing medication as indicated [ ]Patients with portal hypertension should be treated with nonselective betablockers []The antihypertensive agent should be carefully considered owing to potential inhibition of CYP3A4 [ ] Supplementary Table a0 a0Thiazides angiotensinconverting enzyme inhibitors and angiotensin receptor blockers may be used to treat hypertension and are not known CYP3A4 substrates [“ ] a0Thiazide diuretics should be prescribed with caution owing to the associated risk of diarrhea [] a0Diltiazem and verapamil are moderate inhibitors of CYP3A4 [] a0Amlodipine felodipine lercanidipine nisoldipine and nifedipine are not considered to be CYP3A4 inhibitors []BP blood pressure CYP3A4 cytochrome P450 3A4and monitored at least weekly for the first “ months of treatment and monthly thereafter [ ] Close monitoring in the early stages of treatment need not involve weekly visits”phone calls from a clinician nurse or pharmacist may facilitate monitoring in between scheduled appointments [] Patients should be encouraged to report early signs of PPE to their healthcare provider [] it may also be reassuring for patients to know that early reporting and management of AEs

Thyroid_Cancer

Patients with resected oral cavity squamous cell carcinoma OCSCC are often treated with adjuvantradiation RT ± concomitant chemotherapy based on pathological findings Standard RT volumes include all surgicallydissected areas including the tumour bed and dissected neck RT has significant acute and longterm toxicitiesincluding odynophagia dysphagia dermatitis and fibrosisThe goal of this study is to assess the rate of regional failure with omission of radiation to the surgically dissectedpathologically node negative pN0 heminecks compared to historical control and to compare oncologic outcomestoxicity and quality of life QoL profiles between standard RT volumes and omission of RT to the pN0 neckMethods This is a multicentre phase II study randomizing patients with T1“ N0“ OCSCC with at least one pN0hemineck in a ratio between standard RT volumes and omission of RT to the pN0 heminecks Patients will bestratified based on overall nodal status nodal involvement vs no nodal involvement and use of concurrentchemotherapy The primary endpoint is regional failure in the pN0 heminecks we hypothesize that a 2year regionalrecurrence of or less will be achieved Secondary endpoints include overall and progressionfree survival localrecurrence rate of salvage therapy toxicity and QoLDiscussion This study will provide an assessment of omission of RT to the dissected pN0 heminecks on oncologicoutcomes QoL and toxicity Results will inform the design of future definitive phase III trialsTrial registration Clinicaltrialsgov identifier NCT03997643 Date of registration June Current version onJuly Keywords Head and neck cancer Oral cavity Radiotherapy Recurrence Survival Quality of life Randomized controlledtrial Deescalation Correspondence pencillalanglhsconca1Division of Radiation Oncology London Health Sciences Centre Commissioners Rd E London ON N6A 5W9 CanadaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLang Radiation Oncology Page of BackgroundPatients with resected squamous cell carcinoma of the oralcavity OCSCC are at risk of locoregional failure recurrence LRR at either the tumour surgical bed or in theneck Postoperative radiotherapy PORT is often addedafter surgical resection to reduce the risk of local and regional recurrence in patients with highrisk featuresGuidelines generally recommend PORT in patients withmore than one lymph node involved pT3 or pT4 primarydisease lymphovascular invasion LVI perineural invasion PNI close or positive margins extranodal extensionENE and sometimes for patients with lymph node involvement of neck levels IV or V [“]ENE and positive surgical margins are considered thehighestrisk pathological features whereas the other adverse features are considered ˜intermediate risk™ Patientswith highrisk features are generally offered concurrentchemotherapy with PORT based on a posthoc analysistwo of randomized trials [“] In patients with otheradverse features there is limited randomized evidence ofthe benefits of PORT alone [“] Retrospective seriesand comparisons with historical controls have shown reductions in LRR and improvement in overall survivalOS with PORT [“]RT treatment volumes after surgery generally includethe entire surgical bedincluding the dissected heminecks and may include the contralateral undissectedneck at the discretion of the treating physician []However PORT is associated with significant acute andincluding dysphagia mucositis xerostolate toxicitiesmiavoicechanges ototoxicity and hypothyroidism [ ] LargerRT treatment volumes are associated with increasingtoxicity These large treatment volumes are based onhistorical practice without guidance from randomizedevidence The benefit of treating the nodal regions in thepathologically node negative pN0 neck is unknownosteoradionecrosisdermatitisfibrosisRetrospective studies looking at series of oral cavitycancer patients in whom PORT was omitted altogetherhave also demonstrated low rates of isolated nodal recurrence ranging from to [“] In their seriesof patients with mostly T1T2 oral tongue tumourswith a pN0 neck treated with surgery alone PORTto the primary site or PORT to the primary siteand neck So reported a rate of isolatednodal recurrence of “ in all groups [] Mizrachi described a series of patients with T1T4 disease and pN0 neck with not receiving PORT with aneck recurrence rate of [] In Ganly patients with pT1T2 pN0 oral tongue cancer withoutPORT had an isolated regional recurrence rate of with an additional having a simultaneous locoregional recurrence In this study depth of invasion was predictive of neck recurrence []In a series of patients with a pN1 neck the neck recurrencefree survival was in those receiving PORT vs in thosewithout PORT [] It is difficult to draw comparisonsbetween groups receiving or not receiving PORT inthese retrospective studies since those receiving PORThad more risk featuresRetrospective data from a small number of PORT volume studies also demonstrate good oncologic outcomesin patients where radiation was omitted to the contralateral clinically or pathologically N0 neck [ ] In Vegeer patients with welllateralized oral cavity ororopharynx squamousSCC weretreated with unilateral PORT with of patients N0and N1 or N2 Contralateral metastases developedin only of patients with most successfully receivingsalvage therapy []carcinomacellA recent nonrandomized prospective phase II trialeliminated PORT to the pN0 neck in patients withhead and neck squamous cell carcinoma HNSCCdemonstrating excellent results with no isolated failuresand control in the unirradiated pN0 neck Twentypercent of patients included in the study had oral cavitytumours []Taken together the existing retrospective and prospective data suggests that omitting PORT in patientswith a pN0 neck likely has a recurrence rate less than“ However no randomized studies have directlyexamined the omission of PORT in the pN0 neck Thereis also a paucity of data examining the effects of omission of PORT on QoL We hypothesize that omittingPORT to the heminecks that have been dissected andshown to be pN0 will be associated with acceptable ratesof regional recurrence and will improve quality of lifeQoL The goal of this randomized phase II study is toassess oncologic outcomesfunctional outcomes andQoL in patients treated with PORT to the historicallystandard volumes usually including the primary site andall dissected neck areas vs PORT only to the primarysite and pathologically involved hemineck omitting radiation to the pN0 heminecksMethods designObjectivesThe objectives of this trial are to Compare regional recurrence rate to historicalcontrols with omission of PORT to the pN0 neck Compare oncologic outcomes toxicity and QoL forPORT ± chemotherapy based on standardtreatment volumes including the primary sitetumour bed dissected neck ± elective nodalregions vs PORT [± chemotherapy] that avoidstreating the dissected pN0 neck 0cLang Radiation Oncology Page of anisation for Research and Treatment of CancerEORTC Quality of Life Cancer Patients generalQLQC30 [] and head neck HN35 scales[] the EuroQOL 5Dimension 5Level EQ5D5L [] and the Neck Dissection Impairment IndexNDII [] 0f QoL measured at treatment completion and and monthsmeasured with MDADI EORTC QLQC30HN35 EQ5D5L and NDII 0f Rate of feeding tube insertion after start of radiationeither gastric gastrojejeunal or nasogastric andrate of feeding tube use at 1year postrandomization 0f Swallowing function at 1year assessed by ModifiedBarium Swallow MBS study and measured by theModified Barium Swallow Impairment ProfileMBSImP„¢ score [] the Dynamic ImagingGrade of Swallowing Toxicity DIGEST„¢ score []and the Functional Oral Intake Scale FOIS [] 0f Toxicity assessed using the National CancerInstitute Common Toxicity Criteria NCICTCversion 0f Rate of failure in the clinically node negative neck ifapplicable ie the undissected nodenegative neckfor welllateralized tumoursInclusion criteria 0f Age years or older 0f Willing to provide informed consent 0f Eastern Cooperative Oncology Group ECOG 0f Resected OCSCC with at least an ipsilateral selectiveneck dissection The oral cavity includes lips buccalperformance status “It is generally accepted that a risk of nodal recurrenceof “ is sufficient to warrant radiation to a nodalbasin Our hypothesis is that for patients with SCC ofthe oral cavity T1“ N0“ as per AJCC 8th editionwith at least one surgically dissected pN0 hemineck theregional failure rate will be or less at years whentreated with PORT omitting the pN0 neckStudy designThis is an label phase II multicentre randomizedtrial Patients will be randomized between current standard of care treatment Arm vs omission of radiationto the pN0 dissected heminecks Arm in a ratioFig The required sample size is patients Stratification factors include neck nodal status pN0 vs pNand use of chemotherapy Patients will be recruited fromtertiary care centres full list of participating sites available on clinicaltrialsgov NCT03997643Primary endpoint 0f Regional failure in the pN0 heminecksSecondary endpoints 0f OS 0f Diseasefree survival DFS 0f Local recurrence 0f Regional recurrence 0f Locoregional recurrence 0f Rate of salvage treatment surgery ± radiotherapy inthe pN0 neck and freedom from unsalvageable neckrecurrence 0f QoL at year assessed with the MD AndersonDysphagia Inventory MDADI [] the EuropeanFig Study schema 0cLang Radiation Oncology Page of mucosa oral tongue floor of mouth gingivaretromolar trigone and hard palate 0f Patient has at least one pathological feature that isan indication for PORT positive or close ‰¤ mmmargin presence of LVI or PNI pT3 or pT4 diseasepositive lymph nodes or ENE 0f PORT is recommended by the treating physician 0f Pathologically lymph node negative in at least onedissected hemineck with at least nodes recovered in each pN0 hemineck after a dissection thatat minimum includes nodal levels “ in the pN0heminecks 0f Radiation contours have been peerreviewed andapprovedExclusion criteria 0f Patients with an ipsilateral neck dissection only withpositive lymph nodes unless they undergo acontralateral neck dissection that is pN0 0f Patients with bilaterally involved neck nodes 0f Patients with pT3T4 tumours involving midlinewho undergo an ipsilateral neck dissection unless acontralateral neck dissection is performedcontraindications to radiotherapy 0f Serious medical comorbidities or other 0f Prior history of head and neck cancer within years 0f Any other active invasive malignancy except noncavity or neckmelanotic skin cancers lowrisk prostate cancer andStage IIVA papillary or follicular thyroid cancer 0f Prior head and neck radiation at any time 0f Prior oncologic head and neck surgery in the oral 0f Known metastatic disease 0f Locoregional disease recurrence identified followingsurgical resection but prior to start of radiotherapy 0f Inability to attend full course of radiotherapy or 0f Unable or unwilling to complete QoL questionnaires 0f Pregnant or lactating womenfollowup visitsPretreatment evaluation 0f History and physical examination by a radiationoncologist within weeks prior to randomization 0f Staging prior to randomizationCT or MRI of the neck with contrast unlesscontraindicated within weeks ofrandomization This can include the preoperativeCT or the radiation therapy CT or MRI simulation if reviewed by a radiologist–ª In some instances suspicious lymph nodes arevisible on the scan after surgery In suchinstances recurrence must be ruled outpathologically before enrollment either with aneedle biopsy or resection of these nodesCT of the chest or whole body PETCT usually prior to surgery must be within weeks ofrandomizationedentulous patientswithin weeks of randomization 0f Histological confirmation of SCC 0f Pregnancy test for women of childbearing age 0f Dental evaluation prior to starting treatment except 0f Assessment of dysphagia using NCICTC version 0f Completion of QoL scoring prior to initiation of 0f Prior to randomization radiation contours are to be 0f MBS at baseline prior to initiation of treatment with within weeks of treatment initiationpeerreviewed and approvedtreatmentdocumentation of the MBSIimP„¢ DIGEST„¢ andthe FOIS scoresresectionTreatment planSurgicalandchemotherapy will be delivered in accordance with National Comprehensive Cancer Network NCCN ClinicalGuidelines []and adjuvantradiotherapyPrimary tumours should be resected en bloc wheneverpossible with the goal of achieving clear margins Patients with midline involvement of the primary tumourshould receive a bilateral neck dissectionAdjuvant cisplatinbased chemotherapy concurrentwith radiotherapy is at the discretion of the treatingmedical oncologist and is recommended for patientswith positive margins or ENE for patients who can tolerate chemotherapy For patients who are deemed unfitor too elderly years of agefor cisplatinbasedchemotherapy the standard dose andor schedule canbe modified alternative systemic therapy regimensmaybe used eg weekly carboplatin Calais regimen orsystemic therapy can be omitted at the discretion of thetreating physicians It is strongly recommended that radiation starts within weeks of the date of surgery andit is mandatory to start no more than weeks after thedate of surgeryDuring treatment supportive care should be in accordance with local standard of care which often includes speech language pathology SLP assessment AnySLP interventions eg providing swallowing exercisesshould be the same in both arms and conform to localstandard of careDosefractionation In both arms a dose of Gy in fractions will be delivered to the operative bed targetvolumes Centres that normally treat dissected nodenegative levels to Gy in fractions will be permitted 0cLang Radiation Oncology Page of to do so if used consistently for all patients on trialAreas of positive margins or ENE should receive Gyin fractions if those areas can be localized Undissected areas that require coverage in the opinion of thetreating radiation oncologist eg low neck retrostyloidspace should receive Gy in fractions Table Immobilization and localization All patients will beimmobilized in a custom thermoplastic shell and willundergo a planning CT simulation with or withoutIV contrast encompassing the head and neck tobelow the clavicles The planning CT will be fusedwith other diagnostic imaging eg MRI scans or preoperative CT where necessary Bite blocks tonguedepressors jaw separators may be used as per institutional protocolthetime of planning CT prior to contour generation andpatient randomizationthese must be determined atRadiotherapy volume definitions A randomization volume will be defined as the nodal volume in the dissectedpN0 heminecks The randomization volume will depend on the laterality of the neck dissection performedipsilateral vs bilateral and whether pathologically involved nodes are present in the neck dissection definedin Fig Patients with bilaterally involved neck nodesare ineligible Patients with a unilateral neck dissectionwith positive lymph nodes are ineligible unless theyundergo a staged neck dissection of the opposite sidethat is pN0Standard treatment volumes Arm If randomizedincluding thecontoured volumesto Arm randomization volume will be treatedallOmission of pN0 neck Arm If randomized toArm the randomization volume will be omitted fromtreatment planningClinical Target Volumes CTVThe following radiation volumes will be contoured forall patients prior to randomization Table The suffixœpos denotes CTVs in the nodepositive hemineck andthe suffix œneg denotes the CTVs in the nodenegativehemineck Highrisk volume CTV64 regions correspondingto positive margins or ENE if present and if thoseareas can be localized Primary tumour operative bed CTVp60surgically dissected areas corresponding to theresected primary tumour typically including thepreoperative tumour area and any flap reconstructions and clips with a margin Involved necka Surgically dissected involved neck nodalvolume CTVn60pos surgically dissected necklevels in the nodepositive hemineck if applicable In the dissected neck some centres routinely treat only involved nodal areas to Gyand the remainder of the dissected neck to Gy For centres where this is standard this approach is acceptable but all patients enrolledfrom these centres must be treated with this approach The areas treated to Gy would be included in a CTVn54posb Optional uninvolved lowrisk neck on the involved side CTVn54pos lowrisk undissectedneck nodal volume on the involved side if applicable at the discretion of the radiation oncologist This may include the standardlymphatic drainage sites not dissected at thetime of surgery in the node positive heminecksuch as the nodal levels below or above the dissected areas eg level IVb retrostyloid space œRandomization Volume corresponding to theUninvolved necka Surgically dissected involved neck nodalvolume CTVn60neg surgically dissectedneck levels in the node negative hemineck Incentres that routinely treat the pN0 neck to Gy that dose is acceptable but all patients mustbe treated with that approach In such centresthese areas treated to Gy would be includedin a CTVn54negb Optional uninvolved lowrisk neck on theuninvolved side CTVn54neg lowrisk undissected neck nodal volume on the uninvolvedside if applicable at the discretion of theTable Radiation treatment volumes and dosesPTV VolumePTV64Areas of positive margin or ENEPTV60Dissected neckPTV54optionalNot surgically dissected elective nodal regionsCTV Volumes IncludedArm CTV64CTVp60 CTVn60posCTVn60negCTVn54posCTVn54negArm CTV64CTVp60 CTVn60posCTVn54posDose infractions Gy Gy Gy 0cLang Radiation Oncology Page of Fig The œRandomization Volume corresponds to the pN0 heminecks The neck volumes included in the œRandomization Volume dependon whether the patient had an ipsilateral vs bilateral neck dissection and the pathological findings in each heminecks Patients with bilaterallyinvolved neck nodes are ineligible Patients with an ipsilateral neck dissection with positive lymph nodes are ineligible unless they undergo acontralateral neck dissection that is pN0 If randomized to standard treatment volumes Arm all contoured volumes including theœRandomization Volume will be treated If randomized to omission of the pN0 neck Arm the œRandomization Volume will be omitted fromtreatment planningradiation oncologist In a patient who has had aunilateral neck dissection that was pN0 thismay include the contralateral clinically nodenegative cN0 neck if that is standard institutional practiceIf randomized to omission of PORT to pN0 neckArm the CTVn60neg and CTV54neg contours aredeleted after randomization prior to treatment planningTo prevent bias contours cannot be changed afterrandomization Local peer review of contours must takeplace before randomization occurs An overview of theprotocol timeline is shown in Fig Planning Target Volumes PTVA “ mm expansion is used around the combinedCTVs Table to create the PTV as per institutionalsetup and protocolRadiotherapy planning Intensity modulated radiotherapy IMRT photon therapy or proton therapy will beused for all patients in this study IMRT can be deliveredusing staticbeam techniques or rotational techniqueseg Tomotherapy or Volumetric Modulated Arc Therapy [VMAT] If protons are used the dose will be reported in Gy relative biological effectiveness [RBE]where the proton dose is multiplied by an RBE of All reported doses in Gy are considered equivalent Centres with proton therapy will use their institutionalstandard planning and delivery techniquesAll plans will be normalized to ensure that ofeach PTV is covered by of the prescription dosefor that volume An exception will be allowed forcentres that normally prescribe in such a manner that of the PTV be covered by of the prescription dose and such centres must prespecify this before enrolling the first patient and all subsequentpatients must be planned in the same manner Amodified PTV cropped mm from the externalcontour for dose evaluation may be used as per institutional guidelines 0cLang Radiation Oncology Page of 0f All dose delivery for intensitymodulated plans including arcbased treatments will be confirmed before treatment by physics staffConebeam CT andor orthogonal xrays will be usedon a daily basis to verify treatment positioning as per institutional standard practiceCredentialling Prior to enrolling patients each centrewill be given a sample CT dataset through secure filetransfer protocol FTP for contouring planning andphysics QA Enrollment can begin once the plan andQA have been approved at the London Regional CancerProgram Centres who have been accredited for ORATOR [] or ORATOR2 [] are exempt from thisrequirementParticipant discontinuation withdrawalParticipants may voluntarily discontinue participation inthe study at any time If a participant is removed fromthe study the clinical and laboratory evaluations thatwould have been performed at the end of the studyshould be obtained If a participant is removed becauseof an adverse event they should remain under medicalobservation as long as deemed appropriate by the treating physicianFollowup evaluationDay of followup will be the first day of radiotherapyFollowup will consist of history and physical examination with laryngopharyngoscopy CT imaging of theneck ± thorax QoL assessments and MBS The followup schedule is shown in Additional file Additional imaging or laboratory investigations should be carried outat the discretion of the oncologist based on findings inthe history or physical examination Additional treatment eg salvage treatment with surgery or furtherradiotherapy is at the discretion of the treating physicians but will be recorded in the case report formMeasurement of outcomes 0f pN0 neck failure measured as time fromrandomization until disease recurrence in theinitially pN0 heminecks Patients with prior orsimultaneous recurrence at the primary site or inthe initially pN hemineck will be censored for thisoutcome as of that timepoint The primary endpointis a comparison of pN0 neck failure in Arm vshistorical controls this endpoint will also be compared between the two arms as a secondaryendpoint 0f OS measured as time from randomization untildeath from any causeFig Flowchart showing timing of randomization with respect topeer review and treatment planning Contours must be finalizedbefore randomization and may not be changed after randomizationThe maximum dose to PTV64 or PTV60 if no PTV64present should not exceed of the prescribed doseand no volume cc outside of these PTVs should receive of the prescription doseans at Risk OAR definitions dose constraints andplanning priorities are adapted from the following protocols RTOG protocols [] Arm and RTOG [] Arm NCICCTG HN6 ORATOR [ ] andORATOR2 [] and are described in Additional file Quality assurance QAIn order to ensure patientsafety and effective treatment delivery a robust QAprotocolis incorporated The following requirementsmust be completed for each patient 0f Prior to randomization each set of contours will bepeerreviewed either by another individual radiationoncologist or at a team head and neck QA rounds 0cLang Radiation Oncology Page of 0f DFS measured as time from randomization toeither recurrence at any location or deathwhichever occurs first New primary tumours willnot count as DFS events 0f Local recurrence measured as time fromrandomization until disease recurrence at theprimary site 0f Regional recurrence measured as time fromrandomization until disease recurrence anywhere inthe neck 0f Locoregional recurrence measured as time fromrandomization until disease recurrence anywhere inthe neck or at the primary site whichever occursfirst 0f Recurrence in the pN0 neck without otherlocoregional recurrence measured as time fromrandomization until disease recurrence in the pN0neck alone without recurrence at the primary site orpN neck 0f Rate of salvage surgery andor radiation in pN0neck measured as time from randomization tosalvage intervention surgery ± radiation in the pN0neck Freedom from unsalvageable neck recurrencewill be reported as the time from randomization todevelopment of a neck recurrence in the pN0 neckthat could not be salvaged 0f Feeding Tube Insertion Rate of feeding tubeinsertion after start of radiation either gastricgastrojejeunal or nasogastric and rate of feedingtube use at 1year postrandomization Patients withfeeding tubes inserted prior to randomization will becensored for this endpoint 0f Rate of failure in the clinically node negativeneck for patients who have unilateral neckdissections the cN0 neck may be treated withradiation or observed at the discretion of thetreating oncologist see section This is measuredas the time from randomization to failure in the cN0neck and will be reported for the whole group ofpatients who had unilateral dissections and alsostratified by whether radiation was delivered to thatareaEnrollment randomization and allocationPatients will be enrolled by dedicated clinical trials staffandor the investigator at each participating institutionPatients will randomized in a ratio to standard radiation volumes Arm vs omission to the pN0 neckArm A permuted block design with two stratification factors will used with the size of the blocks knownonly to the statistician stratified based upon overall necknodal status pN0 vs pN and use of concurrentchemotherapy yes vs no Randomization sequences aregenerated for each strata separately with a randomnumber generator based on permuted block design Thisgets formatted as a CVS file which gets uploaded intoREDCap []Statistical considerationsSample size The primary endpoint is the rate of relapsein the pN0 neck ie the regional relapse rate in the pN0neck in Arm compared to historical controls Allother endpoints are a comparison between Arm andArm It is generally accepted that a risk of nodal recurrenceof “ is sufficient to warrant radiation to a nodalbasin and therefore we wish to exclude a risk of regionalrecurrence in the pN0 neck of at 2years Using aonesided onesample binomial test allowing for dropout a sample size in Arm of patients provides power at a significance level to detect a regional control rate in the pN0 neck of compared toan unacceptable level of This sample size in Arm and in Arm alsoallows for power to detect a 10point difference inthe total MDADI score at year a secondary endpointassuming the scores are normally distributed with astandard deviation of in each arm It is generally believed that a 10point difference in standardized QoLscores represents a clinically significant difference inQoL [] It is assumed that the QoL scores will be normally distributed with a standard deviation of in eacharm The MDADI will be calculated as the compositescore but will also be reported for each of the subscalesAnalysis planPatients will be analyzed in the groups to which they areassigned intentiontotreat Oncologic outcomes andOS will be calculated from date of randomization usingthe KaplanMeier method A onesided onesample binomial test will be used for the primary endpoint as described above For actuarial comparisons between armsthe stratified logrank test will be used stratified bystratification factors An twosample Ttest will be usedto compare QoL scores at 1year The percentage of patients in each arm who experience a clinically significantQoL decline on the MDADI points will also bereportedMBSImP„¢ scores will be compared at 1year including the total score and the scores on the oral andpharyngeal subscales using a twosample Ttest or aWilcoxon rank sum test as appropriate DIGEST„¢ andFOIS scores will be compared using the Chisquare testPreplanned subgroup analysis will occur based on thestratification variables nodal status [pN0 vs pN] anduse of concurrent chemotherapy as well as based onthe neck dissection performed unilateral vs bilateral 0cLang Radiation Oncology Page of and Tstage T1“ vs T3“ There will also be a preplanned analysis based on the extent of nodes harvestedin the pN0 neck vs or more depth of invasionof the primary tumour mm vs mm or more andphoton versus proton treatmentMultivariable Cox proportional hazards or logistic regression analysis as appropriate will be used to determine baseline and pathologic factors predictive of pN0neck failure DFS OS locoregional recurrence and salvage therapy For the secondary endpoints involvingQoL scales linear mixed effects models will be used forthe MDADI the total scores will be compared betweenthe two arms whereas for the EORTC QLQC30 andHN35 scales each of the subscales eg pain swallowing etc will be compared between the two armsRates of grade ‰¥ toxicity and use of feeding tubes willbe compared between arms using the Chisquare orFisher™s exact test as appropriateUtilities will be calculated from the EQ5D5L whichwill be administered at baseline and at month intervalsQuality adjusted life years QALYs will be assessed asthe area under the preferenceweighted survival curveOverall costs of each treatment strategy will be abstracted from the available literature The incrementalcost effectiveness ratios ICERs between treatment armswill be compared through the standard method of ratiobetween differences in costs and QALYs Point estimatesfor these differences can be derived from multivariablegeneralized estimating equations GEE or generalizedlinear model GLM analysesData safety monitoring committeeThe Data Safety Monitoring Committee DSMC consisting of at least one radiation oncologist and one medical oncologist not involved in the study will meet every months after study initiation Toxicity outcomes willbe monitored but since the experimental arm involvessmaller radiation volumes it is extremely unlikely thattoxicity would be higher in the experimental arm andtherefore no stopping rules for toxicity are includedInterim analysis The DSMC will conduct one interimanalysis once patients have been accrued and completed the 6month QoL questionnaires For this analysis the DSMC will be blinded to the identity of eachtreatment arm but QoL recurrence in the pN0 neckOS and DFS estimates at 2years will be presented foreach armThe DSMC will recommend stopping the trial if thereis an OS difference that is statistically significant with athreshold of p using the stratified logrank testbased on the HaybittlePeto stopping rule this retainsan overall alpha of Ethical considerationsThe Principal Investigator will obtain ethical approvaland clinical trial authorization by competent authoritiesaccording to locallaws and regulations The WorldHealth anization WHO Trial Registration Data Setis shown in Additional file Institutional review board IRB research ethicsboard REB The protocol and any

Thyroid_Cancer

thyroid cancer diagnosis by Raman spectroscopyMarco Sbroscia15 Michael Di Gioacchino1 Paolo Ascenzi1 Pierfilippo Crucitti2 Alessandra di Masi1 Isabella Giovannoni3 Filippo Longo2 Davide Mariotti1 Anda Mihaela Naciu4 Andrea Palermo4 Chiara Taffon3 Martina Verri3 Armida Sodo1 Anna Crescenzi3 Maria Antonietta Ricci1Over the last years the incidence of human thyroid cancer disease has seen a significative increment This comes along with an even higher increment of surgery since according to the international guidelines patients are sometimes addressed to surgery also when the fine needle aspiration gives undetermined cytological diagnosis As a matter of fact only of the thyroid glands removed for diagnostic purpose have a post surgical histological report of malignancy this implies that about of the patients have suffered an unnecessary thyroid removal Here we show that Raman spectroscopy investigation of thyroid tissues provides reliable cancer diagnosis Healthy tissues are consistently distinguished from cancerous ones with an accuracy of ˆ¼ and the three cancer typology with highest incidence are clearly identified More importantly Raman investigation has evidenced alterations suggesting an early stage of transition of adenoma tissues into cancerous ones These results suggest that Raman spectroscopy may overcome the limits of current diagnostic toolsDetection of nodular thyroid pathology is increasing worldwide paired with an increased number of thyroid cancer diagnosis1 Whether this œepidemic spread is real or is due to an exaggerated use of imaging tools in clinical practice is object of a vivid debate within the scientific community Anyway management of patients with thyroid nodule causes a clinical problem and a social burden as well2 To avoid overdiagnosis and overtreatment of these patients physicians need additional information about the biology of each thyroid nodule In particular there is need for a tool able to distinguish between benign and malignant follicular lesions and between indolent and aggressive tumors3 The genomic profile of thyroid tumors has been extensively investigated4 and several immunocytochemical and molecular markers have been proposed for a more accurate classification of thyroid nodules5 However none of these markers reaches a reliable sensibility and specificity to be routinely used in clinical setting6 Moreover although the major genetic alterations of thyroid tumors are known only little information if any is available about changes in protein lipids and other cellular constituents due to thyroid oncogenesisTo correctly manage patients with thyroid nodules and to avoid unnecessary surgery we strongly need a method of analysis that overcomes the limits of current diagnostic tools by identifying specific tumoral markers possibly with high spatial resolution and low impact on the patient Cells and tissues are characterized by a specific biochemical composition Each pathology or cellular abnormality is accompanied by biochemical molecular changes Optical and spectroscopic techniques that correlate the biochemical composition molecular structure and their variations with the morphological alterations represent a powerful diagnostic and potentially clinical tool8Among the experimental techniques available for chemicalphysical studies of matter at the molecular level Raman spectroscopy RS is a good candidate for our needs This technique offers a wealth of molecular information while being not invasive and achieving spatial resolutions as high as a few μm in microRaman apparatuses RS probes the vibrational dynamics of the sample through a fast and not destructive analysis It does not require any sample preparation and can consequently be applied in a0vivo For these characteristics it has been recently applied on a variety of biological and medical samples to identify specific metabolic states of cells tissues and 1Dipartimento di Scienze Universit¡ degli Studi Roma Tre Rome Italy 2Unit of Thoracic Surgery Campus BioMedico University Rome Italy 3Pathology Unit Campus Biomedico University Hospital Rome Italy 4Unit of Endocrinology and Diabetes Campus BioMedico University Rome Italy 5Present address Dipartimento di Fisica Sapienza Universit¡ di Roma Rome Italy email armidasodouniroma3itScientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Raman spectra A Typical Raman spectra of the examined thyroid tissues labelled according to the histology report Stars label the Raman characteristic peaks of cytochrome c triangles those of carotenoids The black vertical dashed lines allow an easier comparison among the spectra B Centroids Raman spectra for the four identified clusters by Kmeans analysisbacteria and in particular to differentiate between healthy and cancer tissues9“ Preliminary studies on thyroid tissues by RS suggest that this technique may have high impact in thyroid cancer diagnosis13“In a RS experiment a laser beam is focalized on the sample and the scattered light is analyzed in frequency Typical spectra after background subtraction are shown in Fig a01A These are characterized by peaks and bands with frequency centered at the vibrational frequency of the intramolecular bonds characteristic of the main sample componentsWe have applied RS augmented by cluster analysis to investigate histological samples from human thyroid Assignement of the RS peaks to carotenoids or cytochrome c in different amounts has allowed to distinguish healthy tissues from cancerous ones and to discriminate for the first time among the three categories of carcinoma with larger incidence namely Papillary carcinoma PTC follicular variant of Papillary carcinoma FVPTC and Follicular carcinoma FC We have also analyzed samples labelled after histology as follicular adenoma FA We found that these samples are spread over four categories reported above by the cluster analysis Subsequent more specific analyses of protein expression have confirmed an anomalous level of tumoral or benign molecular markers in the adenoma samples labeled in the non healthybenign categories by RS All the samples analyzed by RS are reported in a table as Supplementary informationResultsFigure a01A shows from bottom to top the Raman fingerprint region ˆ’ cmˆ’ of four samples with a clear histology diagnosis namely HealthyBenign average of the spectra of samples Hea49 and Hea64 PTC average of the spectra of samples TIR51 and TIR58 FVPTC sample TIR56 and FC average of the spectra of samples TIR47 and TIR55 carcinoma tissues Each spectrum has been normalized to its integrated intensity We notice that each tissue has its own characteristic spectral lineshape According to Rau et a0al15 and references therein this spectral range shows the bands ascribed to proteins lipids and aminoacids Here we will not report a detailed assignment of each band to a particular biomolecule as this is sometimes tricky and not relevant to our aim We will instead put the attention on the distinctive set of peaks which characterize each histological sample We notice that the spectrum of the healthy tissues is dominated by an intense broad and structured band centered around ˆ¼ cmˆ’ possibly due to the presence in the sample of amide I and lipids19 The sharpest structure of this band falls at cmˆ’ and along with the bands at · and cmˆ’ Scientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Agglomerative hierarchical clustering analysis Dendrogram of the Raman spectra of human thyroid tissues as extracted from the AHCA analysis Individual samples are represented by the label Hea for healthy or TIR for not healthy followed by the patient anonymous ID code Those plotted in Fig a01A are labelled healthy FC FVPTC and PTC respectively Dashed squares identify the four clusters namely healthybenign orange Follicular carcinoma or FC black follicular variant of papillary carcinoma or FVPTC magenta and papillary carcinoma or PTC blue The arrows indicate the adenoma samples All samples within the light blue shaded area are carcinomasrepresets the fingerprint of cytochrome c20 This is a protein localized in the mitochondrial intermembrane space under normal physiological conditions The spectrum of a classical papillary carcinoma labeled PTC in Fig a01A is totally different and unambigously shows the presence of carotenoids bands at and cmˆ’ not present in healthy tissues as already reported in the literature10 In the follicular carcinoma spectrum labeled FC in Fig a01A we observe a marked enhancement of the cytochrome c bands As a matter of fact the broad band at ˆ¼ cmˆ’ that dominates the HealthyBenign spectra is barely visible in this case after spectral intensity normalization Interestingly the spectrum of the Papillary Follicular carcinoma labeled FVPTC in Fig a01A looks like a superposition of the PTC and FC spectra showing the fingerprints of carotenoids along with a clear enhancement of the bands ascribed to cytochrome c with respect to the spectra of HealthyBenign tissuesThe presence of carotenoids in both PTC and FVPTC carcinoma is peculiar and worthy of interest The cellular process of carotenoids uptake not directly synthetized by human anisms and their proper identifications is under biochemical investigationAs Raman spectroscopy is able to detect the differences between healthy and cancerous tissues it may be considered as a promising tool for in a0situ cancer diagnosis We have analyzed a total of histological samples of human thyroid tissues samples diagnosed as adenomas including one inadequate TIR59 for RS and not further considered for statistical analysis healthy ones and carcinomas ones namely PTC FVPTC FC and Hyalininzing Trabecular Tumor HTT All samples are labelled as TIR or Hea followed by the patient anonymous ID codeWithin each histology class spectra may differ for noise background or fluorescence contributions although keeping unaltered the characteristic fingerprints commented above This observation suggests that the experimental spectra can be analyzed on quantitative grounds by cluster analysis after backgrounds and fluorescence subtraction This aims at classifying each Raman spectrum into homogeneous groups based on its characteristic features in other words on the presence and relative intensity of specific bands The analysis has been performed following two distinct approaches see œMethodsThe first approach called agglomerative hierarchical clustering analysis AHCA builds a dendrogram of the entire data set by iteratively selecting pairs of the closest spectra or groups of spectra as a function of their Euclidean distance The results of this analysis are reported in Fig a0 where each sample is represented by means of the label Hea or TIR depending on whether the tissue has received a healthy or not healthy histological diagnosis followed by a progressive number This figure tells us that our spectra are consistent with a classification of the dendrogram into clusters assuming a distance threshold of compared to a maximum distance of The four clusters have been labelled according to the diagnosis of the four spectra shown in Fig a01A a priori selected as the most representative of each histology report namely the less noisy with a clear and confirmed diagnosis HealthyBenign and PTC tissues are very well distinct each other and have the smallest dispersion in terms of Euclidean distance within each cluster Indeed the smaller is the dispersion of the Euclideian distances the better is the homogeneity of the group in the present case HealthyBenign and PTC tissues are the most homogeneous The other two clusters FC and FVPTC are more dispersed although equally distinguishable Scientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Kmeans analysis Samples distribution within the four KM clusters as a function of their Euclidean distance from the associated centroid The same labels and colours as in Fig a0 have been used Black triangles refer to adenomasCluster Cluster Cluster Cluster Cluster Cluster Cluster Cluster Table Euclidean distance between the cluster centersTotal of samplesMedical diagnosisRamanTrue negative False negative ClassificationFalse positive True positive AccuracyTable Confusion matrix based on samples healthy and carcinomas tissues This shows that RS has identified False negative diagnosis among the negative Medical diagnosis and different samples among the with a positive diagnosis which fall in a cluster different from those expected on the basis of the medical diagnosisfrom the others The FC cluster is the closest to the HealthyBenign one as both are characterized by the presence of cytochrome c and distinguished only on the basis of its Raman fingerprint relative intensity The FVPTC tissues fall between FC and PTC clusters as predictable from copresence of carotenoids and citochrome c peaks in their Raman spectraThe second unsupervised statistical approach considered is called Kmeans KM its results are reported in Fig a0 This confirms that four clusters properly describe our data set Clusters contain samples from the histology reports of PTC HealthyBenign FVPTC and FC respectively The agreement between the clustering results of the two approaches is very good as we notice only one difference concerning sample TIR60 which switches from a Raman diagnosis of PTC to that of FVPTC Fig a0 shows that healthybenign tissues spectra have on the average the shortest distance from their centroid and the smallest dipersion On the contrary the spectra of FVPTC tissues are the most dispersed possibly due to a different balance of concentration between carotenoids and cytochrome c in each sample Table a0 reports the distance matrix among the clusters centroids Cluster showing the largest centroid distance from the others is readily isolated its Raman spectrum is dominated by the carotenoid fingerprint while discrimination among the others requires consideration of more spectral detailsKM analysis returns also the Raman spectrum calculated for each cluster centroid these are reported in Fig a01B for direct comparison with the prototypical spectra of the four classes of tissues The similarity of the spectra reported in Fig a01AB is astonishing and proves that our clustering is solidBased on these results we have computed a confusion matrix providing the accuracy of our analysis in discriminating between healthybenign and pathological tissues regardless of the specific pathology As reported in Table a0 our accuracy is of ˆ¼ Scientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0cTotal of samplesMedical diagnosisRamanTrue negative False negative ClassificationFalse positive True positive AccuracyTable Confusion matrix based on samples including adenomasThis shows that RS has identified false negative diagnosis among the negative medical diagnosis and different samples among the with a positive diagnosis which fall in a cluster different from those expected on the basis of the medical diagnosisFigure a0 Immunohistochemistry images Microscopic pictures of A TIR43 sample stained with immunohistochemistry for Galectin3 high power field Hematoxylin counterstained and B TIR64 sample stained with immunohistochemistry for HBME1 medium power field Hematoxylin counterstained Panel A shows a mosaic pattern with negative cells intermingled with cells showing positive reaction in cytoplasm as well as in nuclear matrix Panel B shows strong positive reaction due to a microfocus of papillary carcinoma within the follicular adenomaTotal of samplesMedical diagnosisRamanTrue negative False negative ClassificationFalse positive True positive AccuracyTable Confusion matrix based on samples including adenomas after immunohistochemistry revisionThis shows that RS has identified false negative diagnosis among the negative medical diagnosis and that the number of false positive diagnosis goes down to Finally we have applied the same analysis including the spectra of the samples classified as adenoma by the histological diagnosis Their position is highlighted by arrows in the AHCA dendrogram of Fig a0 and in the distributions of Fig a0 These figures show that the spectra of adenoma tissues spread over all four clusters defined by healthy and cancerous tissues This is a quite unexpected result as adenomas are classified as benign tissues and therefore should fall into the healthybenign cluster Accordingly the confusion matrix after inclusion of adenoma samples reported in Table a0 returns an accuracy of the statistical analysis of After the RS analysis four adenoma samples TIR43 TIR44 TIR64 TIR71 falling within the cancerous clusters have been in depth reanalyzed as far as their expression of benignity and cancerous markers are concerned by immunohistochemistry test The lowering of benignity markers CD56 or sometimes the onset of tumoral markers Galectin3 HBME1 are revealed in all these cases The immunohistochemistry images of TIR43 and TIR64 samples are shown in Fig a0 as representative of the all four cases These results could be symptomatic of early stages of progression of adenomas into a specific type of carcinoma Such type of transformation has never been demonstrated and our Raman observation s a new light about the malignant potential of thyroid FA and about the possibility to assess which of these lesions may constitute a clinical concern Interestingly the confusion matrix after this revision gives an accuracy of ˆ¼ A few anomalies visible in Figs a0 and a0 deserve a comment Sample Hea56 and sample Hea63 apparently fall into a wrong cluster namely the FVPTC cluster but interestingly very close to their TIR counterpart FVPTC and TIR63respectively This may happen when the Raman spectrum of the healthy portion is collected from a tissue region too close to the pathologic one where normal and altered cells are almost intermingled giving the superposition of healthy and cancerous Raman spectra In particular the case of sample Hea72 falling within the FVPTC cluster may be due to the diffusion of carotenoids in normal parenchyma at the border Scientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Biochemical analysis Expression levels of cytochrome c have been assessed in thyroids of five patients for each patient the healthy Hea and pathological TIR slices have been analysed A Levels of cytochrome c have been normalized to actin Data represent the mean values ± SDs derived from three replicates normalized to healthy counterparts Student™s t test P a0 a0 P a0 a0 compared with control B Exemplificative images of filters blotted with cytochrome c and actin primary antibodies Images have been gathered at the same timeof the PTC nodule as already observed in previous Raman chemigram map of thyroid carcinomas15 This gives in the Raman spectrum the superposition of the fingerprint of carotenoids with fingerprint of healthy tissues containing cytochrome c thus explaining the œwrong assignment obtained by Raman analysisAdditionally levels of cytochrome c have been further evaluated by biochemical analysis Fig a0 on a few cases Indeed only for patients and PTC and and FC a sufficient amount of thyroid tissue was available to perform immunoblot analyses As shown in Fig a0 cytochrome c levels significantly increase in TIR45 134fold induction compared to its healthy counterpart Hea45 P a0 a0 TIR46 27fold induction compared to Hea46 P a0 a0 and TIR47 456fold induction compared to Hea47 P a0 a0 In agreement with Raman results cytochrome c levels are similar in TIR and Hea slices of patients and In conclusion this study demonstrates the capability of Raman spectroscopy as diagnostic tool for thyroid cancer Indeed through a multivariate statistical analysis of the spectra it is possible to readily separate healthy from cancerous tissues and in the majority of the cases to discriminate among the most common cancer typologies see Supplementary information for details on the individual cases Last but not least our findings highlight the possible usefulness of Raman spectroscopy as a tool for in a0situ and early diagnosis of thyroid pathologyScientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0cMethodsStudy enrollment From January to January we enrolled consecutive subjects females and males with thyroid nodular pathology referred to the Endocrinology Unit of Campus BioMedico University Hospital Their age distribution is centered at a0years with a standard deviation of a0years All the study population had received a cytological diagnosis of indeterminate suspicious or malignant lesion with a formal indication to surgery total thyroidectomy according to the international guidelines24 After signing the informed consent these patients underwent total thyroidectomy at the Surgical Unit of the same InstitutionNeck ultrasound and clinical evaluation All subjects were submitted to thyroid US evaluation using a frequency range of “ MHz on a MyLab Esaote Genova Italy US scan of thyroid gland and neck area were performed by experienced endocrinologists at the Endocrinology Unit Nodules were classified according to ACR TIRADS risk stratification criteria25 without prior knowledge of the cytological results When there was a disagreement the endocrinologists conducted a separate session to reach an unified consensus We collected demographic data including age gender family history of thyroid cancer and using the medical records we evaluated the thyroid function and the presence of autoimmune thyroiditisThyroid tissues preparation At the time of surgery the removed specimens were immediately submitted unfixed to the Pathology Unit in an appropriately labelled container The pathologist valued the gross anatomy of the samples and a tissue slice of about cm wide — cm length — mm of thickness was cut including both healthy and neoplastic areas avoiding surgical margins The slice was snap frozen on a metallic coldplate inside a cryostat A μm section was stained with haematoxylineosin in order to confirm the presence of healthy and neoplastic tissues After this step four consecutive cryostatic sections were cut at μm of thickness collected on separate slides and stored at ˆ’ —¦C until the Raman evaluation Our Raman study was exclusively performed on these frozen unfixed samples The residual slices were defrosted formalin fixed and paraffin embedded with the paired surgical samples for definitive histology Final diagnosis was reported in agreement with current edition of WHO classification of endocrine tumours26 Immunohistochemical analysis for Galectin3 Gene Tex CD56 Agilent and HBME1 Agilent was performed in each case on paraffin sections from neoplastic areas using an automatized immunostainer Omnis AgilentNodules with diameter wider than cm were submitted for biochemical analysis In such cases at the time of gross dissection further slices of about — a0cm were cut from the surgical specimen sampling both healthy and neoplastic tissue Slices were snap frozen and stored at ˆ’ —¦C until biochemical evaluationRaman spectra collection Raman spectra have been collected by means of a Renishaw InVia MicroRaman spectrometer In this setup unpolarised spectra are collected through a Leica DM2700 M confocal microscope equipped with a Leica — LWD and an Olympus — objective The required highcontrast rejection for the elastically scattered light is provided by an holographic edge filter A diffraction grating with a0groovesmm disperses the Raman inelastic scattering contribution providing a spectral resolution of about cmˆ’ A Peltier cooled — pixel CCD detector collects the dispersed light A solidstate diode laser source at nm with a nominal output power of nearly mW has been used as excitation source To prevent photodamage neutral density filters have been used for lowering the laser power at the sample The spot size has been shrink down to few microns when necessary to isolate the contribution of cells about ten microns in size from the surrounding cytoplasm thus reducing spectral interference Spectra have been collected in the extended scan mode covering the “ cmˆ’ frequency shift range and accumulating scans with an equivalent integration time of nearly s per scan Measurements have been collected on points per sample Wire Renishaw software has been used to collect the raw spectra Wire and LabSpec software has been used to perform the preliminary data reduction eg background and fluorescence subtraction while Origin OriginLab Corporation software has been employed to perform the statistical analysis After background and fluorescence subtraction all the spectra have been normalised to their integral in order to avoid any bias due to fluctuations of the experimental conditionsCluster analysis of Raman spectra In order to gain better confidence on the clustering analysis we have selected two unsupervised multivariate statistical approaches to analyze the Raman spectra the agglomerative hierarchical clustering analysis AHCA and the Kmeans one KM Both methods are based on the Euclidean distance among spectra as a measure of their similaritydissimilarity In detail in these analyses spectra are represented by their distance with respect to an origin thus generating a dissimilarity matrix Among the possible choices for the spectral distance we have chosen the Euclidean one since it maximizes the similarities among the spectra The choice to use these two methods was made to exploit their complementarity of hierarchical and nonhierarchical approaches and consequently to give solidity to the obtained results13 “The AHCA method aims at classifying observations each Raman spectrum into homogeneous groups the clusters based on the measured characteristics Euclidean distance associated to each Raman spectrum In particular we have used the completelinkage algorithm in order to enhance differences among clusters It works by pairing the most similar spectra those showing the lowest dissimilarity value in the matrix and then looking for the largest distance between such pair and the rest of the data By iteratively repeating such an approach a classification of spectra into wellseparated groups is obtained and visualised as a dendrogramThe KM method minimizes the variance within each cluster with respect to an a priori number of centroids randomly distributed This protocol starts by associating each data entry Euclidean distance associated to each Raman spectra to the cluster with the closest centroid The centroids are iteratively updated towards convergence The number of clusters k is an input of the routine In order to choose the proper value of k the most Scientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Spread of the individual Kmeans clusters as a function of their number Data are reported by using the same colors as in Fig a0 for the individual clusters Notice that four clusters give the largest spread among the clustersused strategy is the œelbow curve based on the explained variance within the entire dataset This analysis in our case gives only two centroids one for samples containing mainly carotenoids and one for all other cases Thus following the suggestion of the histology diagnosis we have set k a0 a0 In order to verify the reliability of this choice we have calculated the spread of each cluster with respect to its centroid as a function of k Intestingly we find that k a0 a0 is the highest kvalue with the lowest spread as shown in Fig a0We have tested the predictive accuracy of both AHCA and KM methods by generating a confusion matrix This reports the comparison between the medical and spectroscopic diagnosis The accuracy is defined asAccuracy True negative True positivenumber of total samplesBiochemical analysis Frozen thyroids have been weighted homogenized and sonicated in lysis buffer mM TrisHCl pH mM NaCl mM EDTA glycerol vv Triton X100 vv and protease inhibitors Twentyfive micrograms of protein extracts previously quantified with the Bradford assay BioRad Hercules CA have been resolved by SDSPAGE and transferred onto PVDF membranes BioRad After blocking with BSA wv dissolved in TBS buffer Tween20 vv membranes have been probed overnight at —¦C with anticytochrome c mouse mAb sc13560 Santa Cruz Biotechnology Dallas TX USA and antiactin mouse mAb sc47778 Santa Cruz Biotechnology primary antibodies Membranes have been then incubated for h at room temperature with antimouse HRPconjugated secondary antibody BioRad All the experiments have been repeated trice Blots have been acquired and processed using the ChemiDoc Imaging system BioRad Cytochrome c quantification has been performed using the Image Lab software version BioRad Laboratories Results are shown as mean ± standard deviation SD the statistical significance of differences has been tested using the Student™s t test GraphPad Prism version ethics statement The study was approved by the Ethical Committee of the University of Rome œCampus Biomedico UCBM prot TS ComEt CBM and prot PAR ComEt CBM The informed consent was collected from patients before surgery Enrolled patients were recorded in a codified file with an anonymous ID code which was registered in the software database of the Pathology Unit of the UCBM All experiments were performed in full accordance with the principle of Good Clinical Practice GCP and the ethical principles contained in the current version of the Declaration of HelsinkiReceived March Accepted July References Vaccarella S et al Worldwide thyroidcancer epidemic The increasing impact of overdiagnosis N Engl J Med “ Udelsman R Zhang Y The epidemic of thyroid cancer in the United States the role of endocrinologists and ultrasounds Thyroid Rusinek D et al Current advances in thyroid cancer management Are we ready for the epidemic rise of diagnoses Int J Mol 101056NEJMp “ 101089thy20130257 Sci Scientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0c TCGAR Network Integrated genomic characterization of papillary thyroid carcinoma Cell “ 101016jcell201409050 Cantara S et al Molecular signature of indeterminate thyroid lesions current methods to improve fine needle aspiration cytology fnac diagnosis Int J Mol Sci 103390ijms1 Haugen B R et al American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer the American Thyroid Association guidelines task force on thyroid nodules and differentiated thyroid cancer Thyroid “ 101089thy20150020 Paschke R et al European thyroid association guidelines regarding thyroid nodule molecular fineneedle aspiration cytology diagnostics Eur Thyroid J “ 10115900046 Krafft C Popp J Raman4clinics the prospects of Ramanbased methods for clinical application Anal Bioanal Chem Kallaway C et al Advances in the clinical application of Raman spectroscopy for cancer diagnostics Photodiagn Photodyn Ther “ 101007s0021 “ 101016jpdpdt Li Z et al Surfaceenhanced Raman spectroscopy for differentiation between benign and malignant thyroid tissues Laser Phys Lett 1010881612201111404560 Krafft C Popp J The many facets of Raman spectroscopy for biomedical analysis Anal Bioanal Chem “ 101007s0021 Palermo A et al Raman spectroscopy applied to parathyroid tissues a new diagnostic tool to discriminate normal tissue from adenoma Anal Chem “ 101021acsanalc hem7b036 Harris A T et al Raman spectroscopy and advanced mathematical modelling in the discrimination of human thyroid cell lines Teixeira C S B et al Thyroid tissue analysis through Raman spectroscopy Analyst “ 101039B8225 Rau J V et al Raman spectroscopy imaging improves the diagnosis of papillary thyroid carcinoma Sci Rep “ Head Neck Oncol 78H 101038srep3 Rau J V et al Proofofconcept Raman spectroscopy study aimed to differentiate thyroid follicular patterned lesions Sci Rep “ 101038s4159 MedeirosNeto L et al In a0vivo Raman spect

Thyroid_Cancer

"analyse the quality of information included in websites aimed at the public on COVID19Methods Yahoo Google and Bing search engines were browsed using selected keywords on COVID19first websites from each search engine for each keyword were evaluated Validated tools wereThe used to assess readability [Flesch Reading Ease Score FRES] usability and reliability LIDA tool andquality DISCERN instrument Nonparametric tests were used for statistical analysesResults Eightyfour eligible sites were analysed The median FRES score was range 00 Themedian LIDA usability and reliability scores were range 00 and 37range14 00 respectively Alow overall LIDA score was recorded for n of the websites The median DISCERN score 14 was found in websites The DISCERNwas range “ The DISCERN score of score was significantly associated with LIDA usability and reliability scores p and the FRES scorep Conclusion The majority of websites on COVID19 for the public had moderate to low scores with regardsto readability usability reliability and qualityPractice Implications Prompt strategies should be implemented to standardize online health informationon COVID19 during this pandemic to ensure the general public has access to good quality reliableinformationï¾ Elsevier BV All rights reserved Introductionfor The coronavirus COVID19 pandemic has become the greatestglobal health crisis of the st century [] During this pandemicthe demand information on COVID19 has skyrocketedInformation such as latest news updates on the pandemic itssymptoms prevention and mechanism of transmission are highlysought by the public [] On the other hand free access toinformation especially through social media which is accessed bythe majority [] has led to an increase in misinformation and panicassociated with COVID19 [] Although high quality healthinformation is known to be related to lower stress levels andbetter psychological health [] previous studies have shown thatonline information on many medical disorders to be of substandard quality []A previous study done on websites related to COVID19 hasreported substandard quality information that could potentiallymislead the public [] However this study has used a limitedsearch strategy and had not assessed some important areasincluding usability and reliability of the information Thereforethis topic remains a knowledge gap in COVID19 research []Therefore we conducted this study to analyse the current COVID websites targeting the general public in terms of qualityusability readability and reliability using a wide search strategyand validated instruments MethodsAbbreviations USA United States of America FRES Flesch reading ease scoreHONcode Health on the net Code of Conduct SPSS Statistical package for socialsciences NICE National Institute for Health and Care Excellence WHO WorldHealth anization Corresponding author at Department of Surgery Faculty of MedicineUniversity of Colombo PO Box Kynsey Road Colombo Western ProvinceSri LankaEmail addresses ravindrijayasinghegmailcom R Jayasingheranasigmcgmailcom S Ranasinghe umeshejayagmailcom U Jayarajahsanjeewasrgcmbaclk S SeneviratneYahoo Google and Bing were searched using the keywordsœsevere acute respiratory synœnovel coronavirusSARSCoV2 and œcoronavirus The searchdrome coronavirus2 œCOVID19was performed during the first week of May The details ofthe search strategy and the piloting process are provided in thesupplementary material File S1 []Two independent investigators with previous experience ofconducting similar studies assessed the selected websites []Prior to the assessment a pilot run was conducted to ensure101016jpec20200800107383991ï¾ Elsevier BV All rights reservedPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxxuniformity and accuracy The information on symptoms investigations public health measures and available treatmentmodalities were collected The accuracy of the content wasassessed using the national institute for health and care excellenceNICE guidelines on COVID19 [] A rating was given as all ornone based on congruence with the guidelinesValidated instruments were used to assess the quality ofwebsites Readability was assessed using the Flesch Reading EaseScore FRES [] The LIDA Instrument 2007Version12 wasused to analyse the content and the design of the websites usingthe usability and reliability domains [] The quality of thecontent was assessed using the DISCERN questionnaire which has questions in two separate groups [] The detailed assessmentcriteria and the scoring system is included in the supplementarymaterial File S1A website was classified as governmental if it was maintainedby the country™s public health authority If managed by privateinstitutions nongovernmental anizations or voluntary institutions independent from the government they were consideredas nongovernmental The online healthrelated websites arestandardized in terms of their credibility and reliability by onlinecertification sites We chose the Health on the Net code of conductHONcode which is the oldest and widely used out of the qualityevaluation tools available []Data analysis was performed using SPSS Version20 softwareand the associations were determined with non parametric testsA pvalue of was considered statistically significant ResultsOf the retrieved websites were excluded and thein the analysis Theremaining websites were characteristics of the websites are mentioned in Table included Half were governmental websites and only n were HON accredited websites The median FRES was range 00 10th12th grade readability level which is classified asfairly difficult to read Only three websites had a readabilityscore of above equivalent to 7th grade which is therecommended standardThe overall median LIDA score was range “ whilethe median LIDA usability and reliability scores were range 00 and range 00 respectively The median DISCERNscore was range “ which classifies websites as being ofœfair quality Excellent “ Good “ Fair “Poor “ Very poor “ However the top websitesTable A were of excellent qualityTable Website characteristicsWebsite Characteristics Frequency PercentageUsability Governmental websites Notforprofit and private websites HONcode accredited Readability score of above equivalent to 7th grade Readability Date of publication stated References mentioned Disclosure statement by authors Infographics Moderate Low Moderate 00 Low score Moderate 00 Low Used Textonly Reliability Table Correlation between DISCERN scores and other factorsDISCERN SCORELow Mean High 00 Mean Range 00 00 00 00 Range 00 00 00 00 N P valueP0001P0001P0001P P P LIDA Usability LIDA Reliability LIDA Overall FRES Score Government HON Certification No N No Yes Yes Significant correlations were observed between the DISCERNscore and the overall LIDA score as well as LIDA usability andreliability scores Table p HONcode certified websitessites obtained significantly higher DISCERN scores p Pertaining to the currency of information only publishers stated the date of the publication Most websites n did not declare the sources of evidence This was furtherestablished by the median reliability score of Nevertheless the authors have included a disclosure statement in mostn websitesœlowFigures A1 and A2 summarize the rating of websites onindividual criteria assessed by the DISCERN tool The specificinformation provided regarding COVID19 is shown in Fig More than half of the websites failed to discuss the treatmentoptions available n benefits or risks n and effects of no treatment n Furthermore potentialcomplications and prognosis were stated only in and websites respectively Discussion and conclusion DiscussionThis study has shown that still most of the websites on onlinehealth information on COVID19 are of suboptimal quality exceptfor a few credible sources of goodquality health informationNevertheless the websites ranked among the top according tothe DISCERN score Table A2 had high scores indicating thepotential for publishing credible highquality information onlinewhich would benefit the publicin turn causes panic which ranges Misinformation is a major concern during this pandemic aspeople fail to spend adequate time to critically analyse the onlineinformation This fromhoarding medical supplies to panic shopping and using drugswithout prescription with negative social and medical consequences [] Therefore measures implemented to ensure the qualityand accuracy of online information by the responsible authoritiesmay help negate these adverse consequencesinformation Stating the methods of content production with names of thecontributing authors may help increase the credibility of onlinehealth information while displaying the date of the publicationprovides an idea of the currency of the information Absence ofin over half of the websites was a majorsuch drawback especially for COVID19 where new information isgenerated almost daily Health authorities should therefore ensurethat the patient information websites provide the above information and certify websites based on such details so that the publiccan get information from trusted sources []Most users of the worldwide web only have an average level ofeducation and reading skills [] Guidance from the NationalPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxx Fig Website characteristics evaluated outside the DISCERN toolInstitute of Health NIH had shown that the readability should bebelow the level of seventh grade for the lay public to adequatelyunderstand the content [] However the median readabilitylevel was found to be equivalent to 10th12th grade readabilitySuch complexities with the readability of information mayincrease the risk of misunderstandings or misinterpretation Usingshort sentences in writing using the active voice using 12point orlarger font size using illustrations and nontextual media asappropriate and accompanying explanations with examples wouldbe helpful to overcome this problem []So far only a limited number of studies have been done to assessthe quality of health information websites related to COVID19 Thestudy by CuanBaltazar et al prior to February reported poorquality information with approximately of included websiteswith low DISCERN scores [] Our study done three months latershows similar results with only a minimal improvement in thequality of information Furthermore the Cuan Baltazar study hadseveral limitations which includes the limited search strategy andnoninclusion of key quality parameters including readabilityFurthermore out of the sites they had includedwere online news sites that are not considered as patientinformation websites In that study the HONcode seal waspresent only in n websites whereas in our study ofthe sites were HONcode certifiedThere were several limitations in this study Although mostpopular search engines were used in this study under defaultsettings they may produce variable results depending on manyfactors including geographical location and popularity of websitesat a given point of time The algorithms unique to those searchengines are subjected to constant change and therefore the exactresults of our study may not be reproducible However we believethe general patterns observed in our study are validproviding health information to the general public are to be ofsubstandard quality Practice implicationsTo improve the credibility of the content the websites shouldstate methods of content production and display the date of thepublication to give an idea about the currency of the informationTo improve the readability of the content the websites shouldincorporate more nontextual media write in short sentencesusing the activevoice and use larger font sizes The patientinformation websites should display scores of reliability qualityand readability as a guidance for its users Furthermore it is vitalfor medical regulatory authorities and the government to imposeregulations to ensure quality and to prevent the spread ofmisinformationAvailability of data and materialsOn reasonable request from the corresponding author the dataused in the above study can be made availableEthics approval and consent to participateUnnecessary in this type of studyInformed consent and patient detailsNot applicable in this type of studyFundingNone ConclusionCRediT authorship contribution statementThis study has shown the quality readability usability andreliability of the information on COVID19 on majority of websitesRavindri Jayasinghe Conceptualization Methodology Datacuration Writing original draft Visualization InvestigationPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxxValidation Formal analysis Resources Sonali RanasingheConceptualization Methodology Data curation Writing originaldraft Visualization Investigation Validation Formal analysisResources Umesh Jayarajah Conceptualization MethodologyData curation Writing original draft Visualization InvestigationValidation Formal analysis Resources Sanjeewa SeneviratneConceptualization Methodology Writing review editingSupervision Project administrationDeclaration of Competing InterestThe authors report no declarations of interestAcknowledgementsNone declaredAppendix A Supplementary dataSupplementary material related to this can be found in101016jversion at online the pec202008001References[] IJ Bes do Nascimento N Cacic HM Abdulazeem Novel Coronavirusinfection COVID19 in humans a scoping review and metaanalysis JClinical Med [] HT Le DN Nguyen AS Beydoun XTT Le TT Nguyen QT Pham NTK Ta QT Nguyen AN Nguyen MT Hoang Demand for health information onCOVID19 among Vietnamese Int J Environ Res Public Health [] C Wang R Pan X Wan Y Tan L Xu RS McIntyre FN Choo B Tran R Ho VK Sharma A longitudinal study on the mental health of general populationduring the COVID19 epidemic in China Brain Behav Immun [] CS Ho CY Chee RC Ho Mental health strategies to combat the psychologicalimpact of COVID19 beyond paranoia and panic Ann Acad Med Singapore “[] C Wang R Pan X Wan Y Tan L Xu CS Ho RC Ho Immediate psychologicalresponses and associated factors during the initial stage of the coronavirus disease COVID19 epidemic among the general population inChina Int J Environ Res Public Health [] RH Waidyasekera U Jayarajah DN Samarasekera Quality and scientificaccuracy of patientoriented information on the internet on minimallyinvasive surgery for colorectal cancer Health Policy Technol “[] R Jayasinghe S Ranasinghe U Jayarajah S Seneviratne Quality of patientoriented webbased information on oesophageal cancer J Cancer Educ In press[] JY CuanBaltazar MJ MuñozPerez C RobledoVega MF PÃrezZepeda ESotoVega Misinformation of COVID19 on the internet Infodemiology studyJMIR Public Health Surveill 2020e18444[] BX Tran GH Ha LH Nguyen GT Vu MT Hoang HT Le CA Latkin CS HoR Ho Studies of novel coronavirus disease COVID19 pandemic a globalanalysis of literature Int J Environ Res Public Health [] G Eysenbach C Köhler How do consumers search for and appraise healthinformation on the world wide web Qualitative study using focus groupsusability tests and indepth interviews Brit Med J “[] AS Prasanth U Jayarajah R Mohanappirian SA Seneviratne Assessment ofthe quality of patientoriented information over internet on testicular cancerBMC Cancer [] V Udayanga U Jayarajah SD Colonne SA Seneviratne Quality of thepatientoriented information on thyroid cancer in the internet Health PolicyTechnol [] National Institute for Health and Care Excellence Coronavirus COVID19 Accessed April wwwniceukcovid19[] Readable The Flesch Reading Ease and FleschKincaid Grade Level Accessed February readablecomblogthefleschreadingeaseandfleschkincaidgradelevel[] Minervation The Minervation Validation Instrument for Healthcare WebsitesLIDA Tool Accessed February httpwwwminervationcomwpcontentuploads201104MinervationLIDAinstrumentv12pdf[] Minervation Is the Lida Website Assessment Tool Valid Accessed February httpwwwminervationcomdoeslidawork[] Discern Online The DISCERN Instrument Accessed February httpwwwdiscernukdiscern_instrumentphp[] E Fahy R Hardikar A Fox S Mackay Quality of patient health information onthe Internet reviewing a complex and evolving landscape Australas Med J “[] J Kluger As Disinfectant Use Soars to Fight Coronavirus So Do AccidentalPoisonings Accessed April timecom5824316coronavirusdisinfectantpoisoning[] BX Tran AK Dang PK Thai HT Le XTT Le TTT Do TH Nguyen HQPham HT Phan GT Vu Coverage of health information by different sourcesin communities implication for COVID19 epidemic response Int J EnvironRes Public Health [] National Institutes of Health How to Write EasyToRead Health Materials Accessed April wwwscribdcomdocument261199628HowtoWriteEasyToReadHealthMaterialsMedlinePlus[] DM D™Alessandro P Kingsley J JohnsonWest The readability of pediatricpatient education materials on the world wide web Arch Pediatr AdolescMed “Please cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0c"

Thyroid_Cancer

"Immigrant statusfamily relationsACP contemplationACP discussionburial planninga b s t r a c tObjectives To examine how immigrant status and family relationships are associated with advance careplanning ACP engagement and endoflife EOL preference in burial planning among older ChineseAmericans the largest subgroup of Asian AmericansDesign Crosssectional surveySetting Communities in Honolulu Hawai™iParticipants Participants were older Chinese Americans aged years and olderMeasures Measures included ACP contemplation ACP discussion and EOL preference in burial planningimmigrant status family cohesion family conflict demographic information and health statusResults Results show that in comparison to foreignborn Chinese Americans USborn Chinese Americanswere more likely to have ACP contemplation [odds ratio OR confidence interval CI ] ACP discussion OR CI and preferences for burial plans at the end of life OR CI Family conflict increased the possibility of having ACP contemplation OR CI ACP discussion OR CI and EOL preference in burial planning OR CI whereas family cohesion was not associated with these study outcomesConclusions and Implications This study suggests that ACP should be adapted to be more culturallyappropriate especially in a time of coronavirus and xenophobia such as framing ACP as a tool to helpfamilies reduce stress while fulfilling filial obligations in order to ensure equitable access to ACP“ AMDA e The Society for PostAcute and LongTerm Care MedicineAdvance care planning ACP is a process of understanding andcommunicating individuals™ values goals and preferences regardingendoflife EOL care12 Contemplation of individuals™ EOL wishes anddiscussions with families can be as important as discussions withphysicians and completion of an advance directive in guiding care34ACP is a social process built on relationships and alleviation ofburden on others a means to prepare for death and a measure toexercise the ethical principle of patient autonomy5 Burial planningcan ensure individuals™ wishes are executed and relieve the burden ofloved ones to determine what the deceased would have wantedduring the time of grief In this sense burial planning is an importantThis study was supported by a research grant from the Rory Meyers College ofNursing at New York UniversityThe authors declare no conflicts of interest Address correspondence to Bei Wu PhD Rory Meyers College of Nursing NewYork University First Avenue Room New York NY USAEmail address beiwunyuedu B Wu101016jjamda20200604015258610“ AMDA e The Society for PostAcute and LongTerm Care Medicineelement of ACP6 Therefore it makes sense to examine ACP contemplation ACP discussion with family and EOL preference in burialplanning togetherACP can improve quality of EOL care for individuals including lessinhospital death and increased hospice use7 Despite the benefits ofACP the participation rate of ACP remains low especially among olderadults of racial and ethnic minorities Studies found that in the UnitedStates Blacks and Hispanics are less likely to have an EOL discussion adurable power of attorney and an advance directive than their Whitecounterparts89 but there is a lack of knowledge on ACP engagementamong Chinese Americans the largest subgroup of Asian Americansand the fastestgrowing minority group in the USA10Compared with nativeborn Chinese AmericansforeignbornChinese Americans may face more cultural and logistical challengesin ACP engagement because of their limited English proficiencygreater cultural burden in discussing death and dying and acceptingindividual autonomy and lack of ACP knowledge1112 In addition theeffectiveness of ACP may rely on the involvement knowledge and 0cY Pei JAMDA xxx 1e4cooperation of family members13 however because of the lack of richand comprehensive measures of family relationships in previousresearch on ACP few studies have examined the extent to whichfamily relationships ‚uence individuals™ ACP engagement To fill thisknowledge gap this study aimed to examine how immigrant statusand family relationships are associated with ACP contemplation ACPdiscussion with family and preference in burial planning among olderChinese AmericansMethodsDataData were derived from a survey conducted in Honolulu Hawai™iwhere approximately of the total population is composed ofChinese Americans and of the adult population are immigrants14We used snowball sampling and convenience sampling to identify andrecruit key informants from local Chinese groups social anizationsbusinesses and faithbased agencies based on their capacity ofaccessing Chinese communities and their willingness to assist inrecruiting Chinese older adults in the community We collaboratedwith key community leaders This is a common and effective strategyto recruit respondents from minority populations15 as random sampling is challenging because of the unfeasibility of constructing acompleted sampling frame cultural appropriatenesstime andexpense16 The inclusion criteria for the survey participants includedHonolulu residents aged years and older who selfidentified asChinese The detailed recruitment and data collection methods werereported in previous studies17 The participants provided informedconsent prior to the data collection This study was approved by theinstitutional review board at the university with which the secondauthor was affiliated A total of participants were recruited fromJanuary to September MeasuresDependent variables ACP engagement and EOL preference in burialplanningACP engagement includes ACP contemplation and ACP discussionACP contemplation and ACP discussion was assessed by asking respondents if they previously had thought about their endoflifecare plan with family and had discussed the plan with familyrespectivelyEOL preference in burial planning was measured by a hypothesizedquestion Respondents were asked whether formulating a burial planwas one of the most important things for them to consider if theywere diagnosed with a terminal illness and only had months to liveamong several other options Other mentioned options includedhaving religious beliefssupport alleviating pain reducing care andfinancial burden on family and extending their lifeIndependent variablesImmigrant status was measured by asking respondents whetherthey were US or foreignbornFamily relationships were measured by reliable and valid existingscalesdfamily cohesion and family conflict The index of familycohesion was assessed by asking respondents whether familymembers like to spend free time with each other family membersfeel very close to each other and family togetherness is veryimportant18Family conflict was measured using the 5item Family CulturalConflict scale which assesses cultural and intergenerational conflictperceived by respondents in their family19CovariatesSociodemographic variables included gender age marital statuseducation financial strain living arrangement and social activityparticipation Health need factors included selfrated health comorbidity a continuous variable that examines the existence of at least chronic conditions including heart diseases stroke cancer diabeteshypertension high cholesterol thyroid disease arthritis liverrelateddiseases and others disabilities in activities of daily living andpsychological distress Psychological distress was assessed by theKessler Psychological Distress Scale K1020AnalysisFirst we summarized the sample characteristics Then we usedlogistic regression models and calculated odds ratios ORs to testwhether immigrant status family cohesion and family conflict wereassociated with ACP engagement and EOL preferences All the analyses were conducted using Stata version The missing rates for ACP contemplation ACP discussion and EOLpreferences in burial planning were and respectively Toreduce sampling errors and attain more stable analytical results weconducted multiple imputations MIs for each model All thedependent variables were imputed and the imputed values wereretained in the analysis We used imputed data sets as there werehigh levels of missingness on the dependent variables21 For sensitivity analysis a dependent variable was imputed and imputed valueswere deleted for analysis MID The MID method produced ORs thatwere almost identical to those in the model where the imputed valueswere retainedResultsTable summarizes sample characteristics It shows that less thanhalf of the participants had ACP contemplation and ACP discussion Only had EOL preference in burial planning inthe hypothesized situationTable shows ORs with confidence intervals CIs from logisticregressions The USborn Chinese Americans were more likely to haveACP contemplation OR CI ACP discussion OR CI and preference in burial planning OR CI than the foreignborn Higher levels of familyconflict were associated with higher likelihood of ACP contemplationOR CI ACP discussion OR CI and preference in burial planning OR CI whereasfamily cohesion was not significantly related to these outcomesDiscussionThis study aimed to examine the roles of immigrant status andfamily relationships in the associations between ACP engagement andgiving EOL preferences to burial planning among older ChineseAmericans The USborn Chinese Americans were more likely to haveACP contemplation and ACP discussion than the foreignborn Thismay be because the foreignborn Chinese Americans have lower socioeconomic status less English proficiency lower levels of acculturation and less knowledge about ACP and the US healthcare systemthan their USborn counterparts111222 In addition these individuallevel differences may be mixed with other systemlevel barrierswithin the US healthcare system to worsen the disparities in ACPengagement23 For example Chinese American immigrants may havea stronger belief that family and society are held in higher regard thanindividuals and attribute a higher value to collectivism of family andsociety rather than patient autonomy in EOL decision making12Moreover because traditional Chinese culture expects children tocarry the role of protecting their parents™ health safety and generalwellbeing many Chinese children may construe this responsibility as 0cTable Characteristics of the Study Sample of Chinese Americans N ¼ or Mean SDCodingY Pei JAMDA xxx 1e4ACP engagementACP contemplationACP discussionEOL preferences in burial planningUSbornFamily RelationshipsFamily cohesionFamily conflictFemaleAgeMarriedEducationFinancial strainLiving aloneParticipation in social activitiesSelfrated health as excellentgoodNumber of chronic diseaseADL disabilityPsychological distressADL activities of daily living never and don™t want tonever but want toreluctant to yes never and don™t want tonever but want toreluctant to yes no yes no yes least cohesivee12 most cohesive least cultural conflicte10 most cultural conflict male female unmarried married not at alle3 a great deal no yes no yes no yes no help needed needs help least distressfule5 most distressfulmaking every effort to prolong their older parents™ life which maysometimes be in opposition to their parents™ own wishes24 Thesepotential factors surrounding older Chinese immigrants may helpexplain this population™s lack of engagement in ACP Healthcare providers in turn should pay closer attention to these factors in order tothoroughly evaluate patients™ EOL wishes It is noted that the USbornChinese Americans were far more likely to have preferences in burialplanning than the foreignborn The finding is consistent with a previous study in that decisions such as EOL care and funeral and burialpreplanning are impacted by similar factors25 Indeed EOL care decision making and burial planning are integrated processes at theend of life26 and burial plan is included in some advance directivedocuments in practice Future studies on ACP need to consider burialplanningSecond family cohesion was not associated with ACP contemplation ACP discussion and EOL preference in burial planning whereasfamily conflict increased the possibility of ACP contemplation ACPdiscussion and EOL preferences in burial planning The finding isinconsistent with previous study conducted among White olderadults revealing that the positive family relationship encourageswhereas problematic family relationship hinders ACP engagement27The inconsistency is likely due to the fact that Chinese Americansvalue family in the process of EOL decision making2829 The lack ofassociation between family cohesion and ACP engagement may bebecause older adults with higher levels of family cohesion have tobalance between the potential benefit and harm of ACP engagementOn the hand older Chinese Americans may have positive attitudesabout ACP engagement and believe that ACP engagement is importantand necessary because it allows them to witness their loved ones™death and dying experience12 On the other hand closeknit familialrelationships may make both older Chinese Americans and theirfamilies feel more uncomfortable to start a conversation on EOL carebecause discussions about death and dying are often considered ataboo in Chinese culture30 In this sense strong family ties may havelimited impact on ACP engagement An explanation for the significantrelationship between family conflict and ACP engagement could bethat higher levels of family conflict may indicate a greater need for ACPengagement This is because the members in these families are lesslikely to know about the EOL care preferences of older adults and betrusted in the EOL decision making13 These findings suggest thatculture may play an important role in the complex association between family relationships and ACP engagementSeveral limitations of the study deserve mentioning First thecrosssectional data from a small region limit our ability to generalizefindings to older Chinese Americans living in other parts of the UnitedStates as well as to make causalinferences Second the ACPengagement in our study only included ACP contemplation ACP discussion and preference in burial planning Future studies need toinclude more ACP options such as the completion of living wills oradvance directives and the selection of a durable power of attorneyfor health care to understand more about ACP engagement in ChineseAmerican families Third ACP knowledge is an important confoundingvariable for both immigrant status and ACP engagement Futurestudies on ACP engagement need to consider this variableConclusions and ImplicationsDespite these limitations this study sheds light on how immigrantstatus and family relationships shape ACP engagement among olderChinese AmericansIt is found that immigrant status decreaseswhereas family conflict increases the likelihood of having ACPcontemplation ACP discussion and preference in burial planningTable Factors Associated With Advance Care Planning Engagement Results of Logistic Regression N ¼ USborn ref ¼ foreignbornFamily relationshipsFamily cohesionFamily conflictACP ContemplationOR CI ACP DiscussionOR CI EOL Preferences inBurial Planning OR CI All models adjusted for age gender marital status education financial strain living alone social activity participation selfrated health number of chronic disease activitiesof daily living and psychological distress 0cY Pei JAMDA xxx 1e4Health care providers may consider patients™ immigrant status andfamily relationships to better serve ethnically diverse populationsGiven that cultural factors play an important role in ACP engagementACP should be adapted to be more culturally appropriate amongChinese Americans especially in a time of coronavirus and xenophobia such as framing ACP as a tool to help families reduce stresswhile fulfilling filial obligations in order to ensure equitable access toACPAcknowledgmentWe thank Katherine Wang for her editorial assistance We wouldalso like to thank the research team at the University of Hawaii fortheir data collectionReferences Rietjens JAC Sudore RL Connolly M Definition and recommendations foradvance care planning An international consensus supported by the EuropeanAssociation for Palliative Care Lancet Oncol 201718e543ee551 Sudore RL Lum HD You JJ Defining advance care planning for adults Aconsensus definition from a multidisciplinary Delphi panel J Pain SymptomManage 201753821e832e821 Sudore RL Schickedanz AD Landefeld CS Engagement in multiple steps ofthe advance care planning process A descriptive study of diverse older adultsJ Am Geriatr Soc 2008561006e1013 Sudore RL Knight SJ McMahan RD A novel website to prepare diverseolder adults for decision making and advance care planning A pilot studyJ Pain Symptom Manage 201447674e686 Dahlin C Giansiracusa D Communication in palliative care In Ferrell BCoyle N editors Textbook of Palliative Nursing New York NY Oxford University Press p 67e96 KataokaYahiro MR Conde FA Wong RS Advance care planning amongAsian Americans and Native Hawaiians receiving haemodialysis Int J PalliatNurs 20101632e40 Bischoff KE Sudore R Miao Y Advance care planning and the quality ofendoflife care in older adults J Am Geriatr Soc 201361209e214 Kale MS Ornstein KA Smith CB Kelley AS Endoflife discussions with olderadults J Am Geriatr Soc 2016641962e1967 Harrison KL Adrion ER Ritchie CS Low completion and disparities inadvance care planning activities among older Medicare beneficiaries JAMAIntern Med 20161761872e1875 Pew Research Center Key facts about Asian Americans a diverse and growingpopulation Available at wwwpewresearchfacttank20170908keyfactsaboutasianamericans Accessed November Gao X Sun F Ko E Knowledge of advance directive and perceptions ofendoflife care in ChineseAmerican elders The role of acculturation PalliatSupport Care 2015131677e1684 Lee MC Byon HD Hinderer K Alexander C Beliefs in advance care planningamong Chinese Americans Similarities and differences between the youngerand older generations Asian Pac Isl Nurs J 2017283e90 Parks SM Winter L Santana AJ Family factors in endoflife decisionJ Palliat Med making Family conflict and proxy relationship179e184 Tong M Sentell T Insights in public health Challenges investigating healthoutcomes in Chinese Americans using populationbased survey data Hawaii JMed Public Health Ibrahim S Sidani S Strategies to recruit minority persons A systematic reviewJ Immigr Minor Health 20145882e888 Spring M Westermeyer J Halcon L Sampling in difficult to access refugeeand immigrant communities J Nerv Ment Dis 2003191813e819 Zhang W Liu S Zhang K Wu B Neighborhood social cohesion resilience andpsychological wellbeing among Chinese older adults in Hawai™i Gerontologist201960229e238 Rivera FI Guarnaccia PJ MulvaneyDay N Family cohesion and its relationship to psychological distress among Latino groups Hisp J Behav Sci 30357e378 Alegria M Vila D Woo M Cultural relevance and equivalence in theNLAAS instrument Integrating etic and emic in the development of crosscultural measures for a psychiatric epidemiology and services study of LatinosInt J Methods Psychiatr Res 200413270e288 Kessler RC Andrews G Colpe LJ Short screening scales to monitor population prevalences and trends in nonspecific psychological distress PsycholMed 200232959e976 Johnson DR Young R Toward best practices in analyzing datasets withmissing data Comparisons and recommendations J Marriage Fam 926e945 Carr D The social stratification of older adults™ preparations for endoflifehealth care J Health Soc Behav 201253297e312 Shen MJ Prigerson HG Tergas AI Maciejewski PK Impact of immigrant statuson aggressive medical care counter to patients™ values near death amongadvanced cancer patients J Palliat Med 20192234e40 Bowman K Singer P Chinese seniors™ perspectives on endoflife decisions SocSci Med 200153455e464 Kelly CM Masters JL DeViney S Endoflife planning activities An integratedprocess Death Stud 201337529e551 Tanaka M Takahashi M Kawashima D Endoflife activities amongin Japan Omega Westport communitydwelling older adults Carr D Moorman SM Boerner K Endoflife planning in a family context Doesrelationship quality affect whether and with whom older adults planJ Gerontol B Psychol Sci Soc Sci 201368586e592 Hinderer KA Lee MC Chinese Americans™ attitudes toward advance directivesAn assessment of outcomes based on a nursingled intervention Appl Nurs Res20194991e96 YonashiroCho J Cote S Enguidanos S Knowledge about and perceptions ofadvance care planning and communication of ChineseAmerican older adultsJ Am Geriatr Soc 2016641884e1889 Chi HL Cataldo J Ho EY Rehm RS Can we talk about it now Recognizing theoptimal time to initiate endoflife care discussions with older ChineseAmericans and their families J Transcult Nurs 201829532e539 0c"

Thyroid_Cancer

Aprospective blinded multicenter studyDear editorThyroid nodules are common and the major point oftheir clinical evaluation rests with the differentiation ofthyroid cancer1“ Herein we present a twostep study todevelop and validate an effective model based on circulating tumor cells CTCs signatures for papillary thyroid cancer PTC diagnosisA total of subjects were clinically evaluated and of which were included in the final analysis A In the first step patients with PTC from a single hospital and healthy subjects formed the test groupMultimarkers reported in the literatures that were relatedto malignancy and thyroid epithelium CK19 SurvivinGalectin3 Tg and TSHR were evaluated and selectedto form a most effective combination for the diagnosticmodel Next the model was evaluated in the validationgroup that was consisted of participants from four different hospitals in China who had one or more thyroidnodules that yielded an ultrasound diagnosis of ThyroidImaging Reporting and Data System45 TIRADS or and measured cm long in diameter Postoperativepathology was considered as the reference standard andonly patients who received surgery and had definite pathological reports were enrolled in the final analysis Thyroidnodule was excluded by ultrasound in healthy subjectsThe process of CTCs separation and enrichmentincluded CD45 negative Dynabeads„¢ M450 CD45 panLeukocyte 11153D Invitrogen by Thermo Fisher Scientific USA and EpCAM positive Invitrogen byThermo Fisher Scientific USA immunomagnetic selections The reliability of CTCs separation and enrichmentwas verified with fluorescence staining in a cell line model PTC cells added into white blood cells separationfrom mL peripheral blood from a healthy subject B mRNA was isolated from the cell fractions Invitrogen by Thermo Fisher Scientific USA and cDNAwas then reverse transcribed with μL of mRNA total μL using the Sensiscript RT Kit QIAGEN Germany mRNA levels of CK19 Survivin Galectin3 Tgand TSHR were analyzed by the QuantiNova SYBR GreenPCR Kit QIAGEN Germany Thermo FisherScientific USA Three independently isolated RNAsamples were measured to determine the threshold cyclevalues CT mRNA level of these markers were measuredwith relative expression level RQ and presented afternormalization against the reference actin and wascalculated with “ΔΔCT method ΔCt Ct target “ Ctactin ΔΔCT ΔCt13 RQ “ΔΔCT The values werepresented as the mean ± standard deviation SDOf all participants recruited in the study the medianage was years range years and were female participants Among patients with thyroid nodules the median size of the nodules was cm range cm and had a tumor ‰ cm According to thepostoperative pathology patients had PTC of which and patients had central compartment and lateral necklymph nodes metastasis respectively In the test group patients and healthy subjects the median mRNA levelsof the markers were significantly higher in patients thanin healthy controls except for TSHR C CK19 andSurvivin mRNA was not detected in any healthy subject sopositive result of the two markers was defined as detectablemRNA level Tg and Galectin3 mRNA were detected in and healthy subjects respectively Based on theROC curves a cutoff value of ngμg was selected falectin3 with a sensitivity of and a specificity of while a cutoff value of ngμg for Tg giving an optimal sensitivity of and a specificity of The areaunder curve AUC of CK19 Survivin Galectin3 and Tgwere and respectivelyAfter evaluating different combinations of the markers Supporting Information CK19 Survivin and Tgwere selected to form a diagnostic model when positiveresult was recognized when any marker was positive itsThis is an access under the terms of the Creative Commons Attribution License which permits use distribution and reproduction in any medium provided theoriginal work is properly cited The Authors Clinical and Translational Medicine published by John Wiley Sons Australia Ltd on behalf of Shanghai Institute of Clinical BioinformaticsClin Transl Med 202010e142101002ctm2142wileyonlinelibrarycomjournalctm2 of 0c of LETTER TO EDITORF I G U R E Flow chart of the study and diagnostic model establishing A Flow chart of the study B Fluorescence staining of EpCAMCD45 and DAPI after immunomagnetic enrichment of a CTC model PTC cells added into white blood cells separation from ml peripheralblood from a healthy subject CTCs white solid arrow white blood cells white dotted arrow C CK19 Survivin Galectin3 Tg and TSHRmRNA levels of healthy subjects n and patients with PTC n in the test group D HE a and immunohistochemical staining forCK19 b Tg c and Survivin d of a PTC patient enrolled in the test group E Expression of markers in cell lines of PTC by western blotpositive predictive value PPV negative predictive valueNPV sensitivity specificity and AUC value were and respectively and the diagnostic model correctly classified of subjects yielding anoverall accuracy of The expression of selected markers CK19 Survivin and Tg was verified with immunohistochemical analysis in formalinfixed paraffinembeddedpostoperative specimen of PTC patients D andwestern blot in two cell lines of PTC E and thenthese markers were evaluated in the validation groupIn the validation cohort n the median mRNAlevels of CK19 Survivin and were significantly higherin patients with PTC than in patients with benign thyroid nodules P A The clinical sensitivity specificity PPV and NPV of the diagnostic model were and respectively with a diagnostic accuracy of and the AUC for predicting malignancy was CI For detailed performance of individual markers the highest sensitivity of single marker Tg was which was significantlylower than the diagnostic model of 3marker combinationAll patients with positive Survivin n had malignant pathology PPV with a sensitivity of which was consistent with the results of the test groupBIn the diagnostic model based on threeCTCs signatures established in the study has a highdiagnostic performance for patients with suspiciousthyroid nodules multimarker analysissignificantlyimproved sensitivity comparing with single marker 0cLETTER TO EDITOR of F I G U R E Validation of the diagnostic model in a prospective cohort n A CK19 Survivin and Tg mRNA levels of benign andmalignant patients in the validation group B Detailed diagnostic performance of the diagnostic model in the validation groupanalysis Moreover it is discovered in this study thatthe CTCs signature Survivin had a potential value forconfirming PTC diagnosis PPV C O N F L I C T O F I N T E R E S TThe authors have no conflicts of interest to disclosureS TAT E M E N T O F E T H I C SThe study was approved by the ethic committee of CancerHospital Chinese Academy of Medical sciencesAU T H O R S ™ C O N T R I B U T I O N SSiyuan Xu and Jingning Cheng participated in the designof the study data collection and paper writing Bojun Weiand Yang Zhang participated in the data collection andhelped to draft the manuscript Yang Li and Rui Zhangparticipated in the data analysis and validation ZongminZhang Yang Liu and Kai Wang participated in the datacollection and quality control of data Ye Zhang and YingHuang participated in the statistical analysis Xiaolei Wangand Shaoyan Liu participated in the manuscript reviewJie Liu and Zhengang Xu participated in the design of thestudy and helped to revise the manuscript All authors readand approved the final manuscriptSiyuan Xu1Jingning Cheng2Bojun Wei3Yang Zhang4Yang Li5Zongmin Zhang1Yang Liu1Ye Zhang6Rui Zhang7Kai Wang1Xiaolei Wang1Shaoyan Liu1Ying Huang1Zhengang Xu1Jie Liu1 Department of Head and Neck Surgical OncologyNational Cancer CenterNational Clinical Research Centerfor CancerCancer Hospital Chinese Academy of MedicalSciences and Peking Union Medical College Beijing P RChina Department of Otorhinolaryngology ChinaJapanFriendship Hospital Beijing P R China Department of Thyroid and Neck Surgery BeijingChaoyang Hospital Capital Medical University Beijing PR China Department of Endocrinology Peking University FirstHospital Beijing P R China Department of General Surgery Hebei Petro ChinaCentral Hospital Langfang P R China Department of Radiation Oncology National CancerCenterNational Clinical Research Center forCancerCancer Hospital Chinese Academy of MedicalSciences and Peking Union Medical College Beijing P RChina Department of Ultrasound National CancerCenterNational Clinical Research Center forCancerCancer Hospital Chinese Academy of MedicalSciences and Peking Union Medical College Beijing P RChinaCorrespondenceJie Liu and Zhengang Xu Postal address No Panjiayuan Nanli Chaoyang District Beijing PRChinaEmail liujcicamsaccn xuzhg06126com 0c of LETTER TO EDITORSiyuan Xu Jingning Cheng Bojun Wei and Yang Zhangcontributed equally to this workTrial Registration Clinicaltrialsgov IdentifierNCT03772496Funding informationCAMS Innovation Fund for Medical Sciences BeijingHope Run Special Fund of Cancer Foundation of China2016I2m1002 Beijing Hope Run Special Fund of CancerFoundation of China LC2018A26 LC2016A01O RC I DJie Liuorcid0000000339757978R E F E R E N C E S Haugen BR Alexander EK Bible KC et al American Thyroid Association Management Guidelines for adult patients withthyroid nodules and differentiated thyroid cancer the American Thyroid Association Guidelines Task Force on thyroidnodules and differentiated thyroid cancer Thyroid Burman KD Wartofsky L Clinical practice Thyroid nodules NEngl J Med Popoveniuc G Jonklaas J Thyroid nodules Med Clin North Am Cheng SP Lee JJ Lin JL Chuang SM Chien MN Liu CL Characterization of thyroid nodules using the proposed thyroid imagingreporting and data system TIRADS Head Neck Zhang J Liu BJ Xu HX et al Prospective validation ofan ultrasoundbased thyroid imaging reporting and data system TIRADS on thyroid nodules Int J Clin Exp MedS U P P O RT I N G I N F O R M AT I O NAdditional supporting information may be found onlinein the Supporting Information section at the end of the 0c'

Thyroid_Cancer

RUFYs a Family of EffectorProteins Involved in IntracellularTrafficking and CytoskeletonDynamicsRmy Char1 and Philippe Pierre123 Aix Marseille Universit Centre National de la Recherche Scientifique Institut National de la Sant et de la RechercheMdicale Centre d™Immunologie de MarseilleLuminy Marseille France Institute for Research in Biomedicine and IlidioPinho Foundation Department of Medical Sciences University of Aveiro Aveiro Portugal Shanghai Instituteof Immunology School of Medicine Shanghai Jiao Tong University Shanghai ChinaIntracellular trafficking is essential for cell structure and function In order to performkey tasks such as phagocytosis secretion or migration cells must coordinate theirintracellular trafficking and cytoskeleton dynamics This relies on certain classes ofproteins endowed with specialized and conserved domains that bridge membraneswith effector proteins Of particular interest are proteins capable of interacting withmembrane subdomains enriched in specific phosphatidylinositol lipids tightly regulatedby various kinases and phosphatases Here we focus on the poorly studied RUFY familyof adaptor proteins characterized by a RUN domain which interacts with small GTPbinding proteins and a FYVE domain involved in the recognition of phosphatidylinositol3phosphate We report recent findings on this protein family that regulates endosomaltrafficking cell migration and upon dysfunction can lead to severe pathology at theanismal levelKeywords RUFY cancer neurodegenerative diseases immunity RUN FYVE phosphatidylinositol 3phosphatecytoskeletonINTRODUCTIONThe anization of cells into multiple membranous compartments with specific biochemicalfunctions requires complex intracellular traffic and sorting of lipids and proteins to transport themfrom their sites of synthesis to their functional destination Intracellular transport involves lipidvesicles or tubules with the capacity to fuse with one another or to be secreted They collectivelyparticipate in the dynamic exchanges necessary for cell homeostasis Rothman S¸reng Membrane traffic is tightly coordinated with protein synthesis signal transduction ofenvironmental stimuli and cytoskeleton anization allowing the implementation of key cellularfunctions such as endocytosis exocytosis or migration McMahon and Gallop Habtezion VegaCabrera and PardoLpez MacGillavry and Hoogenraad Margaria Tapia Buratta Stalder and Gershlick Several families of molecular components required for orchestrating membrane vesicle exchangeand transport during this process are conserved They include adaptor and coat proteins smallGTPbinding proteins GTPases as well as Synaptosome Associated Protein SNAP ReceptorEdited byRoberto BotelhoRyerson University CanadaReviewed byDaniel G S CapellutoVirginia Tech United StatesBrian Paul CeresaUniversity of Louisville United StatesCorrespondenceRmy CharcharcimlunivmrsfrPhilippe PierrepierrecimlunivmrsfrSpecialty sectionThis was submitted toMembrane Traffica section of the journalFrontiers in Cell and DevelopmentalBiologyReceived June Accepted July Published August CitationChar R and Pierre P TheRUFYs a Family of Effector ProteinsInvolved in Intracellular Traffickingand Cytoskeleton DynamicsFront Cell Dev Biol 103389fcell202000779Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingSNARE proteins and SNARE binding proteins Juliano The vast superfamily of GTPases is involved in the establishmentor regulation of virtually every step of intracellular membranetrafficking They behave as molecular switches that can alternatebetween active and inactive states through GTP binding andhydrolysis into GDP Takai Stenmark Thelargest group of GTPases involved in intracellular membranetraffic is the Rab proteins family Lamb Rab GTPasesspecifically localize to diï¬erentintracellular compartmentsregulating vesicle formation and sorting as well as transportalong the cytoskeletal network Each Rab protein can be recruitedto specific membrane subdomains of a defined anelle andis associated to multiple eï¬ectors controlling membrane fusionand trafficking Rab interaction with the membrane fusioncomplexes and cytoskeleton regulators is therefore crucial forcellular functions including endocytosis and autophagy Chenand WandingerNess Bruce Geng Thomas and Fromme Yuan and Song Here we review the literature concerning a lesswell knownfamily of proteins involved in the complex biochemical crosstalkestablished between the cytoskeleton and intracellular vesiclesThis small group of proteins was named RUFY for œRUN andFYVE domaincontaining RUFYs share a common structuraldomain anization including an Nterminal RUN domainone or several coiledcoil CC repeats and a Cterminal FYVEdomain A The molecular structures of the diï¬erentRUFY proteins has been described Dunkelberg and GutierrezHartmann Mari KukimotoNiino Kitagishi and Matsuda but their function in endocyticregulation and their physiological relevance at the anismallevel are still poorly characterized Kitagishi and Matsuda Terawaki We revisit here how the rufy genefamily was annotated and propose the addition of a novelmember the fyco1 FYVE and Coiledcoil containing domain gene given its sequence and functional similarities withthe other rufy genes Pankiv Terawaki We also highlight recent findings on the implication ofRUFY proteins in the regulation of cytoskeleton and endosomedynamics and their contribution to immunity cancer andneurodegenerative diseasesEndocytosis and AutophagyEndocytosis and autophagy are membrane traffic pathwaysrequired for degradation and recycling ofextracellularand intracellular components respectively Birgisdottir andJohansen These pathways have a common endpoint at thelysosome where their cargo is degraded These both pathwaysintersect at several stages throughout vesicle formation transportand fusion and share some of the components of their molecularmachineries BThere are numerous coexisting endocytic pathways whichinitiate by the formation of nascent endocytic vesicles formedfrom plasma membrane invaginations and scissions Theseendocytic vesicles undergo homotypic fusion and are rapidlytargeted to sorting endosomes SE Sorting events initiated inSE determine the fate of internalized cargo molecules such asrecycling to plasma membrane degradation in lysosomes orother trafficking events Naslavsky and Caplan BOn their way to degradation sorted cargo accumulate inearly endosomes EE that further mature into late endosomesLEthrough multiple events of cargo and lipid sortingLate endosomes adopt a membrane anization termedmultivesicular bodies that are enriched in lysobisphosphatidicacid and contain intraluminal vesicles Gruenberg NextLE potentiate their hydrolytic competence by fusing withlysosomes Pillay resulting in the degradationoftheir contents providing nutrients and key factors tothe cell Doherty and McMahon Kaksonen and Roux Notably endosomes play a role in signal transductionby serving as signaling platforms either for surface activatedreceptors like Tolllike receptors and epidermal growth factorreceptor or metabolic sensors such as mechanistic targetof rapamycin complex mTORC1 Arg¼ello Often they promote the degradation of their targets leadingto signaltermination Chung The endocyticpathway has also specialized functions in diï¬erentiated cellssuch as neurotransmitter release and recycling in neurons orantigen processing and presentation in professional antigenpresenting cellsArg¼ello SolDom¨nech Hinze and Boucrot Endocytosis events and endosomes positioning ishighly dependent on the dynamic and spatial reanizationofinclude actinintermediate filaments or microtubules Fletcher and Mullins Pegoraro the diï¬erent cytoskeleton networks thatlike B cells or dendritic cellsComplementary to endocytosis autophagy is an intracellularprocess by which cells degrade and recycle their own cytoplasmicmaterials Mizushima and Komatsu Autophagy plays acentral role in many physiological processes including stressmanagement development immunity and aging Puleston andSimon Zhong F®lfan Moretti Doherty and Baehrecke Autophagy ispartially controlled though mTORC1 activity and is responsiblefor degradation and recycling of misfolded proteins as wellas obsolete anelles Galluzzi The endpoint ofautophagy is to deliver cytoplasmic material to lysosomes wherelike for endocytosed cargo it is degraded Several autophagyprocesses can be distinguished based on the entry mode ofthe cytosolic components destined for degradation BMacroautophagy involves engulfment of cytoplasmic contentsinto a double membrane vesicle termed the autophagosomeThe autophagosome fuses then with lysosomes becomingin which its cargo is degraded Galluzzian autolysosome The presence ofspecific phosphoinositideslipidstogether with Rab GTPases at a given membranecompartment is often directly correlated with compartmentfunction One of the common mechanism regulating endocytosisand autophagy is an accumulation of phosphatidylinositol phosphate PtdIns3P at surface of EE and on intraluminalvesicles of multivesicular endosomes and on autophagosomesNascimbeni B PtdIns3P is also observedat sites of LC3ˆ’associated phagocytosis another pathway ofinternalization used by the cells to ingest large particulatematerial or microbes PtdIns3P is therefore a beacon used by theFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingFIGURE RUN and FYVE domain containingproteins in the endolysosomal pathway A Schematic representation of the RUFY proteins family B Description ofthe endolysosomal and autophagy pathways and presumed functional locations of RUFY proteins Extracellular material is ingested by endocytosis orphagocytosis The action of different endosomes allows cargo to be sorted recycled or degraded in a complex and regulated process involving fusion maturationand transport along the cytoskeleton Alternatively during autophagy obsolete components present in cytosol are captured in autophagosomes prior fusion withlysosomes and degradation macroautophagy or directly internalized through endosomal invagination microautophagy SE sorting endosome EE earlyendosome TGN trans golgi network LE late endosome MVBs multi vesicular bodies RE recycling endosome MT microtubule CT centrioles ER endoplasmicreticulum The location of PI3P and RUFY proteins known activity is shown Created with BIoRendercomFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and Traffickingcellular machinery to regulate endosomal sorting and autophagyBirgisdottir and Johansen RUN DomainsThe presence of a single copy of a RUN and a FYVE domainat their extremities is the key characteristic defining the RUFYfamily members RUN domains were named after three proteinsbearing similar peptide motifs RPIP8 UNC14 and NESCAnew molecule containing SH3 at the carboxyˆ’terminus Ogura Matsuda RUN domains are present inmultiple proteins RUN proteins in a large panel of anismsFigure and principally allow direct interactions with smallGTPases of the Rap and Rab families Callebaut Yoshida RUN domains adopt a hydrophobicglobular structure bearing six conserved blocks named A to FFigure 3A These blocks correspond to eight αhelices andsome 310helices The first helix is crucial to limit hydrophobicexposure and maintain protein solubility of RUNcontainingproteins Callebaut KukimotoNiino In spite of strong conservation among the domains presentin RUNcontaining proteins the proteins they interact withtheir eï¬ectors are highly variable Mari and thestructural features of the RUN domain alone are not sufficientto define binding specificity for one or several members of theGTPase superfamily Fukuda Most RUN domainbearing proteins bind small GTPases but interactions with othermolecules like kinesin have also been described Boucrot A direct physical link between RUN proteins withactin filaments and microtubules has been also demonstratedTorti reinforcing the idea that these molecules arealso critical for cellular functions requiring actin remodelingsuch as migration or phagocytosis Price and Bos Bos Miertzschke Xu Figure 4AAdditional functions for RUN domains have been describedfor example for the RUN domain present in NESCA whichblocks TRAF6mediated polyubiquitination of the NFkappaBessential modulator and consequently induces NFkB activationThis is just one of the ways in which RUN proteins canact in signal transduction and the coordination of membranetraffic with actin dynamics upon external stimulation Yoshida As well as promoting endosomal fusion throughtheir binding to Rab or Rap GTPases Callebaut FIGURE Evolution of RUN and FYVE domain or rufy genes among living anisms Diagram illustrating the evolution of the rufy genes Species representative ofvarious taxonomic groups are listed data were extracted from the Differential Expression Atlas Genes database EMBLEBI Next to each species studied thenumber corresponds to the number of genes having in its sequence a FYVE green RUN blue or both red domain The œX corresponds to the appearance of acommon rufy ancestor geneFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingFIGURE Molecular anization of RUN and FYVE domains from the RUFY proteins family Alignment of the protein sequences of the RUN A and FYVE Bdomains of the RUFY proteins family in human and mouse A RUN consensus blocks are represented by segments A“F Rpip8 sequence is used as RUN domainreference B FYVE conserved motives and zinc fingers are represented by segments In the alignment œx is any amino acid and œ represents positively chargedamino acid Eea1 sequence is used as FYVE domain reference For all alignment amino acids are colored according to their properties Cyan for hydrophobicpositions A V I L M turquoise for aromatic positions F Y W H red for basic residues K R purple for acidic residues D E green for polar uncharged N Q ST salmon for cysteine C orange for glycine G and yellow for proline P Gray numbers below alignment means the amino acids position after alignment Blacknumbers surrounding the alignments represent the start left and end right positions of the domains in the peptide sequence of each protein Alignment wererealized with Seaviewer analyzer software Gouy Accession numbers for protein are following human Rpip8 NP_0011382971 mouse Rpip8NP_0580391 human Eea1 NP_0035573 mouse Eea1 NP_0010019321 human RUFY1 NP_0794343 mouse RUFY1 NP_7661451 human RUFY2NP_0604574 mouse RUFY2 NP_0817012 human RUFY3 NP_0557761 mouse RUFY3 NP_0818061 human RUFY3XL NP_0010325191 mouseRUFY3XL NP_0012767031 human RUFY4 NP_9408852 mouse RUFY4 NP_0011641121 human FYCO1 NP_0787892 mouse FYCO1NP_0011037232Yoshida their interaction with motor proteins likekinesin or myosin suggests a role for RUN domains in regulatingvesicular and anelle transport Callebaut Yoshida Via these diï¬erent mechanisms RUN proteins havebeen implicated in neuronal development Honda 2017bsignaling Sun migration Yoshida and regulation of various cellular function like endocytosis orexocytosis Kitagishi and Matsuda FYVE DomainsFYVEdomainbearing proteins for Fab1 YOTBZK63212Vac1 and EEA1 are specifically found in association withmembranous anelles enriched in PtdIns3P and highlyconserved among eukaryotes including yeast Hayakawa Figure FYVE domains adopt a zinc finger conformationMisra and Hurley Kutateladze and Overduin Inaddition to FYVE ten types of zinc finger folds have beenFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingFIGURE RUFY proteins are important for intracellular trafficking signaling and cytoskeleton dynamics A Schematic representation of the RUN and FYVEdomains activity of RUFY proteins RUN domains act on signaling endosomal protein trafficking and cytoskeletal network dynamics via small GTPase proteins FYVEdomains bind PtdIns3P and regulates autophagy and endosome trafficking B Function of RUFY proteins in homeostatic conditions C Consequences ofalterations in RUFY proteins functions at the cellular and anismal levelcharacterizedincluding conventional Gal4 GATA1 TFIISMetRS LIM RING domain PKC CRD and PHD domainsZinc fingers are structural conformations adopted by peptidechains upon coordination of two Zn2 cations within a cysteinerich region Schwabe and Klug Stenmark Unlike most molecules bearing zinc fingers FYVE proteinsdisplay only one copy of the domain located at any positionalong the peptide chain highlighting its autonomy as a structuralunit FYVE zinc fingers can stabilize proteinprotein or proteinDNARNA interactions Dunkelberg and GutierrezHartmann A œclassical FYVE domain has eight potential zinccoordinating tandem cysteine positions and is characterized byhaving basic amino acids around the cysteines Many membersof this family also include two histidine residues in a sequencemotif including WxxD CxxC RHHCxCG and RVC whereœx means any amino acid and œ a positively charged aminoFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and Traffickingacid Figure 3B Most deviations from this sequence canreduce the domain affinity for zinc and destabilize it Stenmark Misra and Hurley Stenmark and Aasland Kutateladze and Overduin Within this structuralframework specific modifications in the nonconserved residuesof the domain can radically aï¬ect FYVE protein subcellularlocalization and function by forming a œturret loop and adimerization interface Hayakawa With regard to their affinity for PtdIns3P FYVE domaincontaining proteins are mostly found associated to EE orphagosomes Stenmark Gaullier Stenmark and Aasland Figure 4A The presence ofFYVE domains is therefore correlated to the regulation ofmembrane traffic through specific recognition of PtdIns3Pdomains by œRHHCxCG motifs Gaullier and modulation by associated phosphatidylinositol kinasesPtdIns3P is generated from phosphatidylinositol by Class IIIPtdIns 3kinases PI3Klike Vps34 on target membranessuch as nascent autophagosome omegasomes Melia or EE Di Paolo and De Camilli Raib Scott B In turn accumulation ofPtdIns3P recruits and activates eï¬ector proteins containingFYVE domains favoring transport or fusion of target anellesStenmark and Gillooly Axe Burman andKtistakis Schink Affinity for PtdIns3P isdetermined by the pair of histidine residues present in theœRHHCXCG motif of the FYVE domain Stahelin Diraviyam Lee He This affinity can also be harnessed by FYVE proteins to linkendosomes with mRNA ribonucleoprotein ps mRNPand associated ribosomes playing a role in their longdistancetransport in the cell Pohlmann Importantlymany FYVE proteins homodimerize Dimerization multiplies theconserved residues displayed by the diï¬erent signature motifspresent in the FYVE domain and contributes to a network ofhydrogen bonding and electrostatic interactions that providespositive selection for binding several PtdIns3P head groupsPHdependent insertion of FYVE domain into cell membranesHe Pankiv is reinforced by additionalhydrophobic membrane interactions with the turret loop andortandem lysine residues These nonspecific interactions promoteFYVE domain access to phosphate head groupsthat arehindered by the close packing of lipid molecules This bivalentmechanism increases therefore greatly FYVE domains specificityfor PtdIns3Penriched domains and discrimination againstother mono or polyphosphorylated PtdIns species Misra andHurley Stenmark and Aasland Dumas Kutateladze and Overduin FYVE proteins are therefore key players in endocytosis andautophagy and mutations in FYVE domains can alter profoundlythese functions as well as cellular homeostasis Kamentseva For example EEA1 protein early endosome antigen isknown to be crucial for endosome dynamics and any mutation inits conserved residues or the oligomerization site can drasticallyreduce the affinity between its FYVE domain and PtdIns3PStenmark Gaullier In this contextRUFYs proteins by bearing a Nterminal RUN domain oneor several copies of a coiledcoil domain next to a CterminalFYVE domain A have all the features required tocarryout specific adaptor functions to regulate endocytosis orautophagy by impacting on anelle fusion and mobility alongthe cytoskeletonThe RUFY Proteins FamilyThe RUFY family encompass four genes named rufy1 to sharing homologies and displaying specific tissue expressionand alternative splicing Rufy genes are relatively conservedgenes absent from prokaryotes and fungi Upon evolution theemergence of the common ancestor appeared in vertebrates andarthropods which possess one ortholog CG31064 Figure No RUFY protein could be detected In Caenorhabditis elegansand only a FYVEbearing protein T10G35 considered as anortholog of human EEA1 shows some sequence similaritieswith the RUFY family T10G35 exhibits PtdIns3P bindingactivity and is involved in endocytosis being mostly expressedin epidermis and intestine of C elegans Hayakawa In chordates Rubicon RUN domain and cysteinerichdomain containing Beclin 1interacting protein and FYVE AndCoiledCoil Domain Autophagy Adaptor FYCO1 displaystructural and functional features potentially categorizing themas RUFY proteins Rubicon was identified as a componentthe Class III PI3K complex and a negative regulatorofof autophagy and endosomaltrafficking Matsunaga Zhong Like RUFYs Rubicon containsmultiple functional domains that interact with other proteinsincluding a RUN a CC and a FYVElike domains Wong However despite these similaritiesthe poordegree of sequence homology and the lack of conservationof its FYVElike domain which was found not to bind toPI3P Burman and Ktistakis prevented Rubicon™sintegration within the RUFY proteins family conversely toFYCO1 which we propose here to name RUFY5 and detail thecharacteristics belowRUFY1RUFY1 previously named Rabip4 is an kDa proteinmainly expressed in the brain kidneylung placenta andtestis There are two RUFY1 isoforms Rabip4 and Rabip4™that has an additional amino acid upstream oftheNterminal RUN domain A They were both shownto interact with the small endosomal GTPases Rab4 Rab5and Rab14 Fouraux Vukmirica Table RUFY1 inactivation inhibits efficient recycling ofendocytosed transferrin implicating RUFY1 in the regulationof EE functions through cooperative interactions with Rab4and Rab14 Cormont Yamamoto Nag This was further demonstrated by the alteration ofepidermal growth factor receptor endocytic trafficking kineticsin cells depleted of RUFY1 Gosney and thehijacking of RUFY1 by the bacteria P gingivalis to escapelysosomal degradation Takeuchi In melanocytesRUFY1 was found to form a complex with rabenosyn5Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingTABLE Summary of RUFY proteins functional interactionsProtein AliasesBinding partnerFunctionsRUFY1Rabip4 Rabip4™ZFYVE12RUFY2LZFYVE Rabip4rKIAA1537 FYVE13RUFY3Singar1 RIPXZFYVE30 KIAA087RUFY4ZFYVE31FYCO1RUFY5ZFYVE7 RUFY3CTRCT18 CATC2Rab4EtkRab14AP3Rabenosyn5KIF3ABRab4AAP3 complexPODXL1Rab33ARab4ARab6ARETRap2FascinRab5Rab33AGPM6aRap2STEFYial2complexPAK1FOXK1HOXD9Rab7Recycling endosomal traffickingRegulation of endocytosis through its interaction with RUFY1RUFY1™s recruitment endosome tethering and fusionRegulates spatial distribution of lysosomeSorting endosome pathway in endosomal membrane inmelanocytes and segregates tyrosinaserelated protein1Increases cell proliferation migration and invasionEndosome dynamic Golgi complexassociated Rab33 andautophagosome formation on omegasomesLead to a fusion of the RET tyrosine kinase domain to a RUNdomain and a coiledcoil domain appear to be critical fortumorigenesisControl neuronal polarityControl the growth of axons and neuronal growth coneActs on endosomal traffickingFacilitates cell polarityInduce cell migration and invasion in gastric cancerIncreases cells migration RUFY3mediated with metastasis invasionin colorectal cancerHOXD9 transactivate RUFY3 and it overexpression induce gastriccancer progression proliferation and lung metastasisAutophagosome formation and lysosome clusteringMAP1LC3ABAutophagosome formation and elongationRab7Kinesin1Endosomal transport by acting with microtubule plus enddirectiontransportAllows translocation from the late endosome lysosome andautophagosome to the plasma membrane through plusendmicrotubule transportStudyCormont Yang Yamamoto Ivan Nag Zhi Fukuda Kitagishiand Matsuda Staubitz JanoueixLerosey Wei Yoshida Fukuda Honda 2017aWang Xie 2017aZhu Terawaki Cheng Olsvik Wang KrauŸ and Haucke Raib KIF3AB Rab4A and adaptor protein3 AP3 to diï¬erentiallyregulate tyrosinaserelated protein1 and tyrosinase sorting inendosomes contributing to melanosome maturation Nag Table Moreover silencing the Rabip4™isoform ofRUFY1 was shown to promote outgrowth of plasma membraneprotrusions and to regulate the spatial distribution of lysosomesat their tips through an interaction with AP3 Ivan Figures 1B 4B RUFY1 is also capable of controllingcell migration by regulating integrin trafficking Vukmirica presumably via endocytosis In full agreementwith a role of RUFY1 in regulating endosomal dynamics asingle nucleotide polymorphism S705A in the rufy1 gene wasassociated with high blood glucose levels and type diabetesmellitus susceptibility in an exomewide association studyEWAS Yamada This result is consistent with theearly finding that Rabip4 expression leads to Glucose transporter Glut1intracellular retention Cormont Interestingly RUFY1 display a SH3binding motif œPxxPxPembedded in the FYVE domain and is able to interactingwith the epithelial and endothelialtyrosine kinase ETKand possibly regulates endocytosis through this interactionYang Another EWAS aiming to find earlyonsetAlzheimer™s Disease AD susceptibility genes identified RUFY1among genes involved in endolysosomal transport and knownto be important for the development of AD Kunkle Figure 4CRUFY2lungRUFY2 or Leucine zipper FYVEfinger protein LZFYVE is a kDa protein originally identified as an activating transcriptionfactor2 interactor embryogenesis Dunkelberg and GutierrezHartmann preferentially located in the nucleus andexpressed during After development RUFY2 expression remainshigh in the brainliver and the gastrointestinal tractYang RUFY2 displays two Nterminalleucinezipper domains as well as a Cterminal FYVEfinger domainAlthough it is likely to have a nuclear function at early stagesof embryonic development the presence of a FYVE domainsuggests a cytoplasmic role for RUFY2 in regulating membranetraffic in fully diï¬erentiated cells Importantly the RUN domainof RUFY2 was shown to associate specifically with the Golgicomplexassociated Rab33A Fukuda Table GivenFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and Traffickingthe reported interaction of Rab33A and Rab33B with Atg16Land its putative role in regulating autophagy Fukuda and Itoh RUFY2 could contribute to autophagosome formationthrough a dual interaction with Rab33A and PtdIns3P onomegasomes Figures 1B 4B Irrespective of its function rufy2expression is subject to modulation by the micro RNA miR155BofillDe Ros which is an important regulator ofimmune cells development and inflammatory responses Ceppi The rufy2 gene is also frequently found mutatedin cancer cells with the most frequent mutations convertingit into a strong target for nonsense mediated mRNA decaythereby decreasing considerably its expression Shin Figure 4CRUFY3RUFY3 also known as Rap2interacting protein X RIPXKukimotoNiino or Single AxonRelated Singar1Mori is the best characterized member of theRUFY family RUFY3 the smallest of the RUFY proteins witha molecular weight of kDa A is mostly expressedin neurons Kitagishi and Matsuda Neuronal RUFY3 isatypical since it lacks a FYVE domain and is considered as partof the RUFY family based on strong sequence similarities withthe other members notably in the RUN and coiledcoil domainsFigure 2A RUFY3 is distributed between the cytosol and at theplasma membrane but not in intracellular vesicles presumablybecause it lacks a FYVE domain In artificial conditions likefollowing expression of the dominant gain of function mutantform of Rab5 Q79L in U937 cells RUFY3 was found associatedin large vesicle structures and to coimmunoprecipitate withRab5 via an interaction with its carboxyl terminal domain andsurprisingly not its RUN domain Yoshida LikeRUFY2 RUFY3 was also shown in a 2hybrid screen and bycoimmunoprecipitation to bind Rab33 through its coiledcoildomain CC1 Fukuda In 293T and 3Y1 celllines however RUFY3 was shown not to interact with severalsmall GTPasesincluding Rab2 Rab5 Rab7 Rho and RasThis suggests that either RUFY3 requires cell specific partnerproteins or posttranslation modifications to be able to bind tosmall GTPases RUFY3 was first described as interacting withRap2 a small Raslike GTPase via a residue fragment “ located in the RUN domain JanoueixLerosey KukimotoNiino Table Together with Rap1 Rap2interacts with Ras eï¬ectors such as Raf PI3K and Ral guaninenucleotide dissociation stimulator inhibiting activation of theirdownstream targets and thus suppressing Ras oncogenic activityKukimotoNiino Nussinov In the adultnervous system Rap1 and Rap2 also regulate the maturationand plasticity of dendritic spine and synapses By forminga complex together with Rap2 and Fascin RUFY3 interactswith the filamentous actin network and controls the growth ofaxons and neuronal growth cone Wei Table Recent mechanistic studies indicate that RUFY3 accumulates inlipid rafts by forming a Glycoprotein M6A GPM6aRUFY3Rap2STEFYial2 complex Honda 2017a Table This complex activates the Rac guanine nucleotide exchangefactor Honda 2017b impacting actin anization andpromoting neuronal polarity and growth Figure 4B RUFY3seems therefore to have diï¬erent axogenic functions in brainMori Honda 2017b and not surprisingly rolesfor RUFY3 in amyotrophic lateral sclerosis Arosio major depressive disorder Aberg and AD Zelaya have been reported Olfactory dysfunction occurs in of AD cases and is correlated with elevated rufy3 expressionin glomerular and mitral layers of the olfactory bulb Zelaya RUFY3 is cleaved by caspase and critically required forcaspasemediated degeneration of tropomyosin receptor kinaseA positive sensory axons in vitro and in vivo Hertz Figure 4C Removal of neuronally enriched RUFY3 is able toblock caspase 3dependent apoptosis while dephosphorylation ofRUFY3 at residue S34 appears required for its degradation Hertz Analysis of rufy3deficient mice supports a seconddistinct function for RUFY3 in neuronal growth and polaritysince mutant embryos show defects in axonal projection patternsThese occur in addition to the prevention of CASP3dependentapoptosis in dorsal root ganglions RUFY3 appears therefore to bekey for nervous system development remodeling and functionexplaining the embryonic lethality displayed upon rufy3 geneticinactivation in mouse Hertz With the current advance in genomics and single cell RNAsequencing specific gene expression patterns can be revised andmore accurately defined Analysis of several genomic databasesBioGPS NCBI Human Atlas Protein ImmGen Ensembl revealthat in addition to neurons RUFY3 expression can be detectedin other tissues and cell types Moreover the rufy3 gene appearsto have many transcriptional variants leading to the expressionof diï¬erent protein isoforms Two of these isoforms display aCterminal region extended by amino

Thyroid_Cancer

"Effects of lowdose computed tomography LDCT screening on lung cancercontains a that is not consistent with the data presented With reference to the National Lung ScreeningTrial NLST there are several flaws in the methodology overlooked Also there is no significant reduction in deathsfrom all causes following the screening Therefore any claim that the LDCT screening is superior to usual care isinvalidKeywords Lung cancer screening Low dose computed tomography MethodologyMain textYou recently published a paper by Huang KL entitled œEffects of lowdose computed tomography on lungcancer screening a systematic review metaanalysis andtrial sequential analysis [] In that paper the authorsstate in their Conclusion that œLDCT screening hasshown statistically significant mortality benefits in highquality trials In the they further state thatœLDCT screening is superiority over usual care in lungcancer survivalYet in the Section Benefits and adverse outcomesthey state On the contrary LDCT screening demonstrated no statistically significant difference in allcausemortality RR CI “The authors need to explain how a screening technique that produces no statistically significant differencein allcause mortality between LDCT screening andusual care can be superior to usual careThis comment refers to the available at 101186s128900190883xCorrespondence donbenjaminbigpondcomCancer Information Support Society Chandos St St Leonards NSW AustraliaThe authors also assess the risk for the NLST trial asincludingbeing Good Green on allMethodologycriteriaPotential flaws in methodologyIn fact the NLST trial had several methodological flawsrelated to the randomisation process overlooked by theauthors of the paper The NLST trial compared LDCT screening of highrisk smokers with Chest Xray CXR screening andassumed that Chest Xray screening produced thesame outcome as usual care [] as suggested in theProstate Lung Colorectal and Ovarian PLCOTrial [] despite earlier trials showing it resulted inan increase in allcause mortality [] Anticipating the shortcoming in above theauthors of the NLST trial ensured that the PLCOtrial had in addition to comparing average risksmokers selected high risk smokers who wereoffered Chest Xray screening for comparison withhigh risk smokers offered usual care to validatethe assumption referred to in Yet this selection ofhigh risk smokers was done after randomisation so The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBenjamin BMC Pulmonary Medicine Page of the comparison of deaths of high risk smokers afterChest Xray screening with deaths of those receiving usual care was invalid In addition the PLCOtrial published only lung cancer deaths for theNLSTeligible high risk smokers not deaths fromall causes This means the assumption in Point that Chest Xray screening of high risk smokersproduced the same outcome as usual care in termsof allcause mortality was invalidOther irregularitiesReich and Kim observed that the distribution of deathsover time from the NLSTeligible groups selected fromthe PLCO trial showed irregularities suggesting thatthere were some reporting errors in the PLCO trialThey also observed that there were no extra tumoursfound by the screening in the NLSTeligible groups selected from the PLCO trial [] casting further doubt onthis selection process suggesting another flaw in themethodology The PLCO trial identified less than more tumours by screening compared with about more in previous chest Xray trialsThe above potential flaws and irregularities suggestthat a ˜Red “˜ should be applied to the Randomizationprocess the Missing outcome data and the Overall riskrather than a ˜Green ™ On this basis a lower weightingshould be applied to the NLST trial for the purposes ofthe metaanalysisThe main shortcoming of the current metaanalysislike that of many other randomised controlled trialsRCTs is that the authors ignore the most importantoutcome Allcause Mortality and focus on the Deathsfrom Lung cancer If there is no reduction in overalldeaths following the screening it is not valid to claimthat LDCT screening is superior to usual careAs pointed out by Black WC Allcause Mortalityin Randomized Trials of Cancer Screening both trials ofChest XRay screening they reported on in showedan increase in allcause mortality following Chest XRayscreening that they attributed to the harm caused bypostscreening treatments of higher risk smokers Theypointed out that as œdiseasespecific mortality may missimportant harms or benefits of cancer screening because of misclassification in the cause of death this endpoint should only be interpreted in conjunction with allcause mortality In particular a reduction in diseasespecific mortality should not be cited as strong evidenceof efficacy when the allcause mortality is the same orhigher in the screened group []Other issuesThe NLST trial reported major complication rates following invasive procedures for the LDCT and CXRgroups The risk was higher among persons whounderwent LDCT compared with Chest Xray screening vs per screened The earlier CXR screening trials had shown an increase in deaths among thoseoffered screening compared to those not offered screening usual care This is strong evidence in support ofthe suggestion that some of the reduction in deaths fromlung cancer following LDCT screening could have beendue to deaths from other causes resulting from the treatment that as suggested by Black above shouldhave been classified as deaths from lung cancer Thereshould therefore be strong reservations made about anyclaim that the LDCT screening was superior to usualcareFrom the above one possible explanation for the apparently positive result claimed in the NLST trial is thatthe Chest Xray screening had in fact increased thenumber of deaths among those offered screening as hadbeen observed in previous trials [] the LDCT screeninghad reduced the number of deaths by a similar amountcompared to Chest Xray screening the net result beingthat there was no significant reduction in overall deathsas observed Some of the reduction in lung cancerdeaths could have been due to the methodological flawsoutlined aboveFinally the NLST trial is the only large trial to claimbenefits for cancer screening which would make lungcancer screening the only type of cancer screening toproduce significant benefits Randomised trials of breastbowel prostate and ovarian cancer screening have notproduced significant reductions in allcause mortality []and thyroid cancer screening has largely been discontinued due to much evidence suggesting no benefits butsignificant harm from overdiagnosis and overtreatmentAbbreviationsCXR Chest Xray LDCT Low dose computed tomography NLST NationalLung Screening Trial PLCO Prostate Lung Colorectal and Ovarian TrialRCT Randomised Controlled Trial RR Relative Risk CI Confidence IntervalAcknowledgementsNot applicableAuthor™s contributionsThe above letter is completely the work of the author DB The authors readand approved the final manuscriptAuthors™ informationDon Benjamin has previously published papers on the subject of evaluatingthe efficacy of cancer surgery and cancer screeningFundingThe research giving rise to the above letter is being funded by the author™semployer The Cancer Information Support Society Incorporated based ona recommendation from the Society™s Research Director the author Thisresearch is part of an ongoing fouryear project that has identified a flaw inclaims of benefits from lung cancer and other cancer screening The by Huang [] had supported the claim that LDCT lung cancer screeningproduces benefits contrary to the Society™s research findings The current letter commenting on this therefore uses data produced from the original research and funds for writing this letter come from the same project 0cBenjamin BMC Pulmonary Medicine Page of Availability of data and materialsNot applicableEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe author declares that he has no competing interestsReceived October Accepted July ReferencesHuang KL Effects of lowdose computed tomography on lung cancerscreening a systematic review metaanalysis and trial sequential analysisBMC Pulm Med National Lung Screening Trial Research Team Reduced lungcancermortality with lowdose computed tomographic screening N Engl J Med“ 101056NEJMoa1102873Oken MM for the PLCO Project Team Screening by chest radiographand lung cancer mortality The Prostate Lung Colorectal and OvarianPLCO Randomized Trial JAMA “ 101001jama20111591Black W Haggerstrom D Welch HG Allcause mortality in randomized trialsof cancer screening J Natl Cancer Inst “ Author™s responseto discussion June “Reich JM Kim JS The National Lung Screening Trial premise of null andchest radiography equivalence is to question Am J Roentgenol “Benjamin DJ The efficacy of surgical treatment of cancer “ years laterMed Hypotheses “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Protocol Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trialMarcelo Marreira Lidiane Rocha Mota Daniela F¡tima Teixeira Silva Christiane Pavani To cite Marreira a0M Rocha Mota a0L Silva a0DFT et a0al Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trial BMJ 202010e036684 101136bmj 2019036684 –º Prepublication history and additional material for this paper are available online To view these files please visit the journal online http dx bmj Received December Revised July Accepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJBiophotonics Applied to Health Sciences Universidade Nove de Julho Sao Paulo BrazilCorrespondence toDr Christiane Pavani chrispavani gmail comIntroduction The search for non invasive procedures to reduce localised adiposity in aesthetics clinics has recently been increasing In this context procedures such as cryolipolysis ultracavitation photobiomodulation PBM and other techniques have been proposed Some studies have shown that PBM can be used in body contouring However there is no standardisation of the protocol More than that as in other techniques for reducing adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences to the general health of an individual This work will aim to compare the light wavelengths when using PBM as a technique for reducing the abdominal waist circumference while also evaluating the efficacy of the method Changes in the lipid profile in the blood with a long term follow up will also be appraisedMethods and analysis This will be a controlled randomised double blind single centred clinical trial patients will be recruited at the Nove de Julho University Brazil and then divided into three groups Group A”RED PBM Group B”INFRARED PBM Group C”PLACEBO Sham treatment The treatments will consist of eight sessions two times a week for weeks At each session the participants will receive minutes PBM using a radiant exposure of Jcm2 with an abdominal strap containing LED clusters with devices each following the indication of randomisation All of the groups will receive min of Aussie Current at kHz modulated at Hz “ mA The main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness changes in the local microcirculation and the quality of life and self esteem The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Ethics and dissemination The Ethics Committee of the Nove de Julho University Brazil approved the modified version of this project under No on June This study is not yet recruiting The results obtained Strengths and limitations of this study –º The use of the same dosimetry at different wavelengths will allow for a real comparison between red and infrared as being the most suitable wavelength for body contouring –º Analyses of body contouring will be performed by non invasive methods –º The waist circumference measurement will not discriminate the factors underlying the volume modifications –º The habits of the participants such as diet and exercise routines may affect the results –º Gender may affect the results and be dependent on the number of participants of each gender These differences may not be considered by the statistical analysiswill be published in a peer reviewed journal in the related fieldTrial registration number Brazilian Registry of Clinical Trials”ReBec RBR 9bwxcxINTRODUCTIONFat storage is intended to protect the human body in cases of prolonged fasting intense physical activity and temperature regulation Once freed from these situations fat is stored unnecessarily putting the individual at the risk of health problems together with a greater pr sity for pathologies such as systemic arterial hypertension diabetes mellitus metabolic syndrome and even some types of neoplasms1“Another type of negative impact that is related to excessive fat storage is body dissatisfaction This naturally leads to a decrease in the individual™s self esteem4 Studies have shown that aesthetic treatments significantly increase a patient™s quality of life They Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access are associated with improved self esteem6“ There are some traditional surgical methods for the reduction of abdominal adiposity However the methods are invasive techniques which may present a high number of complications such as bruising seroma pain perforated ans and viscera as well as with an increased risk of deep vein thrombosis10“ The demand for minimally invasive procedures that are aimed at reducing abdominal fat has increased by about while the demand for surgical procedures has decreased by around Among the minimally invasive techniques one can mention low level laser therapy which has recently also been called photobiomodulation PBM PBM has many novel advantages when compared with traditional techniques such as surgical procedures since it can guarantee the preservation of the noble adjacent structures such as the nerves the blood vessels and the skin15 PBM has been widely studied for several applications due to its important biochemical cellular consequences and its few side effects16 Some manuscripts have described erythema and oedema as the main side effects of PBM but importantly these side effects may have been higher as a result of the patient using any drug that increased photosensitivitySome other studies have shown that PBM can be used in body contouring18“ Sadly there is no standardisation of the protocol The treatments vary in terms of the number of sessions “ their frequency “ times per week and wavelength nm nm nm nm while other dosimetry information such as irradiance Wcm2 and radiant exposure Jcm2 are frequently not mentioned Recently Croghan and coworkers showed that two times a week was the best frequency when compared with one or three times a week This was in terms of improving the patients™ quality of life and body satisfaction as well as their weight waist circumference body mass index BMI and body fat mass reduction18 However more studies are needed in order to standardise the wavelength the dosimetry and the application time as well as the durability of the results achievedOne of the proposed mechanisms for a PBM effect in adipose tissue is the formation of transient pores in the adipocyte membranes thus allowing for the lipids to escape15 Adipocyte apoptosis activation has also been proposed The production of reactive oxygen species is also possible due to the action of PBM and this is related to the mitochondrial activation on account of the radiation absorption by the cytochrome c oxidase molecules This is followed by an increased ATP synthesis and with an increased cyclic adenosine monophosphate messenger which can trigger the activation of the lipases that perform the hydrolysis of the triglycerides into fatty acids and glycerol23Some reports have affirmed that the results obtained by the use of PBM for reducing waist circumference are modest and that the reduction is temporary which deserves much greater attention from researchers for a better understanding of this factor These effects may be associated with the mechanisms of action and the dosimetric parameters being used24 When taken to the tissues the free fatty acids are used as an energy source during beta oxidation for the production of ATP In some literature reports PBM is associated with aerobic or resistance exercise while other reports have mentioned waist and arm circumference reductions with the use of PBM displaying an absence of diet restrictions or exercise requirements25“ It is also reported that the amount of fat mobilised during a PBM session is similar to the amount that is consumed during a meal in such a way that it can be absorbed by normal body energy requirements andor exercise routine while at the same time the risk of atherosclerosis is not increased by the treatment30 On the other hand if not consumed these fatty acids may be re esterified and redistributed throughout the body30 causing no final changes in waist circumference Since neuromuscular electrical stimulation increases energy expenditure in a similar way to that associated with exercise the protocol will be complemented with the Aussie current application31As for other techniques for reducing the adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences for the general health of the individual Some studies have shown that these important treatments may affect the serum lipid levels while others affirm that there are no changes in the serum lipid levels32 Given these scenarios this work will aim to compare the different light wavelengths when using PBM as a technique for the reduction of abdominal waist circumference while at the same time evaluating the efficacy of the method and by following the changes in the lipid profile in the blood as well as with reviewing the long term follow upMETHODSStudy designThis will be a controlled randomised double blind single centred clinical trial designed in accordance with the criteria as established by the Standard Protocol Items Recommendations for Interventional Trials It will be conducted at the Nove de Julho University located in the city of S£o Paulo Brazil The recruitment will be performed from September to November through the university website Thus the selection of sites includes urban locations the city of S£o Paulo and its neighbourhoods After verbal and written explanations regarding the procedures the risks and the benefits by MM a coauthor of this protocol those individuals who agree to participate in the study will sign an informed consent form Based on an anamnesis questionnaire the researchers will check if the participants meet the inclusionexclusion criteria The anamnesis questionnaire will include identification data anthropometric data clinical history and daily living habits especially dietary intake physical activity assessments and menstrual period appraisals Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cSince dietary intake and physical activity may have direct effects on the results at each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measured The enrolment period will be extended until the sample size is reachedPatient and public involvement statementThe patients andor the public will not be involved in the design the recruitment or in the conduct of the studyInclusion criteriaThis study will include men and women aged “ years with a BMI of between and kgm2 normotrophic and overweight together with hyperplasia of the abdominal fat tissue abdominal skin folds higher than mm Those who agree to participate in this research will sign an informed consent form see online supplementary file Exclusion criteriaThe following people will be excluded from this survey those participants who are undergoing aesthetic treatments to reduce waist circumference those who have been previously submitted to abdominoplasty or liposuction surgeries those who are on a diet in order to reduce their measurements those people who engage in a physical activity more than two times a week those who are using or have taken drugs or food supplements in last days in order to reduce their measurements and their weight which may affect their lipid metabolism appetite or nutrients absorption those who have been submitted previously to oophorectomy those with signs andor symptoms of climacteric at the menopause pregnant or lactating women those participants who are not regular in attending the sessions those participants who present metabolic dysfunctions diabetes and thyroid disorders cardiovascular problems hypertension cardiac insufficiency arrhythmia thrombosis pacemaker use respiratory issues asthma chronic obstructive pulmonary disease haematological disturbances anaemia renal non alcoholic fatty liver disease dermatological or digestive disorders gastritis ulcers those with a history of oncological pathology those with cognitive deficitsSample calculationIn order to calculate the sample size a study showing the therapeutical effects of PBM when associated with aerobic plus resistance training was used26 The researchers used the highest and the lowest abdominal circumference values as well as the SD of the measurements The highest and lowest abdominal circumference values for the PBM group were and respectively and the highest SD of the measurements was with being the number of intervention groups in the study The effect size hence was calculated when using these values as described belowˆ† biggerˆ’smaller σˆšn2 ˆ’ ˆš accessWhen using the effect size value as calculated above the sample size was calculated using GPower software V3192 Dusseldorf Germany Two way Analysis of Variance ANOVA was used for the interactions within and between the groups in order to evaluate the differences between the three groups studied as well as for the five evaluations during the treatments and the follow up The test power was α005 The sample size was calculated on participantsRandomisationThe randomisation will be performed by DFTS a coauthor of this work who is not directly linked to the treatments or the evaluation of the participants by using the Excel program Microsoft USA The participants will be randomised into blocks of and into groups designated as A B and C Opaque envelopes will be identified by sequence numbers and they will receive a paper containing the information about which treatment will be performed on the participant™s abdomen according to the draw The sealed envelopes will be safely kept with the researcher who generated the randomisation DFTS Before the beginning of the procedures the researcher responsible for the procedure LRM a coauthor of this protocol will receive each envelope and proceed with the treatment as indicated according to its allocation group A team of undergraduate students previously trained and prepared is going to be part of the research group and for the treatment or evaluation of the participants This study will be a double blind study since the participants will not be aware of the group in which heshe is participating only the researcher who will perform the procedure will know The data collection and analyses will be performed by another researcher MM a coauthor of this study who will also be unaware of the allocationsInterventionThe abdomen of the participants will be cleaned by using a neutral cleansing soap They will receive eye protection using goggles for safety This will also help with the blindness of the study PBM will be applied when using abdominal straps as developed by Cosmedical Mau¡ S£o Paulo Brazil following the parameters as described in table The abdomen strap will be covered with a sheet and that will also help with the blindness of the study All of the participants will receive min of PBM with an abdominal strap containing LED clusters with devices following the indication of the randomisation being per group Group A”RED ± nm Group B”INFRARED ± nm Group C”PLACEBO The treatment will consist of eight sessions that will occur two times a week totalling month of treatments The placebo group will use a strap with no light emission but it will emit the same sounds like that of the active device In order to increase the oxidation of the free fatty acids the participants will receive min of Aussie Current at kHz modulated at Hz “ mA for min Tensor Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access Table Dosimetry for the studyParameterRed LEDContinuousCentre wavelength nmSpectral width nmOperating modeAverage radiant power”one LED mWAperture diameter”one LED cmPower density at aperture mWcm2Beam spot size at target”one LED cm2Total number of LEDsArea irradiated cm2Irradiance at target mWcm2 Exposure duration sRadiant exposure Jcm2Energy density at aperture Jcm2Radiant energy kJApplication techniqueNumber and frequency of treatment sessionsContact—week for a month a total of sessionsInfrared LEDContinuousContact—week for a montha total of sessionsDGM Electronica Santo Andr© S£o Paulo Brazil33 None of the PBM devices will significantly increase the temperature at the target area causing no burns or skin damage No modifications in the intervention will be performed for any reason However the participants who withdraw their consent or the ones who are not assiduous to the sessions will be removed from the studyThe dosimetric parameters that will be used in this protocol as presented in table were measured andor calculated The centre wavelength and the spectral width of the devices were measured by a Spectrophotometer USB2000XR1 Ocean Optics Florida USA The radiant power of one LED was measured by a Power Meter FieldMaxII TO Coherent Santa Clara California USA The abdominal straps will be composed of LED clusters having LEDs each totalling LEDs units and distributed in a cm— cm area cm2 each cm2 total see figure The effective irradiated area will be cm2 times the beam spot size at the target The irradiance at the target was determined by the ratio between the average radiant power mW and the beam spot site at the target cm2 The radiant exposure was determined by multiplying the irradiance at the target mWcm2 by the exposure duration s The radiant energy was calculated by multiplying the average radiant power of one LED mW by the total number of LEDs and by the exposure duration sFigure Photobiomodulation PBM application PBM device off A and on B patient receiving the experimental protocol in dorsal decubitus min per session C and DOutcomesThe main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness as measured by ultrasound changes in the local microcirculation quality of life and self esteem The participants will be evaluated at the same time of the day at all times throughout the studyThe anthropometric data that will be collected will be body weight height and BMI skin fold thickness and bioimpedance Blood will be collected for analyses of the lipid profile total cholesterol high density lipoprotein HDL Low density lipoprotein LDL triglycerides and liver function serum glutamic oxaloacetic transaminase SGOT serum glutamic pyruvic transaminase SGPT All of this will be processed and analysed at SCS Medicina Diagn³stica S£o Caetano do Sul Brazil a partner laboratory The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Abdominal ultrasound will be performed to assess the fat layer thickness before and after the treatments For the recording of the local temperature a technique that is widely used is infrared thermography using a Compact Thermal Camera C2 FLIR Systems Oregon USA The thermal camera by means of infrared emission from the body or from the material analysed has the ability to calculate the temperature of a given surface It is possible through this method that the study will infer the changes in local microcirculation34The quality of life questionnaire ˜The WHO Quality of Life WHOQOL BREF™ as well as the Body Shape Questionnaire ˜BSQ34 Self Image Scale™ will be used for the participants These questionnaires have been translated and submitted to cross cultural adaption into Brazilian Portuguese37 The Brazilian Portuguese version of these questionnaires will be applied by MM The questionnaires will take around min to be completed The quality of life and the self image questionnaires will be applied at D0”and again at the end of the last session D30 The flow chart of the study is presented in figure Adverse events will be collected during the treatment sessions and they will be reported to the regulatory agency and again Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cRecruitment recruitmen t accessEvaluated for eligibility Elected patients n174 Anamnesis application of the quality of life and selfesteem questionnaire anthropometry blood collection ultrasonography Randomised n Allocation Group A LED with devices ± nm with Wcm2 Group B LED with devices ± nm with Wcm2 Group C Sham Each session minutes being measured at the end of each session waist circumference and IRT Total Sessions Analysis at the end of treatment Application of the quality of life and selfesteem questionnaire anthropometry blood collection IRT ultrasonography FollowUp days Anthropometry and blood collection days Anthropometry and blood collection days Anthropometry blood collection ultrasonography Figure Flow chart describing the study design the sample composition and the experimental protocol IRT infrared thermographyat the final publication of the results Since the participants are enrolled and randomised the investigators will make efforts to keep the participants together during the follow up by making phone calls emailWhatsApp contact with the patients and with relevant instructions regarding healthcare and beautyAs a strategy to improve adherence at each session the participant will schedule the next visit and receive a card with some instructions regarding preparation for the evaluation day and the appointment date of the next visit When a participant misses a session heshe will receive a phone call in order to reschedule the missed sessionData analysis planThe data that will be collected from this study will only be administered by the principal investigators the authors of this document Since the study will be of short duration and with known minimal risks this trial will not need a formal data monitoring committee After the data collection the data will be anised using Microsoft Office Excel by DFTS coauthor of this protocol and then stored on ˜a protected by password™ computer at the university The data will be analysed by descriptive and inferential statistics and then compiled into tables andor graphs using SPSS V240 For testing the normality of the data the Shapiro Wilk test will be performed If the data show a non parametric behaviour a mathematical function will be used in order to normalise the data Two way ANOVA tests followed by the Bonferroni post test will be performed in order to compare the treatments along with the time points being evaluated Some parameters Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access may affect the results of the therapy and they are going to be analysed as co variables for example skin phototype by the Fitzpatrick scale the stage of the menstrual cycle total cholesterol and triglycerides altered or not α005 will be considered the level of significance for all of the tests used Since this trial is part of a PhD thesis study an interim analysis will be performed for the qualification examination and this can be used at trial adaptations such as for a sample size re estimation or for stopping the trial The trial protocol and the full study report will be fully available at the end of the study after the manuscript of the results has been published At the end of the study the participants from the placebo group who received the treatment will experience no adverse effects and they will have received the most effective treatmentDISCUSSIONStudies have shown that lasers used in PBM typically operate at powers of mW or less They can produce energy in the visible spectrum wavelengths “ nm and near the infrared regions “ nm Light penetration in the soft tissues is known to be directly related to wavelength that is the longer the wavelength the greater the penetration The reports on PBM for reducing local adiposity include the use of green nm red and nm and infrared and nm However there is no comparison available regarding the best wavelength for this purpose18“ Based on the localisation of fat tissue more profound when compared with epidermis and dermis this study will choose red and infrared light for the comparisons The use of the same dosimetry at these different wavelengths will allow for the evaluation of the most suitable wavelength for body contouringSince the waist circumference measurements will not discriminate against the factors underlying the volume modifications a placebo group will be included This will allow for the measurement of the differences in waist circumference due to daily habits or hormonal variations as in the menstrual cycle of women The measurements of the skin folds and bioimpedance will complement the evaluation in terms of body fat At each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measuredGender may also affect the results Sexual dimorphism in adipogenesis is already known as well as sex hormones in the white adipose tissue function and in adipose metabolism39“ If the sample consists of a huge difference in men and women this factor cannot be considered in the statistical analysisThe development of a non invasive protocol for PBM together with an Aussie current for the reduction of adiposity may present an important novel tool for the reduction of health risk problems as well as for increasing an individual™s self esteemETHICS AND DISSEMINATIONThe Ethics Committee of the Nove de Julho University S£o Paulo Brazil approved the modified version of this project and the Patient Informed Consent Form under No on June according to the guidelines of the Brazilian National Ethics Committee The protocol of this study has already been registered in the Brazilian Registry of Clinical Trials being first registered on November and modified on August providing full public access to the protocol information including all items from the WHO Trial Registration Data Set MM and DFTS will be the data curators with the data stored on ˜a protected by password™ computer at the university The results acquired within this project will be presented in conferences and published in a journal in the related field The authorship of the results paper and the conference abstracts will include the authors of this protocol together with other researchers who may contribute to the procedures or to the analysis of the data Any modifications of this protocol will require a formal amendment and they will be approved by the Ethics Committee of the Nove de Julho University The modifications will be properly reported and justified in the manuscript for the publication of the results The main results obtained will be sent to the participants by mailAcknowledgements The authors would like to thank the Nove de Julho University UNINOVE S£o Paulo Brazil for the availability of its laboratories the company Cosmedical for the development of the equipment for PBM and the SCS Medicina Diagn³stica Laboratory S£o Caetano do Sul Brazil for their partnership in the analyses of the laboratory testsContributors MM LR M DFTS and CP designed the study MM and LR M will conduct the experiments and will be making the data acquisitions DFTS and CP will perform data analysis and interpretation MM and LR M drafted the work while CP and DFTS revised it critically for important intellectual content All of the authors approved the final version of the manuscriptFunding The authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorsCompeting interests None declaredPatient consent for publication ObtainedProvenance and peer review Not commissioned externally peer reviewed access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See a0http creativecommons licenses by nc ORCID iDsDaniela F¡tima Teixeira a0Silva http orcid Christiane a0Pavani http orcid REFERENCES Silva Figueiredo P Carla Inada A Marcelino G et a0al Fatty acids consumption the role metabolic aspects involved in obesity and its associated disorders Nutrients “ Landecho MF Tuero C Valent­ V et a0al Relevance of leptin and other adipokines in obesity associated cardiovascular risk Nutrients “ Kong Y Zhang S Wu R et a0al New insights into different adipokines in linking the pathophysiology of obesity and psoriasis Lipids Health Dis “ Jim©nez Flores P Jim©nez Cruz A Bacardi Gasc³n M Body image dissatisfaction in children and adolescents a systematic review Nutr Hosp “ Weinberger N A Kersting A Riedel Heller SG et a0al Body Dissatisfaction in individuals with obesity compared to normal weight Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cindividuals a systematic review and meta analysis Obes Facts “ Kouris A Platsidaki E Christodoulou C et a0al Patients™ self esteem before and after chemical peeling procedure J Cosmet Laser Ther “ Stundzaite Barsauskiene G Tutkuviene J Barkus A et a0al Facial perception self esteem and psychosocial well being in patients after nasal surgery due to trauma cancer and aesthetic needs cluster analysis of multiple interrelations Ann Hum Biol “ Ribeiro F Steiner D Quality of life before and after cosmetic procedures on the face a cross sectional study in a public service J Cosmet Dermatol “ Bensoussan J C Bolton MA Pi S et a0al Quality of life before and after cosmetic surgery CNS Spectr “ Appleton SE Ngan A Kent B et a0al Risk factors influencing transfusion rates in DIEP flap breast reconstruction Plast Reconstr Surg “ Lievain L Aktouf A Auquit Auckbur I et a0al [Abdominoplasty complications particularities of the post bariatric patients within a patients series] Ann Chir Plast Esthet “ Sterodimas A Boriani F Nicaretta B et a0al Revision Abdominoplasty with truncal Liposculpting a year experience Aesthetic Plast Surg “ Al Dujaili Z Karcher C Henry M et a0al Fat reduction complications and management J Am Acad Dermatol “ Krueger N Mai SV Luebberding S et a0al Cryolipolysis for noninvasive body contouring clinical efficacy and patient satisfaction Clin Cosmet Investig Dermatol “ Neira R Arroyave J Ramirez H et a0al Fat liquefaction effect of low level laser energy on adipose tissue Plast Reconstr Surg “ Karu T Mitochondrial mechanisms of photobiomodulation in context of new data about multiple roles of ATP Photomed Laser Surg “ Feng J Zhang Y Xing D Low power laser irradiation LPLI promotes VEGF expression and vascular endothelial cell proliferation through the activation of ERKSp1 pathway Cell Signal “ Croghan IT Hurt RT Schroeder DR et a0al Low level laser therapy for weight reduction a randomized pilot study Lasers Med Sci “ Thornfeldt CR Thaxton PM Horn

Thyroid_Cancer

"The coronavirus disease COVID19 is now a worldwide challenge for public health Among million patients about present mild to moderate disease but studies dedicate to these patients are actually scarce The aim of our study is to clarify the characteristics of laboratory test index of COVID19 patient with moderate symptoms during the first wave of the pandemic in Wuhan ChinaMethods In this retrospective cohort study we included adult inpatients with confirmed moderate disease of COVID19 from the Affiliated Hospital of Jianghan University during February and early March All of these patients were recovered from COVID19 and discharged from hospital Demographic clinical and laboratory data of admission and discharge were extracted from electronic medical records and analyzed using SPSS as well as among young middle age and elderly peopleResults The median age of this cohort of patients was years And the median hospitalization time was days Common clinical manifestations included fever cough asthenia and shortness of breath On admission laboratory results showed normal or increased neutrophil ratio low lymphocyte count decreased hemoglobin level and increased inflammatory indicators erythrocyte sedimentation rate ESR and Creactive protein CRP and some patients were complicated with coagulation disorder and myocardial damage Furthermore patients older than years had statistically higher CRP ESR and fibrinogen level As the health condition was improved at discharge the median level of most laboratory results were in the normal range except hemoglobin and related blood cell count as well as Manuscript submitted July accepted July Published online August aWuhan Institute of Biomedical Sciences School of Medicine Jianghan University Wuhan ChinabThe Affiliated Hospital of Jianghan University Wuhan ChinacThe two authors contributed equallydCorresponding Author Binlian Sun Wuhan Institute of Biomedical Sciences School of Medicine Jianghan University Wuhan China Email binlian17jhuneducn 1014740jocmr4293inflammatory indicator ESR And patients older than years showed slower recovery on coagulation parameters when compared to younger patientsConclusions The severe acute respiratory syndrome coronavirus SARSCoV2 infection induces a controllable inflammatory response in moderate disease of COVID19 in Wuhan China Since patients older than years had higher inflammatory state and more dysregulated coagulation condition it might be essential to closely assess their illnessKeywords COVID19 Moderate disease Clinical feature Laboratory findings InflammatoryIntroductionThe novel severe acute respiratory syndrome coronavirus SARSCoV2 was first identified in Wuhan China in December [ ] and now spread all over the world Its infection in human mainly appears as acute respiratory syndrome sometimes along with digestive and nervous disorders [ ] The outbreak of this coronavirus disease COVID19 was declared a pandemic by the World Health anization WHO on March Globally as of June there have been confirmed cases including deaths []A large cohort study showed the spectrum of COVID19 that among patients had mild symptom had severe disease and were in critical condition [] Naturally most studies about COVID19 focus on the severe and critical cases although more than of COVID19 patients show mild to moderate symptoms It should be equally important to understand the average degree of SARSCoV2 infection especially if this virus would become another endemic virus in communities like the influenza virusOur study describes patients that were admitted in a designated hospital in Wuhan the Affiliated Hospital of Jianghan University the sixth hospital of Wuhan city during February and early March These patients showed moderate symptom of COVID19 and were confirmed both by s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmrThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cLiu et alJ Clin Med Res SARSCoV2 RNA test and chest radiography And they were all discharged from hospital after recovery The aim of our study is to clarify the characteristics of laboratory test index of COVID19 patient with moderate symptoms during the first wave of the pandemic in Wuhan These findings may help us extend our understanding of the pathogenicity in SARSCoV2 infectionMaterials and MethodsPatientsThis retrospective cohort study included adult patients ‰¥ years old with confirmed COVID19 admitted to the Affiliated Hospital of Jianghan University the sixth hospital of Wuhan city in Wuhan from February to March According to WHO interim guidance [] patients with moderate disease of COVID19 on admission were enrolled in this study they showed clinical signs of pneumonia fever cough dyspnea fast breathing but no signs of severe pneumonia including oxygen saturation SpO2 ‰¥ on room air All the patients were confirmed by SARSCoV2 RNA test in respiratory secretions for twice as well as by groundglass opacities or bilateral pulmonary infiltration showed in chest computed tomography CT scan During hospitalization patients were kept in regular wards without intensive cares or invasive mechanical ventilation They received supportive therapy effective oxygen therapy antiviral agent and if necessary antibiotics Patients were discharged from hospital when the following criteria were met body temperature normal for more than days respiratory symptoms significantly improved pulmonary imaging significantly improved on CT scan and SARSCoV2 RNA tests showed negative for twice This study was approved by the Ethics Committee of School of Medicine of Jianghan University Wuhan China This study was conducted in compliance with the ethical standards of the responsible institution on human subjects as well as with the Helsinki DeclarationData collectionsThe laboratory tests including blood routine biochemistry coagulation parameters and cardiac injury biomarkers were performed in patients on admission and during the hospitalization The demographic and clinical information laboratory results and outcome data were finally collected from electronic medical recordsStatistical analysisTable Demographics and Clinical Characteristics of Patients on Admission N Female MaleAge median IQR yearSex Hospitalization days median IQR dayMedical history Hypertension Diabetes Heart disease Cancer Other respiratory disease Kidney disease Liver disease Neurological disease Thyroid disease OthersSymptoms Fever Cough Sputum production Nose obstruction rhinorrhea Shortness of breath Headache Chest pain Myalgia Asthenia Vomiting DiarrheaSigns Respiratory rate median IQR bpm Heart rate median IQR bpm Systolic pressure 140mm Hg SpO2 ‰ Data are median IQR n or nN IQR interquartile range SpO2 oxygen saturationsion IBMResultsContinuous variables were expressed as median interquartile range IQR and compared with the oneway analysis of variance ANOVA test between different age groups A twosided α of less than was considered statistically significant Statistical analyses were done using SPSS verPatient demographics and characteristicsA total of patients with moderate disease of COVID19 were recruited The demographic and clinical characteristics s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res Table Summary of Laboratory Findings on Admission and at DischargeResults on admissionResults at dischargeLaboratory indexNormal rangeNeutrophil count — 109L Median IQR Patient †‘†“†‘†“†“†“ †“†“†‘†“†‘†‘†‘†‘†‘†‘†‘†‘†‘Median IQR NA Patient †‘†“†‘†“†“†“†“†“†‘†“†‘†‘ †‘†‘†‘†‘NA†‘†‘ Female MaleNeutrophil ratio Lymphocyte count — 109LLymphocyte ratio Red blood cell count — 1012L Hemoglobin gL Hematocrit Platelet count — 109L Female Male Female MalehsCRP mgLESR mmhDdimer mgLFibrinogen gLFDP mgLNTproBNPa pgmL years years years hscTnL ngmLCK ULLDH ULData are median IQR or nN aThe normal level of NTproBNP is increased with age Three normal ranges for different age groups are listed and results are presented accordingly †‘For increased blood level †“For decreased blood level hsCRP high sensitive Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation product NTproBNP Nterminal prohormone brain natriuretic peptide hscTnL high sensitive cardiac troponin L CK creatine kinase LDH lactose dehydrogenase IQR interquartile range NA not applicableof these patients are shown in Table Of these patients females and males the median age at disease onset was years range years with patients older than years Most patients had fever and cough as their first symptoms some also had asthenia shortness of breath sputum production and diarrhea As for the underlying diseases hypertension heart disease and diabetes were the most common in medical histories of these patients On admission the vital signs of patients were also recorded Notably although the SpO2 of all the patients were ‰¥ at room air of patients were ‰ Patients received symptomatic and pneumonia treatments in hospital and the median of their hospitalization were days IQR Whole blood cell counting findingsThe whole blood cell counting was monitored for all the patients on admission and during their hospitalization median values showed in Table The white blood cell counts were generally in the normal range whereas of patients of patients showed lymphopenia lymphocyte count — 109L Table Among patients showed lymphopenia of had a decrease by less than lymphocyte count s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cLiu et alJ Clin Med Res Figure Illustration of percentage changes of laboratory index according to number of patients Data are presented as number of patients that had changed level in laboratory assessments a Decreased lymphocyte count on admission and at discharge b Decreased hemoglobin level on admission and at discharge c Increased CRP level on admission and at discharge d Increased ESR level on admission and at discharge e Increased fibrinogen level on admission and at discharge f Increased Ddimer level on admission and at discharge g Increased FDP level on admission and at discharge In for the data collected on admission out for data collected at discharge CRP Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation products The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res Table Statistically Significant Biomarkers on Admission and at Discharge With Different AgesCRP inESR inESR outFibrinogen inFDP outD dimer outCK n n n 944a 1073a 983a 53b 41a 082a 798bData are expressed with the median IQR P values comparing different age groups are from oneway ANOVA aP for group and group vs group has statistical difference bP for group vs group has statistical difference In for the data collected on admission out for data collected at discharge CRP Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation product CK creatine kinase IQR interquartile range — 109L Fig 1a while five of of them showed a decrease by more than lymphocyte count — 109L Fig 1a Twentythree percent of of patients Table still had lymphopenia when they were discharged from hospital and the decrease was limited for most Fig 1a The change of neutrophil count affects fewer patients on admission of nine of patients showed neutrophilia neutrophil count — 109L Table and of patients had neutropenia neutrophil count — 109L Table However neutrophil ratio was augmented in of of patients neutrophil ratio Table and the maximum increase was about This ratio appeared to be in the normal range for most patients at discharge Therefore the main change in white blood cell counts for patients with moderate COVID19 was the decrease of lymphocyte And as the health condition improved at discharge lymphocyte count also gradually increased to normal levelThe data showed that more than of all patients had declined level of red blood cell count of hemoglobin of and hematocrit of Table and four female patients were in severe condition hemoglobin gL Fig 1b When discharged from hospital of of patients still had these decreases Table The four female patients mentioned above had their hemoglobin increased to over gL However one female of years had less than gL hemoglobin and that was less than earlier result Furthermore there were five more male patients had hemoglobin declined at discharge Fig 1b These results suggested that SARSCoV2 infection may be accompanied by oxygen transport defect in red blood cellsAs for the platelet counting on admission of patients seven of had decreased platelet level platelet count — 109L Table and of had increased level platelet count — 109L Table At discharge most patients showed platelet level in the normal range Table Inflammatory biomarkersInflammatory biomarkers were also examined on admission and during hospitalization such as erythrocytes sedimentation rate ESR Creactive protein CRP and procalcitonin PCT For most patients PCT levels were in the normal range data not shown For CRP the median value was mgL IQR among patients on admission Table and patients older than years had significantly higher level Table To be specific CRP levels in serum were increased CRP mgL Table in of patients of and of showed an increase by more than CRP mgL Fig 1c At discharge only of Table had elevated level of CRP and the increased level did not exceed mgL except for one patient Fig 1cLevels of ESR were increased in of patients of ESR mmh Table and of Fig 1d had an increase exceeding mmh Similar to CRP patients older than years had statistically higher ESR level Table The median value of ESR on admission was mmh IQR while the value improved to mmh IQR at discharge Table There were still patients had ESR level more than mmh in serum Fig 1dCoagulation parametersThe coagulation parameters were examined on admission for patients Eighty percent of patients of Table had normal serum levels of Ddimer on admission Among the of patients of who had increased Ddimer level Ddimer mgL Table eight showed an increase by more than Ddimer 2mgL Fig 1f At discharge of patients seven of still had abnormal level of Ddimer though the results were very close to the normal range The fibrinogen level on the other hand had increased in of patients of fibrinogen mgL Table and decreased in of fibrinogen 2mgL Specifically patients of had an increase of fibrinogen level by more than fibrinogen mgL Fig 1e while most decreases were slight Furthermore when compared to patients younger than years fibrinogen level on admission was significantly higher in the patients older than years Table At discharge only seven patients still showed high level of fibrinogen Fig 1e Fibrinogen degradation product FDP was also s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cLiu et alJ Clin Med Res cardial damage Since the outcome of this cohort is known as recovery and discharge from hospital we also collected the last available laboratory results Although all the patients met the discharge criteria for COVID19 some still had abnormal laboratory findings Especially we found the levels of the inflammatory biomarker ESR were still elevated for half of the patients tested Hence patients were asked to go back to the hospital for followup examination including the virus RNA test chest CT scan blood routine coagulation function and other biochemical indicators on both the 14th and 28th day after dischargePrevious studies have found that white blood cell WBC procalcitonin PCT aspartate aminotransferase AST lactose dehydrogenase LDH Cr hscTnL and Ddimer could indicate the progression of COVID19 on admission especially for the severe and mortal cases [ ] And in the moderate condition we analyzed most patients had these indicators in the normal range Therefore our findings are consistent with other studiesHowever these biomarkers were not all specific to COVID19 Similar to other virus infections including SARS [] and H1N1 influenza [] the neutrophil count was mostly normal in mild to moderate patients while lymphocyte count was significantly decreased As the major antiviral cells lymphocyte count declined down to — 109L in this cohort on admission And after receiving antiviral and supportive treatment the lymphocyte counts increased among all the patients at discharge Previous study found that lymphocyte counts would continually decrease in severe and critical COVID19 patients [] These results suggested that SARSCoV2 infection can suppress the level of lymphocyte Therefore closely monitoring lymphocyte counts could be one of the best methods when we evaluate the outcome of moderate patientsThe blood routine results also showed that about of patients had low level of hemoglobin red blood cell and hematocrit and the condition was not improved at discharge Previous studies also reported that of patients had hemoglobin below the normal range [] and the hemoglobin level of severe patients was lower although the difference was not statistically significant [] There might be two explanations First the inflammatory state caused by infection may interfere with erythrocytebone marrow metabolism and iron regulation [] and eventually result in a decline of hemoglobin and red blood cell Second patients could suffer from anemia for some time and appear more vulnerable to SARSCoV2 infection Once infected however correction of anemia could not benefit from all the symptoms of COVID19 and psychological stresses The decrease of functioning hemoglobin may contribute to hypoxia and further aggravate the disease in severe casesexamined and of patients had elevated level of FDP mgL Table Fig 1g on admission Twentyseven percent of patients of still showed increased amount of FDP at discharge Table Therefore fibrinogen and FDP were the most altered parameters in coagulation function from our analysisCardiac injury biomarkersCardiac injury biomarkers were monitored for patients on admission Among the patients assessed all had normal serum levels of high sensitive cardiac troponin L hscTnL ngmL The other cardiac biomarker examined was Nterminal prohormone brain natriuretic peptide NTproBNP and of patients of had elevated level NTproBNP and pgmL according to age Table on admission and five of had an increase by more than At discharge the NTproBNP level had decreased into the normal range except two male patients These two patients still had high level of NTproBNP more than pgmL however other cardiac biomarkers such as hscTnL and creatine kinaseMB CKMB were both in the normal range Considering their history of hypertension they were asked to reevaluate their heart function after the COVID19DiscussionAs an emerging infectious disease COVID19 is now a worldwide challenge for public health Among the million patients about present mild to moderate disease but studies dedicate to these patients are actually scarce Therefore we enrolled adult patients with moderate disease of COVID19 to elucidate the clinical manifestation and laboratory findingsOne of the risk factors of critical and mortal COVID19 is male with older age [] Considering the not severe condition of disease it is rational that female patients present more than male in this cohort Previous studies have found comorbidity such as diabetes and hypertension heart disease as other risk factors [] Similar to other study of patients had diabetes and had hypertension Similar to other studies in Asia [ ] patients mainly present fever cough asthenia and shortness of breath In Europe however a recent study showed that patients with mild to moderate disease mainly had headache loss of smell nasal obstruction and asthenia [] Considering the big differences it is possible that olfactory dysfunction might be neglected during the first wave of SARSCoV2 infection in Wuhan Also the virus is phylogenetically distinct from Asia type B to Europe type C [] which could contribute to different clinical outcomesThrough analysis of laboratory findings we found that patients with moderate disease of COVID19 had common characteristics on admission normal or increased neutrophil ratio low lymphocyte count decreased hemoglobin level increased inflammatory indicators ESR and CRP and some patients were complicated with coagulation disorder and myoExamination of inflammatory biomarkers found that PCT was in the normal range for moderate patients while CRP and ESR were increased in and of patients on admission respectively Other studies including mild to moderate disease showed heterogeneous results [] and found three of or of of patients had increased level of CRP [ ] Thus SARSCoV2 infection is involved with the disorder of inflammatory response Furthermore we find that the increase of both CRP and ESR were correlated with patient€™s age which may indicate that high inflammatory state s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res and more severe pneumonia present more often in elderly people In addition the increase of CRP level was more transient as it returned to normal range in most people when they were discharged from hospital Nevertheless it took more time for ESR to return to normal level especially for patients older than years Certainly elder people would need more time to recover and to metabolize the extra inflammatory biomarkersAbnormal coagulation condition is known as associated with poor prognosis of COVID19 In particular serum level of Ddimer and FDP were significantly higher in lethal cases [] Other coronavirus infection such as SARS and Middle East respiratory syndrome MERS showed similar increased coagulation activities in severe cases [ ] The possible mechanism is that local inflammatory response induced by cytokine responses stimulates coagulation cascade and hemodynamic changes [ ] In patients with moderate COVID19 we found about and presented increased level in fibrinogen FDP and Ddimer on admission respectively And when compared to young people patients older than years tend to have higher levels of these coagulation parameters Certainly the hemodynamics and endothelial conditions are more complicate in elder people Furthermore SARSCoV2 infection as well as the inflammatory response it induced could deteriorate the existed dysregulation As the health condition improved at discharge these parameters decreased to normal ranges for most patients Yet again patients over years had significantly higher level of fibrin degradation products In the meantime we found that the platelet level had no obvious decrease in most patients with moderate disease Therefore as reported previously thrombocytopenia happens more often in severe and critical patients [] while abnormal coagulation condition is slight and temporary in moderate patientsThe receptor of SARSCoV2 angiotensinconverting enzyme ACE2 is expressed in myocardial cells and vascular endothelial cells [] It is possible that heart dysfunction is directly targeted by virus infection hence early evaluation and continued monitoring of cardiac damage are important [] We systematically examined the cardiac function biomarkers on admission CKMB and cTnL had no obvious change in these moderate patients while about of patients showed increased NTproBNP This percentage is much lower than previous reported of the severe cases that had elevated NTproBNP Furthermore we only found two patients still had NTproBNP higher than normal range at discharge indicating that moderate patients had little cardiac complicationThis study provides us more information about moderate COVID19 but still has some limitations First this is a relatively small singlecenter study Second due to the retrospective study design a few laboratory tests were not done in all patients In addition several patients were hospitalized during a short period that some tests were not reexamined before they were discharged Third we do not possess any viral kinetic data in these patients further studies are necessary to elucidate the correlation between viral load and laboratory changesIn this ongoing pandemic of infected people showed moderate disease [] it is important to understand more about this moderate spectrum of disease to settle into a longterm problem We conducted a cohort study of adult patients with moderate disease of COVID19 in Wuhan China Based on the clinical characteristics we conclude that SARSCoV2 infection inhibits the immune system of patients and induces a controllable inflammatory response in moderate COVID19 Elderly patients have higher inflammatory state and more dysregulated coagulation condition It is essential to closely assess their condition for a better clinical managementAcknowledgmentsThis study was supported by the Natural Science Foundation of China Hubei Province Department of Education Science and Technology Project B2019232 and the Wuhan Municipal Education Bureau Project Financial DisclosureThere are no financial conflicts of interest to discloseConflict of InterestAll authors declare no competing interestsInformed ConsentAll subjects provided written informed consentAuthor ContributionsYL XZ and BS designed and performed the study YL and BS drafted the manuscript and did critical editing XZ and RG were involved in data collection YL JQ QY JS and WL analyzed the data BS carefully supervised this manuscript preparation and writingData AvailabilityAll data used in the study are available from the corresponding author by request Although some data confidential in nature may only be provided with restrictionsReferences Zhu N Zhang D Wang W Li X Yang B Song J Zhao X et al A novel coronavirus from patients with pneumonia in China N Engl J Med Coronaviridae Study Group of the International Committee on Taxonomy of Viruses The species Severe acute respiratory syndromerelated coronavirus classifying 2019nCoV and naming it SARSCoV2 Nat Microbiol Huang C Wang Y Li X Ren L Zhao J Hu Y Zhang s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cLiu et alJ Clin Med Res L et al Clinical features of patients infected with novel coronavirus in Wuhan China Lancet Wang D Hu B Hu C Zhu F Liu X Zhang J Wang B et al Clinical characteristics of hospitalized patients with novel coronavirusinfected pneumonia in Wuhan China JAMA WHO Coronavirus disease COVID19 Situation Report Available from wwwwhointdocsdefaultsourcecoronavirusesituationreports20200628covid19sitrep160pdfsfvrsn2fe1c658_2 Wu Z McGoogan JM Characteristics of and important lessons from the coronavirus disease COVID19 outbreak in China summary of a report of cases from the Chinese Center for Disease Control and Prevention JAMA WHO Clinical management of COVID19 Available from wwwwhointpublicationsdetailclinicalmanagementofcovid19 Liu K Chen Y Lin R Han K Clinical features of COVID19 in elderly patients A comparison with young and middleaged patients J Infect 2020806e14e18 Zheng Z Peng F Xu B Zhao J Liu H Peng J Li Q et al Risk factors of critical mortal COVID19 cases A systematic literature review and metaanalysis J Infect 2020812e16e25 Li LQ Huang T Wang YQ Wang ZP Liang Y Huang TB Zhang HY et al COVID19 patients' clinical characteristics discharge rate and fatality rate of metaanalysis J Med Virol Cao Y Liu X Xiong L Cai K Imaging and clinical features of patients with novel coronavirus SARSCoV2 A systematic review and metaanalysis J Med Virol Lechien JR ChiesaEstomba CM Place S Van Laethem Y Cabaraux P Mat Q Huet K et al Clinical and epidemiological characteristics of European patients with mildtomoderate coronavirus disease J Intern Med Forster P Forster L Renfrew C Forster M Phylogenetic network analysis of SARSCoV2 genomes Proc Natl Acad Sci U S A Zhou F Yu T Du R Fan G Liu Y Liu Z Xiang J et al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet Velavan TP Meyer CG Mild versus severe COVID19 Laboratory markers Int J Infect Dis Lee N Hui D Wu A Chan P Cameron P Joynt GM Ahuja A et al A major outbreak of severe acute respiratory syndrome in Hong Kong N Engl J Med Cheng Y Zhao H Song P Zhang Z Chen J Zhou YH Dynamic changes of lymphocyte counts in adult patients with severe pandemic H1N1 influenza A J Infect Public Health Chen N Zhou M Dong X Qu J Gong F Han Y Qiu Y et al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet Cavezzi A Troiani E Corrao S COVID19 hemoglobin iron and hypoxia beyond inflammation A narrative review Clin Pract Zeng F Huang Y Guo Y Yin M Chen X Xiao L Deng G Association of inflammatory markers with the severity of COVID19 A metaanalysis Int J Infect Dis Chen G Wu D Guo W Cao Y Huang D Wang H Wang T et al Clinical and immunological features of severe and moderate coronavirus disease J Clin Invest Wang F Hou H Luo Y Tang G Wu S Huang M Liu W et al The laboratory tests and host immunity of COVID19 patients with different severity of illness JCI Insight Tang N Li D Wang X Sun Z Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia J Thromb Haemost Wong RS Wu A To KF Lee N Lam CW Wong CK Chan PK et al Haematological manifestations in patients with severe acute respiratory syndrome retrospective analysis BMJ AlAbdallat MM Payne DC Alqasrawi S Rha B Tohme RA Abedi GR Al Nsour M et al Hospitalassociated outbreak of Middle East respiratory syndrome coronavirus a serologic epidemiologic and clinical description Clin Infect Dis Moore JB June CH Cytokine release syndrome in severe COVID19 Science Giannis D Ziogas IA Gianni P Coagulation disorders in coronavirus infected patients COVID19 SARSCoV1 MERSCoV and lessons from the past J Clin Virol Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Liu L et al Clinical Characteristics of Coronavirus Disease in China N Engl J Med MendozaTorres E Oyarzun A MondacaRuff D Azocar A Castro PF Jalil JE Chiong M et al ACE2 and vasoactive peptides novel players in cardiovascularrenal remodeling and hypertension Ther Adv Cardiovasc Dis Guzik TJ Mohiddin SA Dimarco A Patel V Savvatis K MarelliBerg FM Madhur MS et al COVID19 and the cardiovascular system implications for risk assessment diagnosis and treatment options Cardiovasc Res s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0c"

Thyroid_Cancer

inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the p65 and actin cytoskeleton regulatory pathwaysNaze G Avci1 Sadaf Ebrahimzadeh‘Pustchi1 Yasemin M Akay1 Yoshua Esquenazi2 Nitin Tandon Jay‘Jiguang Zhu Metin Akay1Glioblastoma GBM is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy GBM tumors show nuclear factor‘κB activity that has been associated with tumor formation growth and increased resistance to therapy We investigated the effect of inhibitor BAY ‘ with Temozolomide TMZ on the signaling pathways in GBM pathogenesis GBM cells and patient‘derived GBM cells cultured in 3D microwells were co‘treated with BAY ‘ and TMZ or BAY ‘ and TMZ alone and combined experiments of cell proliferation apoptosis wound healing assay as well as reverse‘phase protein arrays western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells The results revealed that the co‘treatment significantly altered cell proliferation by decreasing GBM viability suppressed pathway and enhanced apoptosis Moreover it was found that the co‘treatment of BAY ‘ and TMZ significantly contributed to a decrease in the migration pattern of patient‘derived GBM cells by modulating actin cytoskeleton pathway These findings suggest that in addition to TMZ treatment can be used as a potential target to increase the treatment™s outcomes The drug combination strategy which is significantly improved by inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatmentGlioblastoma multiforme GBM is the most malignant primary brain tumor in the central nervous system Current standard of care therapy includes surgery followed by radiotherapy and concomitant and adjuvant chemotherapy with the alkylating agent Temozolomide TMZ which provides survival benefits for patients with GBM1 However even with the advances in surgical resection combined with TMZ therapy and irradiation the prognosis for newly diagnosed GBM patients remains poor In fact due to its rapid proliferation increased invasion and migration capacity and chemoresistance to the alkylating agents a0the median survival is only a0months with the ˜Stupp™ regimen radiation with daily TMZ — “ a0weeks followed by cyclic TMZ2 and 5year survival rate is less than which is the lowest longterm survival rate of malignant brain tumors3“ TMZ methylates DNA at the O6 positions of guanine and DNA repair enzyme O6methylguanine methyltransferase MGMT removes alkyl groups from O6 position of guanine in DNA making cells resistant to TMZ6 Therefore new therapies are necessary to prevent cell proliferation and induce apoptosis for GBM patientsNuclear factorkappa B NFκB is a regulatory transcription factor of the Rel gene family including p50 cRel RelB or p65 subunits It is involved in the control of tumor cell proliferation migration immune response and apoptosis7“ Studies have shown that NFκB gene was involved in the regulation pathways of different cancer types such as thyroid cancer head and neck squamous cell carcinoma and colorectal cancer711“ Increased 1Department of Biomedical Engineering University of Houston Cullen Blvd Houston TX USA 2UTHealth Neurosurgery McGovern Medical School Memorial Hermann at Texas Medical Center The University of Texas Health Science Center at Houston Houston TX USA email makayuheduScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cactivation of NFκB has also been identified in GBM tumors where the expression of NFκB was much higher in GBM tissue compared with nonGBM tissue1415 NFκB also promotes chemoresistance to TMZ and regulates MGMT activity in GBM by promoting MGMT gene expression through NFκB binding sites within the MGMT promoter16 NFκB inhibitors such as parthenolide do not completely eradicate tumors therefore they are mostly used in combination with other drugs17 When used in combination with TMZ NFκB inhibitor parthenolide has been shown to activate mitochondrial apoptosis signaling in U87MG and U373 GBM cells which lead to cell death18 and had a combined effect on cell cytotoxicity in LN18 and T98G glioma cells19 NFκB inhibitor CBL0137 has been shown to bind DNA leading the functional inactivation of the Facilitates Chromatin Transcription FACT complex a chromatin remodeling complex regulating transcription replication and DNA repair2021 In a0vitro evaluation of the CBL0137 on FACT p53 and NFκB has been done using U87MG and A1207 GBM cells It was shown that CBL0137 induced loss of chromatinunbound FACT activated p53 and inhibited NFκB dependent transcription21 In a0vivo studies showed that CBL0137 was effective in increasing survival rates in TMZresistant orthotopic mouse models21 Moreover Wang et a0al indicated that NFκB inhibitor BAY suppresses the expression of MGMT and enhances the TMZinduced apoptosis in TMZ resistant U251 cells22 However there is still a lack of characterization of the precise pattern of NFκB activation in combination with TMZ in GBM cell populations that have been a0surgically resected from patientsIn vitro and in a0vivo identifications and validations of molecular targets of GBM are important as they can progress into clinical studies Studies reported that combining multiple gene targets may prevent tumor growth and improve the treatment strategy for GBM23“ Both Bay and TMZ exert antitumoral activities individually in different tumor types28“ Therefore in this study we aimed to analyze functionally the combined effect of Bay and TMZ in different GBM cells For this purpose first we used our 3D PEGDAbased hydrogel microwell platform31“ to provide reliable preclinical models that can recapitulate in a0vivo features of the GBM tumors We cultured GBM cells U87 and LN229 and patientderived GBM cells in 3D microwells for a more precise and personalized treatment approach We then treated GBM cells with Bay and TMZ in combination or alone Our results indicated that the cotreatment of Bay and TMZ significantly reduced cell viability in all three cell lines in correlation with a significant decrease in the spheroid size The levels of NFκB protein and its subunits p65 and p50 were also significantly decreased compared with the control and single drug applications Similar a0decreases in the cell viability and protein levels were observed in all three GBM cells Tumor biopsy samples could give more realistic information about how tumors respond to drugs when they are used for in a0vitro or in a0vivo studies35“ Therefore we decided to continue our experiments with only using our patientderived GBM cells We treated patientderived GBM cells with Bay and TMZ or alone and analyzed specific cellular proteins along with their posttranslational modifications via reversephase protein arrays RPPA to elucidate the mechanism of action of the proteins3839 We observed that several cell signaling pathways including cell metabolism proliferation apoptosis were significantly affected by the combination of the drugs which were consistent with the literature4041 Furthermore our RPPA data revealed that there was a significant change in the modulation of actin cytoskeleton and following experiments including western blot analysis for the expression of FAK protein and wound healing assay for cell migration patterns confirmed the RPPA results We observed a significant decrease in both actin fluorescence intensity and migration pattern in the a0cotreated patientderived GBM cells To the best of our knowledge the effect of cotreatment of Bay and TMZ has never been studied previously on the actin modulation of patientderived GBM cells These results suggested that Bay and TMZ induced alteration in the a0actin filament anization by reducing the level of focal adhesion protein which might implicate in cell apoptosis The effect of Bay with TMZ necessitates further exploration to better understand its mechanism of action in GBM and potential therapeutic tools for GBM treatmentResultsCo‘treatment of Bay ‘ and TMZ reduced viability of GBM cells We used our previously a0published data to select the most effective drug concentrations for this study42 We cultured LN229 U87 and patientderived cells in the microwells for a0days where they formed 3D spheroids and we added a0µM of Bay and a0µM of TMZ in combination or alone Then we cultured the spheroids for more days with or without drug Control group did not receive any treatment The cell viability assay was performed on day after drug administration The results showed that the a0cotreatment significantly reduced cell viability of GBM cells LN229 and U87 and patientderived GBM cells cultured in 3D PEGDA microwells respectively as shown in Fig a01ac When they were used alone TMZ reduced cell viability to and p and Bay reduced cell viability to and in LN229 U87 and patientderived GBM cells respectively compared to control groups Fig a01d However when they were used in combination the viability of the cells significantly decreased to and in LN229 U87 and patientderived GBM cells respectively compared to control groups p Fig a01d Tumor cells are generally less sensitive to drug treatments in 3D cultures than in 2D cultures4344 This could reflect reduced compound access or differences in the response to cell death To confirm that cotreatment was more effective compared to single drug use we quantified the size of the spheroids using ImageJ45 Our data showed that after a0days of drug treatment the spheroids™ sizes were significantly reduced in the cotreatment by and in LN229 Fig a01e U87 Fig a01f and patientderived GBM cells p Fig a01g respectively compared to control group p When we compared the spheroids™ sizes of the cotreatment with TMZ alone there was a reduction of and in LN229 U87 and patientderived GBM cells respectively p Finally the spheroids™ sizes of the cotreatment compared with Bay alone showed a decrease of and in LN229 U87 and patientderived GBM cells respectivelyScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Representative images of the GBM tumor cells cultured in the PEGDA microwells a“c LN229 U87 and patientderived GBM cells were cultured in the microwells for a0days respectively After day Bay and TMZ were applied either alone or in combination onto the cell spheroids Control group did not receive any treatment The cells were cultured with or without drugs additional more days The images were taken on Day Day and Day after the drug application to observe the disruption in the spheroids Dotted black lines represent the edge of the tumor spheroid Scale bars a0µm d Bar graph showing trypan blue staining for cell viability of LN229 U87 and patientderived GBM cells e“g Spheroid size quantification was done using ImageJ for LN229 U87 and Patientderived GBM cells respectively Twotailed ttest followed by Wilcoxon test were done GraphPad Prism v5 Data represent the mean ± SD of three biological replicates p and p Suppression of activity in GBM cells by co‘treatment of Bay ‘ and TMZ As a readout of NFkB activity after drug treatment we first quantitatively assessed the cytoplasmic activation of phosphorylated NFκB p65 subunit in both treated and untreated groups in all GBM cells NFκB pp65 subunit activity was observed in the control groups of all three GBM cells Fig a02a NFκB pp65 subunit activity decreased to and when TMZ applied alone and and when Bay was applied alone in LN229 U87 and patientderived cells respectively However the decrease in NFκB pp65 subunit was reduced to when LN229 U87 and patientderived cells respectively were cotreated p Fig a02a Bay specifically inhibits NFκB activation by blocking phosphorylation of IκBα46 In independent experiments we analyzed the abundance of phosphorylated NFκB p65 NFκB p50 and IκBα in all three GBM cells Qualitative and quantitative western blot analysis revealed that the exposure to Bay with TMZ significantly downregulated the abundance of NFκB p65 NFκB p50 and IκBα compared with control and Bay or TMZ alone Fig a02b Please note that loading controls were used for each experiment but only the representative loading control for p and tP65 and p and tP50 was presented Fig a02b The cell viability assay cells™ size and protein expressions in all three GBM cells revealed similar results without any dramatic change Therefore considering the importance of using patientderived tumor cells to elucidate the mechanism of drugs and respective signaling pathways35“ we further continued our experiments using patientderived GBM cellsApoptosis was promoted by co‘treatment of Bay ‘ and TMZ RPPA technology is designed for multiplexed antibodybased relative quantification where each array is tested with a validated antibody specific to a particular protein along with their particular posttranslational modifications47 In the attempt to elucidate the mechanism of action of Bay with TMZ by which NFκB subunits were modulated and to identify downstream signaling molecules we employed RPPA platform using our drug treated or untreated patientderived GBM cells RPPA results showed that many oncogenic pathways were altered by the drug treatments but more specifically by the cotreatment Fig a03a Decreased expression of NFκB was not only associScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 NF“kB activity in LN229 U87 and patientderived GBM cell lines a NF“kB p65 subunit activity in LN229 U87 and patientderived GBM cell lines respectively The cells cultured with or without drugs for a0days were collected from the microwells and subjected to ELISA Data represent the mean ± SD of three biological replicates p and p b Representative immunoblots LN229 U87 and patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels bottom panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p ated with changes in the a0NFκB pathway but also with apoptosis cell metabolism and proliferation which were confirmed by the analysis of downregulated RPPA proteins in Enrichr KEGG libraries4849 Fig a03c p One of the specific pathways given by RPPA was apoptosis Apoptosis is one of the important mechanisms that regulates cell death and suppress tumorigenesis Studies have demonstrated that Bcl2 family proteins can positively and negatively regulate apoptosis by regulating antiapoptotic protein Bcl2 and proapoptotic protein Bax4050 Our RPPA data using patientderived GBM cells showed that the fold change of Bcl2 relative to control was times higher in cotreated group TMZ alone Bay alone respectively Fig a03b In order to further confirm whether the expression of a0these proteins were downregulated by the cotreatment we performed western blot analysis Our results showed a similar decrease in Bcl2 protein expression in the cotreatment compared with the control and single drug a0treatment Fig a03d In contrast Bax protein fold change relative to control was times higher in cotreated group TMZ alone Bay respectively where we observed a significant increase after the cotreatment of Bay with TMZ compared with the control p Fig a03b Bcl2Bax ratio is a key indicator in susceptibility of the cells to apoptosis Western blot results confirmed the change in Bcl2Bax ratio in the cotreatment compared with the control group and single a0drug treatment Fig a03d Our RPPA data also showed a significant increase in the cleavedcaspase protein Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The effect of Bay and TMZ on signaling pathways in patientderived GBM cells a Heat map presentation of RPPA analysis showing the changes in the protein expression RPPA was performed on lysates treated with Bay and TMZ alone or in combination All relative protein level data points were normalized to the a0control group Red and green indicate up and down regulations respectively in the heat map The samples were run in duplicate n b Fold change of the a0selected proteins relative to the a0control group via RPPA Data represent the mean ± SD of two biological replicates p p Wilcoxon rank sum test c Analysis of downregulated RPPA proteins shows a a0significant activation in numerous Enrichr KEGG pathways The pathways were a0sorted by p value ranking d Representative immunoblot validation of significantly altered proteins involved in different KEGG pathways Patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p fold change relative to control times higher in the cotreatment compared with times higher in TMZ alone and times higher in Bay alone p Fig a03b To confirm if cotreatment triggered apoptosis correlated with caspase activation we performed western blot analysis with procaspase3 cas3 and cleavedcaspase3 Ccas3 We observed that Bay and TMZ induced apoptosis was associated with cas3 Fig a03d Please note that loading controls were used for each experiment but only the representative loading control for Bax cas3 and Ccas3 was presented Fig a03d Moreover another important mechanism of NFκB activation in GBM regulates through AKT phosphorylation of IκB Our RPPA data showed relative fold changes of in the cotreated group TMZ alone and Bay alone respectively p Fig a03b The western blot results also confirmed a significant decrease in the abundance of AKT pT308 Fig a03dTo further investigate whether cotreatment of Bay with TMZ can lead to glioma cell apoptosis and to confirm our RPPA and western blot results we performed apoptosis assay TUNEL The patientderived GBM cells were cotreated with Bay with TMZ or single drug treated and subjected to TUNEL assay to detect DNA damage Fig a04a The results indicated that TUNEL cells in the cotreatment were increased tenfold compared with control and and 24folds compared with TMZ alone and Bay alone respectively p Fig a04b Additionally in some TUNEL cells we observed a typical ring type chromatin aggregation underneath the nuclear membrane which suggested an early stage apoptosis51 Fig a04a red arrows There were also a few TUNEL cells that lacked the typical apoptotic ringlike nuclear structure indicating that they were either at a different stage of apoptosis or alternatively undergoing necrosis52 that we have not investigated furtherCo‘treatment of Bay ‘ with TMZ changed actin anization by inhibiting FAK phosphorylation and cell migration Actin filaments Factin are one of the main components of the cellular cytoskeleton which regulates actin dynamics and migration process in the cells The disruption of the actin cytoskeleton inhibits cell migration and adhesion53 Depolymerization or cleavage of actin lamins and other cytoskeletal proteins have been also found to be involved in cell apoptosis54“ To confirm the RPPA results showing changes in the actin modulation pathway and to understand the mechanism that regulates cytoskeletal Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Apoptosis assay TUNEL a Fluorescent images of TUNEL cells in patientderived GBM cells TUNEL assay was performed on cells treated with Bay and TMZ in combination or alone in the microwells Cells were collected from the microwells trypsinized and replated into 8well chamber slides TUNEL cells green with ringlike nuclear stain are indicated with red arrows Nuclei were counterstained with DAPI blue b Numbers of TUNEL cells are presented as mean ± SD of three biological replicates p and p X20 objective Scale bars a0µmanization we treated patientderived GBM cells co treated with Bay with TMZ or single drug treated 3D spheroids collected from the microwells were stained with phalloidin green and DAPI blue Staining cells with fluorescently conjugated phalloidin is considered the most reliable method of accurately labeling Factin in fixed cells57 In the control group intact cells formed finemeshed networks with a distinct Factin anization on both day Fig a05a upper panel and day Fig a05a bottom panel In single drug treated cells actin was still found to be polymerized to filaments as it can be seen by its interaction with phalloidin at both days and However the cells which were cotreated with Bay and TMZ lost their Factin anization and their shape compared with the control and the single drug treated groups at day Fig a05a bottom panel Changes in the a0actin distribution within the cells were quantified by measuring the staining intensity using Fiji Macro ImageJ as described previously5859 At day we observed a a0significant decrease in the fluorescence intensity of phalloidin when the cells were cotreated with Bay and TMZ compared with the a0control and single drug treated groups p Fig a05b To investigate the drug related Factin mechanism we examined the levels of FAK protein following cotreatment or single drug treatment As shown in Fig a05c cotreatment significantly decreased the level of phosphorylated FAK compared with both control and single drug applications p Furthermore we investigated cell migration patterns of the patientderived cells that were cotreated with Bay and TMZ or single drug treated We collected 3D spheroids from microwells after drug treatment and replated them in 24well plate to perform scratch wound healing assay We noted a significant increase in cell density in the scratch area in both control and Bay alone after and a0h of scratch formation p Fig a06a Although compared with the a0control cells both cotreatment and TMZ alone groups showed a decrease in the cell migration into the scratch area after a0h we observed that after a0h the migration rate of the cotreated cells was significantly slower than the cells that were treated with TMZ alone p Fig a06b These results indicated that the disanization of actin microfilaments was concomitant with the cell apoptosis after the a0cotreatment of Bay with TMZDiscussionDespite the increase in the median survival of GBM patients from to months4 the clinical efficacy of standard of care therapy including TMZ chemotherapy combined with surgery and radiotherapy is still limited Due to challenges in treating GBM significant attempts have been made to develop single or combined drug treatments60“ However given the cost long time frame and risks of failure associated with developing a new drug repurposing available drugs may be the most effective alternative therapeutic strategy Therefore it is important to evaluate potential drug combinations for GBM treatmentScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Changes in the actin cytoskeleton and migration pattern in patientderived GBM cells cotreated with Bay and TMZ or single drug treated in the microwells a Upper panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin green and DAPI blue Bottom panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin and DAPI Scale bars a0µm b Intensity of staining obtained with phalloidin was measured in each cell using ImageJ and displayed as boxplots with to confidence intervals A twoway ANOVA with Dunnett™s multiple comparisons test was performed to determine statistical relevance Three biological replicates n p p c Representative immunoblots show the levels of FAK pTyr397 and total FAK in patientderived GBM cell lysates cotreated with Bay and TMZ or single drug treated for a0days in the microwells The levels of the proteins were quantified using ImageJ right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p Due to the cell repellent property of PEGDA hydrogel tumor cells can form aggregates at the bottom of the microwells and selfassemble into spheroids in each well within a0days following cell seeding313363 Compared with 2D monolayer cell culture 3D spheroids have an important advantage their larger size Thus often drug effects can easily be monitored over time by measuring the size and shape of spheroids4344 Additionally using 3D in a0vitro tumor models can better recapitulate in a0vivo features of the tumors We used PEGDA hydrogelbased microwell platform313363 in order to culture different types of a0GBM cells commercially available GBM cell lines LN229 U87 and a0patientderived GBM cells However we investigated the effect of the drugs on the patientderived GBM cells more in detail since growing tumors from tumor biopsy samples could give very detailed information about how tumors respond to drugs35“ Considering the precious nature of the patient samples this platform which requires fewer cells compared with 2D monolayer cultures provides us with a robust tool to recapitulate in vivo features of GBM tumors and to test our drug combinationsNFκB is one of the major transcription factors associated with GBM and responsible for activating a series of cellular responses including cell proliferation survival invasion and apoptosis6465 Previous studies have shown that NFκB can activate Akt and promote cell survival and proliferation by downregulating the expression of phosphatase and tensin homolog deleted on chromosome ten1866 NFκB pathway can inhibit cell apoptosis by inhibiting a stressactivated protein kinase and a mitogenactivated protein kinase signaling pathway67 It can also be activated in response to treatment with cytotoxic drugs such as vinca alkaloids and topoisomerase inhibitors Several studies have demonstrated the activation of NFκB in GBM patientderived stemlike cells cultures96869 Moreover alkylating agents TMZ can activate NFκB through DNA damage pathway activation7071 The combination effect of Bay and TMZ have been showed in our previous study where we determined the most effective drug concentrations on GBM cells using our microfluidics platform42 Another study that investigated the combined effect of NFκB inhibitor BAY with TMZ showed that combined drug application induced TMZ resistant in U251 GBM cells22 However the characterization of the precise pattern of NFκB activation in different GBM cell populations from surgically resected tissues still remains elusive Therefore in this study we investigated the interaction of Bay with TMZ and their effects on the LN299 and U87 GBM cell lines as well as patientderived GBM cells in order to recapitulate NFκB activation as in a0vivo features of the GBM and its signaling pathways We applied a0µM of Bay and a0µM of TMZ3442 in combination or alone for all three GBM cell types First we observed a significant decrease in both cell viability and size of the spheroids in the cotreatment compared with control and single drug application Then we showed quantitatively and Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Cell migration of patientderived GBM cells by wound healing assay a Patientderived cells were cotreated with Bay and TMZ or single drug treated in the microwells trypsinized and replated in 24well plates After they reached to their confluency a scratch wound was formed with a 200μl tip and cells were incubated for the next a0h Images were taken 4x at a0h a0hr and a0hr Scale bars a0µm b The wound width was measured with ImageJ and the average wound width was shown Data represent the mean ± SD of three biological replicates p and p oneway ANOVA with Tukey™s post hoc testqualitatively the expression of NFκB in all three GBM cell types a0We noted a significant decrease in the cotreated group compared with control and single drug application Our western blot data also confirmed the decrease in the abundance of pP65 pP50 and pIKBa that Bay has been shown to inhibit its phosphorylation46 However in the cotreated group the decrease was significantly higher compared to both control and single drug application This data showed that cotreatment of Bay and TMZ has more effect on the inhibition of NFκB pathway than Bay or TMZ alone and suggests a a0decreased downstream transcription of oncogenic proteins72 Although there were slight differences in the NFκB expression patterns in three different GBM cell types a0we focused on the patientderived cells in the rest of the study due to their ability to better recapitulate the genomic similarities to primary disease7374Proteins that interact with each other activate multiple pathways which can result in apoptosis according to tissue type and pathological condition Glioblastoma tumors express high levels of antiapoptotic BCL2 family proteins such as Bcl2 and BclxL which may cause glioblastoma cells to resist apoptosis75 The proapoptotic members of Bcl2 family such as Bax and Bak are necessary for their proapoptotic effect Interactions and the ratio between antiapoptotic Bcl2 and proapoptotic Bax are decisive factors in the induction of apoptosis7677 Active NFκB can prevent cells from apoptosis by stimulating the expression of genes and promoting cell proliferation Although patientderived GBM samples have been shown to be highly resistant to apoptosis77 our data revealed changes in the expression of various members of Bcl2 family and NFκB signaling pathway after cotreatment of Bay and TMZ Our RPPA results outlined distinct molecular profiles in which apoptotic P53 signaling and NFκB signaling pathways were significantly affected after the a0cotreatment These results supported that the inhibition of NFκB expression could inhibit the expression of Bcl2 and promote the expression of Bax thus promote apoptosis Our data also suggested the possible interaction between Bcl2 and p53 in Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Proposed schematic of the a0signaling pathways involved in Bay and TMZmediated inhibition in GBM patientderived cells The effect of combined therapy of Bay and TMZ was achieved through the inhibition of SrcFAKVinculin which regulate the cytoskeleton anization through MAPKs JNK and PI3KAKT signaling pathways Exposure to both Bay and TMZ also leads to receptormediated activation of Bax but not Bcl2 in the subsequent inhibition of the downstream NFκB transcription factor Inhibition of NFκB in turn causes cell deathregulating cell survival and death7778 The activation of extrinsic and intrinsic molecular pathways can lead to the proteolytic activation caspases The extrinsic pathway is triggered by proapoptotic ligands that activate cell surface death receptors and procaspase8 which in turn leads to the cleavage of caspase3 and apoptosis79 Our results determined that the a0cotreatment significantly inhibited the expression of caspase3 while the expression of cleaved caspase3 was increased Additionally TUNEL assay which detects DNA strand breaks which could occur as an event in the apoptosis showed a dramatic increase in the TUNEL cells after the cotreatment compared with the a0control and single drug application Altogether these results suggested that the inhibition of cell proliferation Bcl2 and caspase3 by a0the cotreatment of Bay and TMZ may occur through the NFκB mediated apoptosis and they might be tightly coupled8081The literature provides evidence that supports crosstalk between PI3KAktmTOR signaling pathway and NFκB which is downstream of Akt NFκB activation in GBM regulates through AKT phosphorylation of IκB resulting in an activated NFκB that translocates to nucleus8283 Our data showed that when Bay was used with TMZ there was a decrease in the abundance of PI3Kp110 AktpS473 AktpT308 and mTORpS2448 This preliminary data is important to suppo

Thyroid_Cancer

" Despite several efforts the development of an effective vaccine for COVID19 may take a much longer timeTraditionalnatural medicine already experienced by humans could be an earlier solution Considering the researchteam™s experience in using nanoclays as highaffinity material for cancer metastasis melanoma treatment andbone regeneration we propose to use these nanoclays for the preventiontreatment of COVID19 Owing to highaffinity nanoclays would capture the viruses before the latter get engaged with human hACE2 In this studymolecularlevel simulations and modeling of the interaction of coronavirus spike and hACE2 proteins wereperformed with and without nanoclays The results showed a very high level of affinitycohesiveness among SARSCoV2 spike and nanoclays as compared to the one between the former and hACE2 We premise that these nanoclays since already being used as drug carriers could also be injected as œclaysalone medicine Recommendationshave also been provided for future in vitro and in vivo studiesBackgroundThe sudden emergence and rapid spread of novel coronavirus SARSCoV2 have significantly affected thehealth and lives of human beings in addition to criticallyaffecting the world economy SARSCoV2 spike S bindswith high affinity to human angiotensinconverting enzyme hACE2 and uses it as an entry receptor to invade target cells Fig 1a b [] The virussurface spikeprotein mediates coronavirus entry into host cellsSARSCoV2 spike protein contains a receptorbindingdomain RBD that recognizes explicitly as its receptorhACE2 [ ] The surface of hACE2 contains two virusbinding hotspots that are criticalfor SARSCoV2 Sbinding Several naturally selected mutations in SARSCoV2 RBD surround these hotspots and regulate theinfectivity pathogenesis and crossspecies and humantohuman transmissions of SARSCoV2 [ ]At present there are no clinically approved vaccinesor drugs that specifically target SARSCoV2 Followingthe real protocol of developing a vaccine it may takemuch longer time to come up with an effective vaccine Correspondence habibrehmankfupmedusa3Engineering Research International ERI Riyadh Saudi ArabiaFull list of author information is available at the end of the There is a lot of interest in the development of therapeutic antibodies against SARSCoV2 Despite many efthese antibodies have not yet beenforts howeverdiscovered [] exceptin a few trials [] One trialshowed the potent neutralization of SARSCoV2 bybinding to the RBD of its S glycoprotein [] In this trial[] antibody cocktails a mixture of different antibodiesis recommended due to the increased neutralization effect it has on SARSCoV2 However use of antibodiesin the past from convalescent patients of SARSCoV totreat SARSCoV infection has shown adverse reactionsin the patients such as AntibodyDependent Enhancement ADE causing increased viral infectivity and otherharmful immune responses [] Moreover based on theexperience with the vaccine development efforts forSARSCoV and MERS chances of the materialization ofthe efforts being made for SARSCoV2 seems quitethin Therefore naturaltraditional medicines that have ahistory of safe consumptioningestion by humans couldbe considered as one of the treatment options for SARSCoV2 Being a natural material and a history of humanuseconsumption we suggest œhighly charged nanoclays to be used as coronavirus blockers and inhibitorsof the spikemediated entry into the human cells The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 0cAbduljauwad Nanoscale Research Letters Page of Fig Schematics of the SARSCoV2 attack on human hACE2 and the subsequent immune system response a b RBD binding hACE2 withoutan interference c RBD complexed with the antibody at receptor attachment site hence competing with hACE2 d RBD complexed with RBD at asite other than where receptor attaches resulting in the alteration of RBD structure and interruption of lock and key binding of RBD to hACE2Nanoclays nanosized natural materials originatingfrom minerals of the sedimentary rocks have got avery high affinity to bacteria and viruses [] Due toisomorphous substitution in their molecular structurethese nanoclays exhibit charge deficiency on theirsurfaces This charge deficiency on their surfaces isneutralized by the water molecules and the dissolvedcations Fig The charged structure and large surface area of clay nanops give them an affinityfor charged entities as found on bacterial surfacesand bacterial toxins Their distinct biomedical properties include high absorption the ability to engulf microbes and no toxicity Each of the electrically activeclay minerals has its distinct morphology characteristics and interaction behavior The most studied biomedical application of nanoclays includes serving ascarriers and complexes for anticancer drugs such as5fluorouracil and trastuzumab [“] They havetherefore been a potential alternate medicine for several diseases [“] Clay nanops due to theiradhesive nature have also been used as carriers forsustainedrelease medicine [ ] Nanoclays havealso successfully been used to adsorb and treat bovinerotavirus and bovine coronavirus [] Researchers[] intercalated methotrexate MTX an anticanceragentinto the anionic clay to create a nanohybriddrug They used the coprecipitation and subsequenthydrothermal methodology to prepare this chemicallystructurally and morphologically welldefined twodimensional drugclay nanohybrid The researchers[] discovered that due to the biocompatibility andhigh loading capacity bentonite nanoclay could beused for the preparation of the drugdelivery vehiclesthey prepared doxorubicinbentoniteIn thisto form ananoclay complex DOXBent complexsustainedreleaseintradrugdeliverystudysystem for 0cAbduljauwad Nanoscale Research Letters Page of Fig a SEM image and b the corresponding molecular structure of Namontmorillonite showing the configuration isomorphous substitutioncharge deficiency and interlayer cations from []tumoral chemotherapy of melanoma As montmorillonite clay is recently being studied to be used as anadditive and drug carrier materialthese nanoclaycomposites appeal their use in various dosing formmainly for controlled release of the drug [] The researchers [] also discovered that nanoclays can beused into recent dual functional drug delivery systemsDDSs to have efficiency in the drug delivery and soreduce the toxicity of doxorubicin DOX that is being used for thyroid cancer treatment Using a libraryof single“single type photo cleavable amphiphilicJanus dendrimers researchers [] developed a selfassembling lightresponsive dendrimersomes vesicleplatform Similar to the nanoclays surface modifiedbioactive virusmimicking anic nanovesicles fromglycodendrimersomes have structural modificationsthat contribute to manifest SARSCoV2 and hostpathogenic molecular interactions that help the virusto escape from the human immune system []Through considerable previous research we developedbasic characterization and behavior modeling ofthecharged clay minerals [“] and their applications in thecontrol of cancer metastasis [] in vitro and in vivo studies on melanoma treatment [] and the calcium depositionbone regeneration studies [] In a previous study bythe authors [] it was demonstrated that clay nanops had got a high affinity to the charged surfaces Thehigh attraction affinity of the nanoclays and the increasednonspecific adhesion attraction of the cancer cells makenanoclays favorable candidates to control cancer metastasis In that study we demonstrated the possible use of twocharged clay minerals to control the metastasis of thecancer cells Namontmorillonite SWy3 and palygorskitePFll Further to the findings of the authors™ previous research [] on the use of these nanoclays for the control ofcancer metastasis we also through in vitro and in vivostudies established that these nanoclays have inhibitory effects on melanoma cancer cells mainly on cell proliferationand viability [] In these previous studies in addition tolaboratory experiments molecularlevel simulations werealso performed on the nanoclay and cells™ interactionsThese simulations provided the assessment of the relativelevel of cohesivenessaffinity in the interactions with andwithout clay nanopsperceptionthroughthisestablishingBased on all the above experience of the authors onthe highaffinity potential of nanoclays we propose thatthe nanoclays could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2 This paper is a first steptowardsamolecularlevelsimulation and modeling approachBased on the results of the molecularlevel simulationsan outline of the recommendations for the next phasesof in vitro and in vivo research is also provided As thesenanoclays are also successfully being used as medicinecarriers we also premise that they can also be injectedingested as œclaysalone medicine and thus we haveproposed a tentative nanoclays administration methodology for this purposeMaterials”MoleculesSelection and Formulation of SARSCoV2 and hACE2Molecules of SARSCoV2 spike S and hACE2 were acquired from the protein data bank website RCSB [“] 0cAbduljauwad Nanoscale Research Letters Page of The molecular models of SARSCoV2 spike S andhACE2 formulated in Materials Studio software [] arerespectively shown in Fig 3a b Before being subject tothe simulations these molecules were charged using thecharge equilibration method QEq of the softwareSelection and Formulation of Nanoclay CrystalliteNamontmorillonite one of the most active members ofthe smectite group of clay minerals was selected for thestudy Namontmorillonite is a layered phyllosilicate claysmectite Fig In the colloidal form the space between neighboring layers can contain free sodium calcium or magnesium cations that are electrostaticallyattracted to external negatively charged surfaces [] Inits dry powdered state Namontmorillonite exists asequidimensionalflakessheets with dimensions of approximately × × microns Fig 2a Thesenegative charges on their interlayer surfaces are balancedby the cations As colloids the interlayer cations get dissociated from the clay ps and associate themselveswith the other negatively charged surfaces These ps also have positively charged edges due to the presence of the broken bonds at their ends Morphology andfurther characteristics of these nanoclays are providedin Table while formulation of their crystallites in Materials Studio software are explained belowIn the software Namontmorillonite crystallites wereformulated based on fundamental properties such asCEC exchangeable cations and interlayer charges Table The size of the molecularcrystallite size was selectedbased on the results of the p size analysis usingthe dynamic light scattering DLS technique [] Thefinal form of clay crystallite created in the software istheseshown in Fig 3c Afterthe preparation ofcrystallites in the design mode of the software using theinherent properties these were charged using the chargeequilibration method QEq of the softwareMethods”MolecularLevel SimulationsThis part of the study consisted of the simulation andassessment of the interactions of the SARSCoV2 spikeS with clay crystallites and with hACE2 Although thesemodels may not be the complete replication of the actual in vitro conditions these have been incorporatedwith all the essentialinteractions and are quite wellsuited for the intended relative and comparative studyIn the software the sorption and simulations of theformulated configurations of SARSCoV2 S Namontmorillonite crystallites and hACE2 were carriedout using Monte Carlo MC and molecular mechanicsMM techniques The enhancement of affinity in all thesimulated configurations was assessed in terms of thecalculated cohesive energy density CED”CED beingconsidered as a measurement of the cohesiveness of themolecular system Due to the largesized computationsinvolved in the simulationsthese calculations werecarried out using the highperformance computing facilities HPC at KFUPM KSA The overall methodologyand the choice of particular methods and the simulationparameters were based on authors™ previous research[“] while it is detailed in the subsequent sectionSARSCoV2 Spike S Interactions with hACE2 and ClayCrystallitesTo simulate the interaction of SARSCoV2 S with claycrystallites various numbers of the crystallites of Namontmorillonite clay were sorbed on SARSCoV2 Smodel For these sorption simulations the MetropolisFig Molecularlevel models of a SARSCoV2 spike b hACE2 and c Namontmorillonite crystallite formulated in Materials Studio software 0cAbduljauwad Nanoscale Research Letters Page of lnoitauccoFlnoitcaretnInoitcaretnInoitcaretnIninoisrepsidretawnoisrepsiDygrenelatotygreneWdvˆ’ygreneBAlraopcilihpordyHˆ’ˆ’ˆ’ˆ’aCaNretaWytiniffasγlaitnetopVmPZateZreyalretnIegrahclardeharteTlardehatcOlebaegnahcxEegrahcegrahcsnoitacqemgCECecafruSNaeragmslarenmirehtOliacmehClaumrofacilisOiSgMlAaCaNecruoSytnuoCASUYWkoorC][yWSyacletinolliromtnomaNfonoitaziretcarahcliacmehcdnalacisyhpfoyrammuSelbaT 0cAbduljauwad Nanoscale Research Letters Page of Monte Carlo method was selected in the Sorption module of the software In each sorption step clay crystallitesoccupy spaces around the spike S model to lower theoverall energy of the complex The required number ofcrystallites were sorbed in a maximum of stepsand then the energy of the system was minimized usingthe Forcite module of the software based on the MDprinciples The similar sorption process was repeated forthe interaction modeling of the SARSCoV2 spike molecule with hACE2 In this process hACE2 moleculeswere sorbed around the RBD of the spike S of SARSCoV2 After the completion of the sorption process theenergy of the formulation was minimized using MDbased module of the softwareThe Forcite module ofthe software incorporatingNPT constant number of ps pressure andtemperature ensemble was used for MD simulationswith a modified universal force field [] The simulations were run for to ps with an interval of 05fs ortill a constant volume is obtained A Berendsen thermostat with a decay constant of ps was used to controlthe temperature during the simulation During the MDsimulations the assumed temperature was kept constantat K °C with an atmospheric pressure kPaA Berendsen barostat with a decay constant of pswas used to control the pressure of the system TheBerendsen methodology was considered as the most appropriate for the single crystallites after several trials involving other thermostats and barostats available in thesoftware In the Monte Carlo method the parametersfor the ratios of exchange conformer rotate translateand regrow were selected as and respectively with the corresponding probabilities as and Amplitudes adapted for rotationand translation were ° and respectivelyCohesive Energy Density CED MeasurementIn this study the assessment of the affinitybindinglevelin the SARSCoV2clay crystallites and SARSCoV2hACE2 complexes was measured through thechanges in the CED After the sorption of clay crystallites and the subsequent performance of moleculardynamics of each of the configurations the CED wasdetermined using the cohesive energy density optionof the Forcite module of the software The authorshave experienced that the CED concept consisting ofthe total van der Waals and electrostatic CEDs canquite closely explain the various molecularlevel processes and interactions and simulate the extent of affinitybinding created among the simulated complexes[“] Quantitatively CED is defined as the amountof energy needed for the transition of mol of material from the liquid to the gaseous phase It is also ameasureofaffinityattractivenessthe mutualofmolecules and is expressed both as electrostatic andvan der Waalsan NPTensembleaveraged overforcesIn the Forcite module van der Waals energies wereevaluated using atombased cutoffsIn this methodnonbond interactions are simply calculated to a cutoffdistance and interactions beyond this distance are ignored To avoid the discontinuities caused by direct cutoffs most simulations use a switching function to turnoff nonbond interactions over a range of distancessmoothly An effective potential is created by multiplyingthe actual potential by the smoothing function Thechoice of the function in the intermediate range is crucial and should be continuously differentiable in this region so that forces can be calculated In this study acubic spline smoothing function was used with a splinewidth of and a cutoff distance of Results and DiscussionsThe final configuration of the SARSCoV2 ShACE2complex is shown in Fig 4a while the complexes between SARSCoV2 spike and different numbers of clayNamontmorillonite crystallites are respectively shownin Fig 4b c For comparison purposes total CEDs ofvarious proportionsnumbers of the clay crystallites onthe SARSCoV2 spike and the interaction of the laterwith hACE2 are plotted in Fig Based on our experience we have hypothesized thatnanoclays due to their high adhesive properties couldalso act as SARSCoV2 inhibitors They can do it bystrongly associating with the spike S present on SARSCoV2 The results obtained from the molecularlevelsimulations of the interactions indicate that due to veryhigh CED between SARSCoV2 and the nanoclays ascompared to the former and hACE2 Fig they couldinhibit SARSCoV2 from getting engaged with hACE2Moreover it could also be concluded from Fig thatthe extent of inhibition due to nanoclays is increased inquantity dosagedependent wayNanoclay Interactions with SARSCoV2 Spike SAuthors in their earlier research have demonstrated therole of nanoclays in promoting adhesion among thecancer cells and their microenvironment and hence controlling metastasis [] Adhesion measurements of mix of Namontmorillonite and palygorskite showedan increase in adhesion by among cancer cells andthe extracellular matrix proteins Fig 6a A corresponding SEM of the nanoclays binding the Raji cells and thefibronectin proteins is shown in Fig 6b Sample imagingwas performed in SEM mode in an FEI ESEMFEG XL atthe Miller School of Medicine University ofMiami Florida Authors also discovered in their previousresearch that electrostatic van der Waals and ZP 0cAbduljauwad Nanoscale Research Letters Page of Fig Molecularlevel simulation results in Materials Studio Software a SARSCoV2 S and hACE2 CED Jcm3 b SARSCoV2 S modelinteracting with twelve crystallites of Namontmorillonite CED Jcm3 and c SARSCoV2 S model interacting with twentyfour crystallites ofNamontmorillonite CED Jcm3”obtained using Sorption technique implemented in the softwareattractions seem to be dominating in the adhesion processes [] We conclude that the same mechanismswould have also facilitated the binding of the adhesivesurfaces of the nanoclays to the spike of SARSCOV2Fig ZP is a measure of the dispersion or flocculationtendency in the colloidal form including the interactions 0cAbduljauwad Nanoscale Research Letters Page of Fig Variation of cohesive energy density CED for SARSCoV2 ShACE2 and the complexes of the former with different numbers ofNamontmorillonite crystalliteswith the other constituents present in the suspensionmedium As a general rule a zeta potential greater than mV either positive or negative indicates dispersiontendency while a zeta potential of less than mV generally results in agglomeration Higher dispersion tendencies ZP of the clay nanops used in the study ˆ’ to ˆ’ mV lead to higher dispersion tendency andhence in the generation of higher surface area amplifyingthe interactions with the SARSCoV2 spike Althoughbased on their ZP Namontmorillonite nanopshave hydrophilic nature they in the presence of saltsalso promote secondary adhesion mechanisms betweenhydrophobic and hydrophilic surfaces [] It should alsobe noted that these clay nanops have high dispersion tendency due to their hydrophilic nature and relatively higher repulsive acidbase AB interactions Table High dispersion in turn results in the generation ofhigh surface area for increasing the attractive interactions Higher surface areas promote larger attractionsdue to the van der Waal attractions and the electrostaticforces among oppositely charged surfaces Besides although of relatively lesser degree positively chargededges of Namontmorillonite ps also get electrically attracted to the spike SThe results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites Fig also confirm the above interaction behaviors It has been observed that the sorption of the claynanops results in the formation of closely interacting strong van der Waals attraction fields These van derWaals attraction fields create higher CED of the claySARSCoV2 configuration Substantial increase in totalCED after the addition of clay crystallites Fig is alsoa testimony of a very high affinity of SARSCoV2 withthese ps as compared to the affinity of the formerwith hACE2systemagglutinationNanoclays as PseudoantibodiesBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays we premise that nanoclays could be mimicked asantibodies and can thus attract and engulf coronavirusesbefore they get engaged with human hACE2 Antibodiesare glycoproteins synthesized by plasma cells as part ofthe adaptive immune response to assist in the clearanceof infection from the body Antibodies aid in infectionclearance in multiple ways such as opsonization of pathogens to facilitate phagocytosis activation of the complementandneutralization of viruses and toxins When bound to theviral surface proteins antibodies prevent the entry of theviruses into the cell by preventing the attachment of viruses to their target receptor on the cell Antibody binding can occur at different sites on the surface proteinleading to various mechanisms that cause the same effect In the case of SARSCoV2 two viral neutralizationmechanisms by antibodies have been observed [ ]and shown in Fig 1c d One of the mechanisms involvesdirect binding of antibodies to the attachment site of theSARSCoV2RBD resulting in the antibody competingwith the target receptor hACE2 Another mechanism involves the binding of antibodies to the other sites onRBD without any competition with the target receptorThe latter is shown to be involved in neutralization byof microbes 0cAbduljauwad Nanoscale Research Letters Page of Fig a Summary of adhesion force measurements among RajiRajiFN assembly using AFM before and after treatment with various proportionsof Namontmorillonite and palygorskite clay nanops [] Error bars represent the variations in the trials b SEM image of the binding of Rajicells and Fibronectin proteins produced by nanoclaysthe most potent Monoclonal Antibody mAb discoveredin the study [ ] Analogous to the antibodies interaction with SARSCoV2 RBD inhibiting the latter toengage with hACE2 a similar molecularlevel model isprepared for nanoclays resulting in a similar inhibitionof the coronaviruses and shown in Fig Owing to theirvery high affinity nanoclays would get attracted tospikes of SARSCoV2 and thus restrict engagement ofRBDs of these spikes with hACE2Proposed Nanoclay Administration MethodologyClay use as drug carriers has been tested multiple timesyielding promising results of little to no cytotoxicity tocells of the human body Kaolinite clay mineral wastested for use in a potential drug delivery system andwas shown to have high biocompatibility and very lowas[]negligiblecytotoxicity [] Poly DLlactidecoglycolidemontmorillonite nanop cytotoxicity in vitro was alsodemonstratedPalygorskitepolyethyleneiminefluorescein isothiocyanate nanocomposites also exhibited almost no cytotoxicity in vitro[] Authors have also experienced injecting nanoclayssubcutaneously for the treatment of melanoma duringin vivo studies [] Based on the use of clay as a cancerdrug carrier and in other sustainedrelease medicine[“] we propose that nanoclays may be injected asœclayalone medicine subject to the verification in vivoand clinical trialsAlthough nanoclays are nonbiodegradable a comprehensive understanding of the design of the similar inanic nanops with their metabolic performancein the body carried out in the study [] could also 0cAbduljauwad Nanoscale Research Letters Page of Fig Three possible mechanisms of interactions of montmorillonite nanoclay with the SARSCoV2 spike S Electrostatic attraction amongpositively charged nanop edges and NaCa ions with negatively charged virus surfaces Van der Waals attractions ZPelectrostatic interactionscategorize these nanoclays as human body clearable inanic agentsConclusions and RecommendationsBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays these could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2The results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites result in the formation of closely interacting strongvan der Waals attraction fields These van der Waals attraction fields create higher CED of the claySARSCoV configuration Substantial increase in total CED afterFig Interaction mechanism of nanoclay ps with SARSCoV2 spike S inhibiting the interaction of the later with hACE2 0cAbduljauwad Nanoscale Research Letters Page of addition of clay crystallites is also a testimony of a veryhigh affinity of SARSCoV2 with these ps as compared to the affinity of the former with hACE2We propose to continue the research by carrying outin vitro interaction studies between SARSCoV2 anddifferent percentage of nanoclays Based on theoptimum dose of nanoclay developed in the in vitrophase we suggest to carry out in vivo studies on the animals The animal study should be carried out both withand without nanoclay to finalize the nanoclay dose andshould lay the foundation for the clinical trialsAcknowledgementsThe authors highly acknowledge KFUPM for providing highperformancecomputing research facilitiesAuthors™ ContributionsAll the authors equally participated at all the research levels The authorsread and approved the final manuscriptFundingNo fundingAvailability of Data and MaterialsAll data generated or analyzed during this study are included in thispublished Ethics Approval and Consent to ParticipateNot applicableConsent for PublicationNot applicableCompeting InterestsThe authors declare that they have no competing interestsAuthor details1Civil Environmental Engineering department King Fahd University ofPetroleum Minerals KFUPM Dhahran Saudi Arabia 2Royal College ofSurgeons in Ireland RCSI Bahrain campus Busaiteen Bahrain 3Engineering Research International ERI Riyadh Saudi ArabiaReceived July Accepted August ReferencesEwen Callaway and Nik Spencer The race for coronavirus vaccines agraphical guide eight ways in which scientists hope to provide immunityto SARSCoV2 News Feature Nature vol April Li F Li W H Farzan M Harrison S C Structure of SARS coronavirusspike receptorbinding domain complexed with receptor Science httpsdoi101126science1116480 Li WH Angiotensinconverting enzyme is a functional receptorfor the SARS coronavirus Nature “ httpsdoi101038nature02145Li F Structural analysis of major species barriers between humansand palm civets for severe acute respiratory syndrome coronavirusinfections J Virol “ httpsdoi101128jvi0044208 Wu KL Peng GQ Wilken M Geraghty RJ Li F Mechanisms of hostreceptor adaptation by severe acute respiratory syndrome coronavirus JBiol Chem “ httpsdoi101074jbcM111325803 Wang C Li W Drabek D Okba NMA van Haperen R Osterhaus ADME A human monoclonal antibody blocking SARSCoV2 infection NatCommun Jiang S Hillyer C Du L Neutralizing antibodies against SARSCoV2and other human coronaviruses Trends Immunol “Pinto D Park YJ Beltramello M Walls AC Tortorici MA Bianchi S Crossneutralization of SARSCoV2 by a human monoclonal SARSCoV antibody Nature “da Rocha Dias S Salmonson T van ZwietenBoot B Jonsson B Marchetti SSchellens JH Pignatti F The European Medicines Agency review ofvemurafenib Zelboraf® for the treatment of adult patients with BRAF V600mutationpositive unresectable or metastatic melanoma summary of thescientific assessment of the Committee for Medicinal Products for HumanUse Eur J Cancer “Sahel N Abduljauwad and HabiburRehman Ahmed Enhancing cancer celladhesion with clay nanops for countering metastasis Nature ScientificReports April httpsdoi101038s4159801942498y Zhang Y Long M Huang P Yang H Chang S Hu Y Mao L Intercalated 2D nanoclay for emerging drug delivery in cancer therapyNano Res “ Chianelli R R Das S US Patent No Washington DC US Patent and Trademark Office Han S Liu F Wu J Zhang Y Xie Y Wu T Y Targeting of fluorescentpalygorskite polyethyleneimine nanocomposite to cancer cells Appl ClaySci “Sun B Ranganathan B Feng SS Multifunctional poly D Llactidecoglycolide montmorillonite PLGAMMT nanops decorated byTrastuzumab for targeted chemotherapy of breast cancer Biomaterials“Lin FH Lee YH Jian CH Wong JM Shieh MJ Wang CY A study ofpurified montmorillonite intercalated with 5fluorouracil as drug carrierBiomaterials “ Bothiraja C Thorat UH Pawar AP Shaikh KS Chitosan coated layeredclay montmorillonite nanocomposites modulate oral delivery of paclitaxel incolonic cancer Mater Technol 29sup3B120“B126Kevadiya BD Thumbar RP Rajput MM Rajkumar S Brambhatt H Joshi GVBajaj HC Montmorillonitepolyεcaprolactone composites asversatile layered material reservoirs for anticancer drug and controlledrelease property Eur J Pharm Sci “ Guo MY Wang AF Muhammad F Qi WX Ren H Guo YJ Zhu GS Halloysite nanotubes a multifunctional nanovehicle for anticancer drugdelivery Chin J Chem “ Martínez C D Cationic clays upon cancer therapy Virtual MultidisciplinaryConference QUAESTI Konta J Clay and man clay raw materials in the service of man ApplClay Sci “ Murray HH Traditional and new applications for kaolin smectite andpalygorskite a general overview Appl Clay Sci “ Volzone C Retention of pollutant gases comparison between clayminerals and their modified products Appl Clay Sci “ Dong Y Feng SS Poly dllactidecoglycolidemontmorillonitenanops for oral delivery of anticancer drugs Biomaterials “ Clarka KJ Sarrb AB Grantb PG Phillipsb TD Woodea GN In vitrostudies on the use of clay clay minerals and charcoal to adsorb bovinerotavirus and bovine coronavirus Vet Microbiol “ Choi G Huiyan P Alothman Z Vinu A Yun C Choy J Anionic clay asthe drug delivery vehicle tumor targeting function of layered doublehydroxidemethotrexate nanohybrid in C33A orthotopic cervical cancermodel International Journal of nanomedicine DOI httpsdoi102147IJNS95611 Hosseini F Hosseini F Jafari S M and Taheri A Bentonite nanoclaybaseddrugdelivery systems for treating melanoma Clay Minerals DOI httpsdoi101180clm201842018 Inamuddin Asiri A M and Mohammad Ali Applications of nanocompositematerials in drug delivery DOI httpsdoi101016C20160050751 Avolume in Woodhead Publishing Series in Biomaterials Zhang Y Long M Huang P Yang H Chang S Hu Y Tang A and MaoL Emerging integrated nanoclayfacilitated drug delivery system forpapillary thyroid cancer therapy doi 101038srep33335 Sci Rep Li S Xia B Javed B Hasley W D MelendezDavila A Liu M Kerzner MAgarwal S Xiao Q Torre P Bermudez J G Rahimi K Kostina N YMöller M RodriguezEmmenegger C Klein M Percec V and Good M CDirect visualization of vesicle disassembly and reassembly usingphotocleavable dendrimers elucidates cargo release mechanisms ACS 0cAbduljauwad Nanoscale Resear

Thyroid_Cancer

RNAs circRNAs are a novel and unique class of noncoding RNAs that are backspliced from premRNAs It hasbeen confirmed that circRNAs are involved in various malignant behaviors of hepatocellular carcinoma HCCHowever the role of circRNA in the regulation of ferroptosis and the underlying mechanism remain unknown HerecIARS hsa_circ_0008367 was found to be the most highly expressed circRNA after sorafenib SF treatment in HCCcells Small interfering RNA against cIARS sicIARS significantly suppressed the cellular sensitivity to SF or Erastinthrough inactivating ferroptosis which may be partially attributed to the inhibition of autophagy and ferritinophagyPrediction analysis and mechanistic identification revealed that cIARS physically interacted with RNA binding proteinRBP ALKBH5 which was a negative regulator of autophagic flux in HCC The dissociation of BCL2BECN1 complexmediated by ALKBH5 silencing was effectively blocked by sicIARS Furthermore the inhibition of ferroptotic eventsautophagic flux and ferritinophagy resulted from sicIARS were significantly rescued by ALKBH5 downregulationOverall cIARS may be an important circRNA positively regulating SFinduced ferroptosis through suppressing theALKBH5mediated autophagy inhibitionIntroductionHCC is a highly refractory and prevalent cancerworldwide with new cases and deathsevery year1 Rapid advances in diagnosis and treatmenthad improved patient outcomes However the survivalgains are stagespecific2 Liver resection transplantationablation ortransarterial chemoembolization benefitspatients in early or intermediate stages3 while treatmentfor latestage HCC has remained challenging The standard firstline systemic drug against advanced HCC issorafenib SF BAY Nexavar which is currentlyCorrespondence Xiaoqing Wei weixqrm163com orJie Li heplijie163com1Department of Hepatobiliary Surgery The First Affiliated Hospital ofShandong First Medical University Jinan Shandong China2Department of Hepatobiliary Surgery Shandong Provincial QianfoshanHospital Shandong University Jinan Shandong ChinaFull list of author information is available at the end of the These authors contributed equally Zhiqian Liu Qi WangEdited by Inna Lavrik The Authors known as a ferroptosis inducer4 exerting only limitedeffects on overall survival and time to tumor progression56 Therefore it is imperative to explore novel andeffective therapeutic target to improve the cellular sensitivity to SFˆ’ was targeted SubsequentlyIn recent years ferroptosis has been confirmed to be anessential mechanism in SF treatment7 It is a kind of finelycontrolled cell death featured with irondependent accumulation of lipid hydroperoxides8 When HCC cells wereexposed to SF the cystineglutamate antiporter systemsystem xcthe cystineuptake was blocked and the biosynthesis of glutathioneGSH was inhibited resulting in accumulation of lipidperoxidation products and eventually inducing ferroptosis Meanwhile SF also induces autophagic flux in cellsWhen autophagy flux is initially induced MTORC1 dissociates from the ULK12ATG13 complex leading todephosphorylation of the complex9 Then phagophorenucleation occursthe ATG14BECN1recruiting Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of PIK3C3PIK3R4 complex10 Nexttwo ubiquitinlikeconjugation systems are involved in phagophore elongation11 When de novo autophagosome formation iscomplete they are delivered to the lysosome initiatinglysosomal degradationActually autophagy is a targetable pathway regulatingcellular sensitivity to ferroptosis12 The crosstalk betweenautophagy and ferroptosis has attracted more and moreattentions in the recent years Autophagy inhibition bybafilomycin A1 or chloroquine or lack of key autophagyassociated gene for example ATG5 ATG7 will partiallysuppressed ferroptotic eventsthrough ferritinophagyinactivation reducing the turnover of ironbinding ferritinand the iron release13“CircRNAs primarily originate from backsplicing eventsof premRNAs The covalently bonded loops are highlystable and abundant16 The deregulation of circRNAs isclosely related to various human diseases including cancer17“ In HCC several studies have revealed that circRNAs are involved in multiple malignant behaviors20However the functions and mechanisms of circRNAs inSF treatment are still not comprehensively understood Inthe current research we found a novel circRNA derivedfrom the IARS gene ID from circBase hsa_circ_0008367named cIARS cIARS was proven to be a promoter offerroptosis in HCC cells after SF treatment which waspartially attributed to the activation of autophagy andferritinophagy The underlying molecular mechanism ofcIARSmediated ferroptosis was also clarified in this studyResultsCircular transcript cIARS hsa_circ_0008367 is significantlyupregulated in SFtreated HCC cellsRNAseq was performed in three pairs of SFtreated anduntreated HCC celllines HepG2 SMMC7721 andHuh7 to screen differentially expressed circRNAs Cellswere treated with μM of SF for h uniquecircRNAs were found Among these circRNAs had already been reported in circBase21 and theother circRNAs were newly discoveredAmong the annotated circRNAs were ofextremely low abundance with fragments per kilobasemillion FPKM values in cellular samples and werethus excluded from this study The other circRNAscomprised two groups presented no significantchanges in expression levels after SF treatment and theother were differentially expressed circRNAs foldchange ‰¥ pvalue The volcano plots demonstrated that there were upregulated and downregulated transcripts Fig 1a The results of hierarchicalclustering are displayed in a heatmap Fig 1b generatedwith Heatmap Illustrator22 and suggest that there weretwo different clusters of transcripts The most highlyexpressed circRNA was cIARS hsa_circ_0008367 andOfficial journal of the Cell Death Differentiation Associationwas marked with black arrow in the volcano plotsAccording to circBase cIARS is an exonic circRNA nt in length originating from the exon and exon of the IARS gene on chr9 “ Fig1c qPCR showed that the relative expression levels ofcIARS were significantly higher in SFtreated cell linesthan in untreated ones Fig 1d Both convergent anddivergent primers of cIARS were applied for amplificationThe band of cIARS was only observed in cDNA samplenot the genomic DNA Fig 1e Sanger sequencing further validated that the sequence around the junction siteabout bp around the site was consistent with theresult of RNAseq and CircInteractome database23 Fig1f In addition cIARS was much more resistant to RNaseR Uμg which degrades linear but not circularthan IARS and GAPDH Fig 1g Whentranscriptsactinomycin D ActD a transcription inhibitor wasadded to HCC cells for the indicated time periods cIARSwas much more stable than its linear counterpart Fig1h These evidences suggested cIARS to be a highlyabundant and stable circular transcript in HCC cellscIARS is found to be a significant regulator of SFinducedferroptosisTo clarify the biological role of cIARS we first knockdown cIARS expression with a junction sitespecificsiRNA vector sicIARS The effects of the sicIARSwas shown in Fig 2a CCK8 assay showed that SFinduced growth inhibition was evidently weakened in sicIARS transfected cells Erastininduced growth inhibitionwas also attenuated by sicIARS Fig 2b c To determinethe underlying mechanism sicIARSintroduced HCCcells were treated with various cell death inhibitorsFerrostatin124 a specific ferroptosissignificantly undermined the therapeutic effects of either SFor Erastin in both sicIARS and NC transfected cellsHowever ZVADFMK an apoptosisinhibitor andNecrosulfonamide a necroptosis inhibitor exerted nosignificant influence on SF or Erastininduced growthinhibition Fig 2c Simultaneously malondialdehydeMDA and the level of Fe2 were significantly reducedwhile intracellular GSH obviously increased in the cIARSsilencing cells following SF or Erastin administration Fig2d These evidences suggested cIARS to be a positiveregulator of ferroptosis in HCC cellsinhibitorcIARS positively regulates SFinduced autophagy andferritinophagycIARS was also found to be an autophagy regulatorWestern blot WB assay showed that cIARS knockdownsignificantly decreased LC3 lipidation and increased p62accumulation Fig 3a Either autophagosomes or autolysosomes were observed via microscopic examinationafter AdmCherryGFPLC3 adenovirustransfection 0cLiu Cell Death Discovery Page of Fig See legend on next pageThis experiment is applied for concurrent observation ofautophagosome and autolysosome The signal of greenfluorescent protein will be quenched during fusion ofautophagosome and lysosome Thus the red signal ofmCherry indicates autolysosome and the merge of greenand red signals yellow puncta indicates autophagosomesicIARS significantly decreased the amount ofredautolysosome and yellow autophagosome puncta percell demonstrating an inhibition of autophagy fluxFig 3b TEM visuallyautophagicsuggested theOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of see figure on previous pageFig Circular transcript cIARS hsa_circ_0008367 is significantly upregulated in SFtreated HCC cells RNAseq was performed in three pairsof SFtreated μM h and untreated HCC cell lines HepG2 SMMC7721 and Huh7 a The differentially expressed circRNAs were visualized withvolcano plots the ˆ’log10 pvalue and the log2 fold change are plotted on the y and x axes respectively The dashed lines signify the filteringcriteria p fold change ‰¥ Upregulated circRNAs are shown in red and downregulated circRNAs are shown in green The most highlyexpressed circRNA was marked by a black arrow b Hierarchical cluster analysis showing the differentially expressed circRNAs The six columnsrepresent the six different cellular samples Each row indicates a circular transcript and the colors represent the abundance of the transcriptsc Schematic diagram of the backsplicing transcript generated from linear IARS Exon and exon are colored red and green respectively dRelative expression of cIARS after SF treatment for h in the HCC cell lines e Divergent and convergent primers for cIARS were applied to amplifyboth cDNA and gDNA Agarose gel electrophoresis visualized the products f The sequence around the junction site was confirmed through Sangersequencing The empty triangle indicates the junction site g RNase R exoribonuclease Uμg °C min was applied to remove the lineartranscripts from cellular extracts leaving circRNAs behind qPCR was applied to assess the resistance of RNAs to RNase R h RNA decay assayevaluating the stability of cIARS and IARS by qPCR after ActD μgml administration The error bars represent the standard deviation SD of at leastthree independent experiments p compartments sicIARS decreased the number of doublemembraned vacuoles to a relatively low level Fig 3cThese results showed that cIARS is a positive autophagyregulator in SFtreated HCC cells Furthermoretheprotein levels of FTH1 and NCOA4 the substrate andcargo receptor of ferritinophagy were determined by WBassay sicIARS resulted in remarkable accumulation ofboth FTH1 and NCOA4 Fig 3d This finding indicatedthatthe ferroptotic events in SFtreated HCC cellspositively regulated by cIARS may be partially associatedwith autophagy and ferritinophagycIARS specifically interacted with RBP ALKBH5 AlkBHomolog RNA demethylaseRecent studies have revealed that circRNA“RBP interaction has important roles in diverse biological processesAccording to CircInteractome database we found six RBPsbearing at least two binding sites matching to cIARSincluding FMRP Fragile X Mental Retardation SFRS1Serine And Arginine Rich Splicing Factor ALKBH5HuR ELAV like RNA binding protein IGF2BP1 InsulinLike Growth Factor MRNA Binding Protein andLIN28A Lin28 Homolog A Fig 4a RBP immunoprecipitation RIP assay in both HepG2 and Huh7 cell linesdemonstrated that the relative levels of cIARS in ALKBH5enriched samples were much higher than in the other fiveRBPs Fig 4b RNA pulldown and RNA EMSA were performed to determine the interaction using an antisenseprobe spanning the junction site of cIARS The presence ofspecific bands demonstrated the physical binding of cIARSand ALKBH5 Fig 4c d Interestingly SF administrationhad no influence on the protein levels of ALKBH5 in bothHepG2 and Huh7 cells Fig 4e but remarkably increasedthe cIARS“ALKBH5 interaction Fig 4f which may be dueto SFinduced expression of cIARScIARS repressed the role of ALKBH5 in the regulation ofautophagyALKBH5 had been previously proven to be an autoprole inin cancer2526 Howeverhagy inhibitoritsOfficial journal of the Cell Death Differentiation AssociationSFtreated HCC cells remains unclear WB assay showedthat siRNA against ALKBH5 siALKBH5 significantlypromoted the transformation of LC3B I to II and degraded p62 Fig 5a This result suggested ALKBH5 to be anegative regulator of autophagy in HCC cells qPCR andWB showed that sicIARS failed to influence ALKBH5mRNA and protein levels Fig 5b c similarly siALKBH5 also had no impact on the relative expressionof cIARS Fig 5d A previous study revealed that siALKBH5 promoted the dissociation of BECN1 and BCL a key step during phagophore nucleation In SFtreated HCC cells we gained similar results via IP assayMore importantly this process can be effectively blockedby cIARS silencing Fig 5e These results demonstratedthat cIARS repressed the biological role of ALKBH5 inautophagycIARS regulates ferroptosis through ALKBH5mediatedautophagyTo explore whether cIARSregulated ferroptosis viaALKBH5 we performed several phenotyperescueexperiments First siALKBH5 successfully rescued theeffects of sicIARS in autophagic flux and ferritinophagyFig 6a b Second ALKBH5 knockdown effectively reintensified the SF cytotoxicity which was remarkablyimpaired by sicIARS Fig 6c Third sicIARSmediateddecrease of MDA Fe2 and increase of GSH can berescued by siALKBH5 Fig 6d“fTaken together a novel circRNA in HCC was revealedin our research We partially clarified its role andmechanism in ferroptosisDiscussionAs a novel class of noncoding RNA circular transcriptshave attracted widespread attention However circRNAregulated ferroptosis in human diseases has not beenwidely investigated A small portion of studies focused onthe circRNAmediated autophagy For instance Chenet al27 reported that circHIPK3 depletion significantlyvia miR1243pSTAT3PRKAAinduced autophagy 0cLiu Cell Death Discovery Page of Fig cIARS is found to be a significant regulator of SFinduced ferroptosis a The expression levels of cIARS after transfection of sicIARS or NCb The evaluation of growth inhibition induced by SF in sicIARS or NC transfected cells at the indicated concentrations for h c The evaluation ofcytotoxicity of SF μM h and Erastin μM h with or without several inhibitors of cell death including ferrostatin1 μM ZVADFMK μM or necrosulfonamide μM d The assessment of MDA Fe2 and GSH during SF treatment μM h p AMPKa axis and there was an antagonistic regulation onautophagy between circHIPK3 and linear HIPK3 Du WWet al28 showed that the oncogenic circDnmt1stimulatedautophagy flux in breast carcinoma via interaction withboth p53 and AUF1 These findings demonstrated thepotential of circRNAsin autophagy regulation andprompted us to explore the role of circRNA in ferroptosiswhich had been identified as an autophagic cell death12research we delineated a mechanism ofcircRNAmediated ferroptosis during SF treatment inHCC cells circRNA cIARS hsa_circ_0008367 wasIn ourOfficial journal of the Cell Death Differentiation Associationscreened from RNAseq analysis Phenotypically cIARSpositively regulated ferroptosis which may be partiallydependent on autophagy and ferritinophagy Mechanistically cIARS physically interacted with RBP ALKBH5and negatively regulated its role in autophagyitis essentialTo comprehensively investigate the complicated cirto deeply evaluate itscRNA networkbinding partners In this study cIARS is found to be aninteractor of RBP ALKBH5 which had previously beenreported to be a N6methyladenosine m6A eraser Itsrole in autophagy regulation is completely different in 0cLiu Cell Death Discovery Page of Fig cIARS positively regulates SFinduced autophagy and ferritinophagy a The expression levels of LC3B and p62 in sicIARSintroduced cellswith or without SF treatment b Microscopic observation of autophagy flux after transfection of AdmCherryGFPLC3 for h mCherryGFPLC3 wasvisualized by fluorescence microscopy The green fluorescent protein is acid sensitive and will be quenched in the acidic condition of lysosome while themCherry is stable at low pH Thus the red puncta represent autolysosomes and the yellow puncta generated from the merge of both green and red signalsrepresent autophagosomes The autophagosomal and autolysosomal abundance was measured by the number of puncta lower density of yellow and redpuncta suggested lower level of autophagic flux c Visualization of autophagic compartments via TEM The red arrowheads indicate doublemembranedvacuoles d The assessment of the protein levels of FTH1 and NCOA4 in sicIARS or NC transfected cells with or without SF treatment μM hlower m6A leveldifferent tissues and diseases252629“ In lung cancer25ALKBH5 upregulation stabilized UBE2C an autophagyinhibitor with maintenance ofInovarian cancer26 ALKBH5 inhibited autophagy throughactivating PI3KAktmTOR signaling pathway stabilizedBCL2 mRNA and promoted the interaction betweenBCL2 and BECN1 Herein cIARS was proven to be apivotal regulator of autophagy ferroptosis and ferritinophagy depending on negatively regulating the biologicalinHCC cellsrole of ALKBH5 an autophagy inhibitibco Carlsbad CA USA and penicillin Umlstreptomycin µgml solution in the medical researchcenter of Shandong Provincial Qianfoshan HospitalShandong First Medical University All of the cell linesmentioned in this research were cultured within tenpassages The transfection experiments were performedaided by Lipofectamine Life Technologies Carlsbad CA USA The RNA oligonucleotides in this workwere designed and constructed by GenePharma Shanghai China including siRNAs against cIARS or ALKBH5and the corresponding negative controls NC and NC™Sequences were shown as follows ²“² sicIARS GACUUU GAG GAG AUC AGA CAC siALKBH5 GGAUAU GCU GCU GAU GAA ATT NCNC² UUC UCCGAA CGU GUC ACG UIn this study the cIARS“ALKBH5 axis was demonstrated to be a key mechanism regulating ferroptosisduring SF treatment Further studies are still needed atthe clinical and mechanistic levelsMaterials and methodsCell culture and transfection assayUnder humidified conditions with CO2 at °C theHCC cell lines HepG2 SMMC7721 and Huh7 boughtfrom the National Infrastructure of Cell Line Resourcewere cultured in Dulbecco™s Modified Eagle™s mediumHyClone Logan UT USA with fetal bovine serumCell Counting Kit8 CCK8 assayThe growth inhibition rate of HepG2 and Huh7 cellswas assessed by the CCK8 Dojindo Laboratories Japanassay The blank or transfected cells × cells perwell were seeded into 96well plates with three replicatewells After the treatment with SF or Erastin or variouscell death inhibitorsFerrostatin1 ZVADFMK orOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig cIARS specifically interacts with ALKBH5 a Prediction analysis of the interaction between cIARS and RBPs through CircInteractome b RIPassay evaluating the physical interaction between cIARS and the six candidate RBPs in HCC cells c d RNA EMSA and RNA pulldown evaluating thephysical interaction between cIARS and ALKBH5 e The protein levels of ALKBH5 in HCC cells in the absence or presence of SF treatment f Theassessment of the physical binding of cIARS and ALKBH5 by RIP assay in the absence or presence of SF treatment p Necrosulfonamidefor h a µl volume CCK8reagent was added to each well Then measuring theabsorbance at nm after incubation with the CCK8solution at °C for hRNA extraction and analysisExpression profiles of genomewide circRNAs in threepairs of HCC cell lines before and after SF treatmentwere explored on an Illumina HiSeq platform byNovogene Beijing China The cDNA from divergentprimers was subjected to Sanger sequencing by Zhonghong Boyuan Biological Technology Jiangxi ChinaAgarose gel electrophoresis was used to detect the qPCRamplification of cDNA and genomic DNA after applyingwith the divergent and convergent primers of cIARSTherelative fold changes in expression were calculated withthe formula ˆ’ΔΔCt The sequences of all the primers werelisted as follows ²“² cIARS F AGC GAT GAC TTTGAG GAG ATC A R CCC AGT AGC ACA GGT CATTG IARS F CAT ATC CAG TTT CTC CAT CGG A RTGG ATT TTC CAG GAG CAA TAC T ALKBH5 FGCA AGG TGA AGA GCG GCA TCC R GTC CACCGT GTG CTC GTT GTA C U6 F CTC GCT TCGGCA GCA CAT A R ATT TGC GTG TCA TCC TTGCG GAPDH F CAG AAC ATC ATC CCT GCC TCTAC R ATG AAG TCA GAG GAG ACC ACC TGRibonuclease R RNase R assayRNase R assay R0301 Uµl Geneseed GuangzhouChina was used for the identification of circRNAAccording to the manufacturer™s guidance µl ×Reaction Buffer and U RNase Rµg RNA were mixedto the total RNA and then added RNaseFree Water toform a µl reaction solution system After digestion withRNase R for min at °C the enzyme then was inactivated at °C for min and then directly performOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig cIARS repressed the role of ALKBH5 in the regulation of autophagy a The expression levels of LC3B and p62 in siALKBH5 or NC²introduced cells with or without SF administration b c The relative expression of ALKBH5 mRNA and protein in sicIARS or NC introduced HCC cellsd The relative expression of cIARS in siALKBH5 or NC² transfected cells e The assessment of the role of sicIARS in ALKBH5mediated interactionbetween BECN1 and BCL2Official journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of see figure on previous pageFig cIARS regulates ferroptosis through ALKBH5 a b The evaluation of the role of siALKBH5 in sicIARSregulated autophagy and ferritinophagyThe WB assay demonstrated that the effect of sicIARS on LC3 lipidation and p62 accumulation can be significantly reversed by siALKBH5 in SF μM h treated HCC cells a the effect of sicIARS on the accumulation of FTH1 and NCOA4 can be evidently rescued by siALKBH5 in either HepG2 or Huh7cells treated by SF μM h b c Identification of the role of ALKBH5 in cIARSregulated growth inhibition by CCK8 The results demonstrated thatsiALKBH5 significantly reversed sicIARSmediated growth inhibition in SF administered HCC cells d“f The rescue experiments evaluating the effects ofsiALKBH5 in the sicIARSmediated ferroptotic events The relative levels of MDA and iron were obviously decreased and the level of GSH increased insicIARS introduced HCC cells these impacts can be effectively reversed by ALKBH5 knockdown The black whiskers indicated the difference between theœsicIARS and œNC groups while the red ones indicated the difference between the œsicIARSNC™ and œsicIARSsiALKBH5 groups p reverse transcription reaction The qPCR assay was usedto determine the relative expression of cIARS IARS andGAPDH compared to the mock groupActinomycin D ActD assayActD assay HY17559 MedChem Express New JerseyUSA was used to detect the stability of RNA μgmlActD reagent was used to treat HepG2 and Huh7 cellsAfter or h of administration the total RNAwas extracted respectively to determine the relativeexpression of cIARS or IARS by qPCR assayWestern blot analysisRadioimmunoprecipitation assay buffer RIPA was appliedto lyse cells Total proteins were then harvested and quantified with bicinchoninic acid assays Beyotime ShanghaiChina The target proteins were separated through SDSPAGE and transferred to polyvinylidene fluoridemembranes Merck Millipore Burlington MA USA Themembranes were blocked with nonfat milk incubated withprimary antibodies and then incubated with secondaryantibody diluted at a ratio of Jackson ImmunoResearch West Grove PA USA The primary antibodieswere antiLC3B Cell Signaling Technology BeverlyMA USA antip62 Cell Signaling TechnologyantiHuR ab28660 Abcam Cambridge MA USA antiSFRS1 ab133689 antiFMRP ab17722 antiALKBH5ab195377antiLIN28Aab46020 antiFTH1 ab65080 antiNCOA4 ab86707antiBCL2 ab32124 antiBECN1 ab62557 and antiGAPDH ab9485 The protein signals were visualized withenhanced chemiluminescence detection reagentsECLMillipore Burlington MA USA and quantified with ImageLab software BioRad Hercules CA USAantiIGF2BP1ab82968based on the 3rd edition of the Guidelines for the Interpretation of Assays for Monitoring Autophagy32ImmunoprecipitationandImmunoprecipitation KitRIP and IP were performed to confirm the RNA“proteinand protein“protein interactions using RIP Kit Milliporeab206996according to the manufacturer™s guidance RIP related detailshad been described in our previous studies3334 The IP assayconsists of four steps including antibody binding beadspreparation bead capture and elution The volume of theantibody binding system was made up to µl with lysisbuffer containing the protease inhibitor cocktail and gentlymixed for h the protein AG sepharose µlreactionwas washed twice with wash buffer centrifuged at × gfor min and aspirated the supernatant between washesRNA pulldown assayPierce„¢ Magnetic RNAProteinPullDown KitThermo was applied to evaluate RNA“protein interaction using biotinlabeled junctionspecific probe and itsnegative control designed and synthesized by ViageneBiotech Jiangsu China Detailed procedure strictly followed the manufacturer™s guide cIARSprobe ²TGTAAA TTA GAG GAG TGT CTG ATC TCC TCA AAGTC3² Negative control ²GCA GCC TGA TCA CGACTG ACT TTA GTG TTT GCA TT3²RNA EMSAThe LightShift Chemiluminescent RNA EMSA KitThermoFisher Scientific was applied to evaluate theinteraction between cIARS and ALKBH5 according to themanufacturer™s guidance The details were described inour as previous research34Observation of autophagy fluxLipid peroxidation assayAdmCherryGFPLC3 adenovirus Servicebio Technology Wuhan China was transfected into HCC cellsAfter h incubation cells were observed and photographed using a fluorescence microscope OlympusFSX100 Tokyo Japan Autophagic compartments werefinely observed through transmission electron microscopyTEM HT7700 HITACHI Tokyo Japan The identification of autophagic vacuoles in TEM images was mainlyThe relative level of MDA was evaluated through Lipidab118970 according to the manuPeroxidation Kitfacturer™s guidanceIron assayThe relative level of intracellular iron was determinedby an Iron Assay Kit ab83366 according to the manufacturer™s instructionsOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Glutathione assayThe relative level of intracellular GSH was assessedthrough a GSH Colorimetric Detection Kit CS0260SigmaAldrich St Louis USA according to the manufacturer™s instructionsStatistical analysisThe statistical analyses in this work were carried outusing Prism The results from at least three independenttests are shown as the mean value ± standard deviationSD The mean values of two groups were compared viaunpaired Student ttests œpvalue  was defined assignificant p p AcknowledgementsThis work was supported by the National Natural Science Foundation of China[Grant No ] Shandong Natural Science Foundation [Grant NoZR201808200282] and Medical and Pharmacological TechnologyDevelopment plan Shandong [Grant Nos 2017WS188 and 2017WS284]Author details1Department of Hepatobiliary Surgery The First Affiliated Hospital ofShandong First Medical University Jinan Shandong China2Department of Hepatobiliary Surgery Shandong Provincial QianfoshanHospital Shandong University Jinan Shandong China 3Department ofReproduction Medicine Jinan Maternal and Child Health Care HospitalAffiliated to Shandong First Medical University Jinan Shandong ChinaAuthor contributionsStudy design ZL XW and JL data collection QW ZX and XW dataanalysis ZL QW and XW cellular experiments QW ZL XW and ZXmanuscript preparation ZL QW ZX and JL figures and manuscriptproofing ZL QW JL and XW project administration XW and JLConflict of interestThe authors declare that they have no conflict of interestPublisher™s noteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsReceived May Revised July Accepted July References Bray F Global cancer statistics GLOBOCAN estimates of incidenceand mortality worldwide for cancers in countries CA Cancer J Clin “ Siegel R L Miller K D Jemal A Cancer statistics CA Cancer J Clin “ Forner A Reig M Bruix J Hepatocellular carcinoma Lancet “ Li J Ferroptosis past present and future Cell Death Dis Llovet J M Sorafenib in advanced hepatocellular carcinoma N Engl JMed “ Cheng A L Efficacy and safety of sorafenib in patients in the AsiaPacificregion with advanced hepatocellular carcinoma a phase III randomiseddoubleblind placebocontrolled trial Lancet Oncol “ Official journal of the Cell Death Differentiation Association Dixon SJ Pharmacologicalinhibition of cystineglutamateexchange induces endoplasmic reticulum stress and ferroptosis Elife e02523 Stockwell B R Ferroptosis a regulated cell death nexus linking metabolism redox biology and disease Cell “ Jung C H ULKAtg13FIP200 complexes mediate mTOR signaling to theautophagy machinery Mol Biol Cell “ Itakura E Mizushima N Characterization of autophagosome formation siteby a hierarchical analysis of mammalian Atg proteins Autophagy “ Geng J Klionsky D J The Atg8 and Atg12 ubiquitinlike conjugation systems in macroautophagy ˜Protein modifications beyond the usual suspects™review series EMBO Rep “ Zhou B Ferroptosis is a type of autophagydependent cell death SeminCancer Biol httpsdoi101016jsemcancer201903002 Torii S An essential role for functional lysosomes in ferroptosis of cancercells Biochem J “ Gao M Ferroptosis is an autophagic cell death process Cell Res “ Hou W Autophagy promotes ferroptosis by degradation of ferritinAutophagy “ Memczak S Circular RNAs are a large class of animal RNAs with regulatory potency Nature “ Bi W CircRNA circRNA_102171 promotes papillary thyroid cancer progression through modulating CTNNBIP1dependent activation of betacateninpathway J Exp Clin Cancer Res Su Y circRIP2 accelerates bladder cancer progression via miR1305Tgfbeta2smad3 pathway Mol Cancer Chen J Circular RNA profile identifies circPVT1 as a proliferative factorand prognostic marker in gastric cancer Cancer Lett “ Wang M Yu F Li P Circular RNAs characteristics function and clinicalsignificance in hepatocellular carcinoma Cancers httpsdoi103390cancers10080258 Glazar P Papavasileiou P Rajewsky N circBase a database for circular RNAsRNA “ Deng W Wang Y Liu Z Cheng H Xue Y HemI a toolkit for illustratingheatmaps PLoS ONE e111988 Dudekula D B CircInteractome A web tool for exploring circular RNAsand their interacting proteins and microRNAs RNA Biol “ Dixon S J Ferroptosis an irondependent form of nonapoptotic celldeath Cell “ Guo J Deregulation of UBE2Cmediated autophagy repression aggravates NSCLC progression Oncogenesis Zhu H A

Thyroid_Cancer

Association of variant on the promoter of cluster ofdifferentiation in graves disease and gravesophthalmopathyYuHuei Liu123 ChiouYuan Shen1 and FuuJen Tsai3451Graduate Institute of Integrated Medicine China Medical University Taichung Taiwan 2Drug development center China Medical University Taichung Taiwan 3Department ofMedical Genetics and Medical Research China Medical University Hospital Taichung Taiwan 4Department of Pediatrics China Medical University Hospital Taichung Taiwan5School of Chinese Medicine China Medical University Taichung TaiwanCorrespondence YuHuei Liu yuhueiliumailcmuedutwThe macrophage migration inhibitory factor MIFcluster of differentiation CD74plays a role in immunological functions The present study aims to investigate whethersinglenucleotide polymorphisms SNPs in the MIF and CD74 are risk factors for developing Graves ophthalmopathy GO in patients with Graves disease GD A case“controlstudy enrolled patients with GD with and without GO and healthy individuals SNPs were discriminated using realtime polymerase chain reaction Hardy“Weinbergequilibrium as well as frequencies of allele and genotype between GD patients with andwithout GO were estimated using the Chisquare test The effects of CD74 on adipocyteproliferation and differentiation were evaluated using 3T3L1 preadipocytes QuantitativeDNAimmunoprecipitation was used to detect the binding capacity of NR3C1 and FOXP3to AG oligonucleotides The results showed that individuals carrying the GG genotype atrs2569103 in the CD74 had a decreased risk of developing GD P3390 — ˆ’ oddsratio OR confidence interval CI “ however patients with GDcarrying the AG genotype at rs2569103 in the CD74 had an increased risk of developing GOP0009 OR CI “ The knockdown of CD74 reduced adipocyteproliferation and differentiation NR3C1 had a higher affinity for A whereas FOXP3 had ahigher affinity for G of rs2569103 The results suggested the existence of a link between thegenetic variation of CD74 promoter and the risk for developing GD and GO which shouldbe considered in clinical practiceBackgroundGraves disease GD a complex autoimmune disorder that occurs more often in women is characterized by the presence of autoantibodies and thyroidstimulating immunoglobulins targeting thethyroidstimulating hormone receptor to stimulate both thyroid hormone synthesis and thyroid glandgrowth and results in hyperthyroidism and its accompanying features [“] Graves ophthalmopathyGO is one common anspecific complication affecting “ of patients with GD [] Activation oforbital fibroblasts through proliferation and differentiation into adipocytes and myofibroblasts is thoughtto play a major role in the generation of the extracellular matrix During inflammatory cell infiltrationand edema the activation augments the volume of tissues surrounding the eyes which in turn leads to anincrease in intraocular pressure []Genetic predispositions epigenetic regulations and environmental factors are risk factors for GD andGO [“] Representative studies shed new light on the pathogenesis of GD such as thyroid antigensthyroidstimulating hormone receptor and human leukocyte antigen HLA class I and II regions []However the genomewide approaches to determining the relative risks of developing GO are relativelyReceived June Revised July Accepted July Accepted Manuscript online August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072limited [] Candidate gene approaches revealed that polymorphisms of genes involved in immune response andinflammation might be linked to the development of GO [“]Cluster of differentiation CD74 encoded by CD74 is an HLA class II histocompatibility antigen gamma chainalso known as HLADR antigenassociated invariant chain and a signaltransducing receptor of macrophage migration inhibitory factor MIF that maintains cell proliferation and survival [] The singlenucleotide polymorphisms SNPs in HLA class II and MIF play a role in the development of GD [“] Conversely the chromosome5q3133 region where CD74 is located 5q32 may play a pivotal role in the development of GD and could be thesusceptibility region for developing GD [] Results from mRNASeq also reveal CD74 as a novel signature fD However to our knowledge there is no study on the putative impact of CD74 locus variations on the risk ofGD or GO In an attempt to contribute to the understanding of the pathogenic processes underlying GD and GO acase“control study was designed to evaluate the association between SNPs in the upstreamdownstream regulatoryregion of the MIFCD74 axis and the risk of developing GD and GOMethodsPatients healthy individuals and DNA isolationThe study followed the Declaration of Helsinki and was approved by the Medical Ethics Committee of China MedicalUniversity Hospital DMR100IRB144 CMUH103REC2071 A total of patients with GD females100males mean age y range “ y at enrollment from the China Medical University Hospital and patients had GO and did not All participants provided written informed consent Detailed descriptions of theinclusionexclusion criteria blood drawing and handling genomic DNA storage and quality assurance have beendescribed [] SNP data for ethnicitymatched healthy individuals were obtained from the Taiwan biobankSNP selection and genotypingSNPs were selected based on the following criteria i a threshold minor allele frequency MAF in the Asian population of ii primerprobe set passed by the manufacturer criteria to ensure a high genotyping success rate andiii SNP data for healthy individuals could be obtained without imputation from the Taiwan biobank Four SNPsnamely rs476240 and rs507715 in the downstream region of MIF which is also the upstream region of MIF antisense RNA [MIFAS1] as well as rs13175409 and rs2569103 in the upstream region of CD74 were analyzedGenotyping using specific primerprobe sets have been described previously []Cell cultureThe human HEK293 cells and mouse 3T3L1 preadipocytes were obtained from Bioresource Collection and Research Center BCRC Hsinchu Taiwan and maintained in Dulbecco™s modified Eagle™s medium DMEM Thermo Fisher Scientific Waltham MA USA with fetal bovine serum Uml penicillin and μgml streptomycin and mM Lglutamine at —¦C in a humidified atmosphere of CO2CD74 knockdownShort hairpin RNAs shRNAs obtained from the RNAi core Academia Sinica Taipei Taiwan were used in CD74knockdown experiments For CD74 knockdown confluent 3T3L1 preadipocytes in sixwell dishes were incubated inOptiMEM Thermo Fisher Scientific and transfected with either CD74 shRNA or nonspecific shRNA using Lipofectamine Thermo Fisher Scientific according to the manufacturer™s protocol After h the medium was replacedwith complete DMEM with a differentiation cocktail μM 3isobutyl1methylxanthine μM dexamethasoneand μM insulin to induce differentiation into mature adipocytes day Western blottingEqual amounts of protein lysates were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis andthen transferred to polyvinylidene fluoride membranes After blocking with skim milk the membranes wereincubated with primary antibodies and subsequently with appropriate peroxidaseconjugated secondary antibodiesPrimary antibodies including targets catalog numbers dilutions and suppliers were as follows antibodies specific toCD74 GTX110477 were from GeneTex Hsinchu Taiwan and antibodies specific to actin MAB1501 were from MilliporeSigma St Louis MI USA The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Adipocyte differentiationThe 2day postconfluency preadipocytes were cultured in complete DMEM with a differentiation cocktail μM3isobutyl1methylxanthine μM dexamethasone and μM insulin On day of differentiation cells wereswitched to complete DMEM with μM insulin for the remaining duration of differentiationCell counting3T3L1 cells were detached from sixwell plates using trypsin Thermo Fisher Scientific resuspended in complete DMEM and counted using a cell counter Millipore every day from day “Oil Red O stainingDifferentiated adipocytes were fixed in formalin and stained for min with Oil Red O MilliporeSigma working solution Oil Red O dye in isopropanol Oil Red O was extracted using isopropanol and theabsorbance was measured at nm using a spectrophotometerCell culture and extraction of nuclear proteins from established NR3C1FOXP3 and CD74 transformantsCells were transfected with the pCMV3ˆ’Cˆ’Mycˆ’NR3C1 pCMV3ˆ’Cˆ’Mycˆ’FOXP3 or pCDNA4CD74 usingthe Lipofectamine kit Thermo Fisher Scientific according to the manufacturer™s protocol The nuclear proteinswere extracted using NEPER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific supplementedwith protease inhibitor cocktail and phosphatase inhibitors Roche Basel Switzerland according to the manufacturer™s protocolQuantitative DNA immunoprecipitation qDNAIP assayqDNA“IP assays were performed on nuclear extracts from established FOXP3 and NR3C1 transformantsDNA binding of FOXP3 or NR3C1 was assessed using the annealed double strand oligonucleotides 5cid3biotinlabeled rs2569103A probes 5cid3CCAAATGGCTGGTTTCAGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCTGAAACCAGCCATTTGG3cid3 as well as 5cid3biotinlabeled rs2569103G probes 5cid3CCAAATGGCTGGTTTCGGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCCGAAACCAGCCATTTGG3cid3 PURIGOBiotechnology Taipei Taiwan For the binding reactions μg of nuclear proteins were incubated with or without labeled oligonucleotides in binding buffer [ mM Tris“HCl pH mM NaCl mM MgCl2 mMEDTA mM DTT mgml polydI“dC and glycerol] for min at —¦C in a final volume of μl FOXP3“ or NR3C1“nucleotide complexes were crosslinked with formaldehyde final concentration for min at room temperature followed by immunoprecipitation with antibodies specific to Myc tag GTX115046 GeneTex and Protein AG magnetic beads GE Healthcare Immunoprecipitated DNA was detected usinghorseradish peroxidaseconjugated streptavidin The reaction was developed with the 33cid355cid3tetramethylbenzidinereagent Sigma and read at nm with a Microplate reader BioRad Hercules CA USAStatistical analysesThe statistical analyses were performed using the PASW Statistics software from IBM Armonk NY USAA ttest was used to evaluate the associations between GO and age A Chisquare test was used to evaluate the associations between polymorphisms and GD or GO Screening for linkage disequilibrium LD was performed usingHaploview ver [] A twotailed Pvalue less than with Bonferroni correction was considered statistically significant [] Logistic regression with a confidence interval CI was used to estimate odds ratiosORsResultsDemographic data clinical characteristics and their correlations withGO in patients with GDThe frequency distributions of clinical characteristics such as goiter nodular hyperplasia myxedema vitiligo andage in male and female groups were compared between the patients with GD with or without GO As demonstratedin Table gender and age were significantly associated with GO in patients with GD Even myxedema was associatedwith GO in patients with GD however due to a limited number of cases the association needs further investigation The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Demographic data and clinical characteristics of graves disease patients with or without graves ophthalmopathyCharacteristicGDnonGO N GDGO N PNumber of patientsFemale genderAge of diagnosis Year Mean ˆ’ SD[Range]Presence of goiterNo1a1bPresence of nodular hyperplasiaPresence of myxedemaPresence of vitiligoWith radioiodine therapy historyWith thyroid surgery historyWith smoke historyFree T3 pgmlFree T4 ngdlT3 ngdlT4 μgdlTSH μIUmlTRAb positive ˆ’ [ˆ’] ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ [ˆ’] ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ Abbreviations GD graves disease GO graves ophthalmopathy N numberaFrequencies of genotypes were determined by the chisquare test using — or — contingency tablesbSignificance of age were evaluated by t testP005P00010039a — 105b 0165a0539a0039a 0743a0273a0227a0527a0900a0692a0146a0310a0479a0482aThese results adhered to other epidemiological results that GO occurred more commonly in the middleaged femalepopulationLD among SNPs of MIF and CD74Four SNPs of the MIF and CD74 were genotyped to determine whether polymorphisms in these genes influencethe development of GO in patients with GD The distribution of the four SNPs fit the Hardy“Weinberg equilibriumHWE in patients with GD and healthy individuals However the strong r208 LD r2 values calculated for thetwo SNPs at the CD74 in healthy individuals were not observed in patients with GD with or without GO suggestingthat there is more variation in the extent of LD within CD74 in patients with GD Figure Allele and genotype distributions of CD74 contribute to GDGOdevelopmentNo significant association was found in the examined SNPs of MIF nor was a significant association found betweenthe polymorphisms and the clinical features or the indicators of thyroid function including free triiodothyronineT3 free thyroxine T4 thyroid stimulating hormone TSH and thyrotropin receptor antibodies TRAbs in patients with GD However allele frequencies showed that individuals carrying a G allele at rs2569103 in the CD74 hada reduced risk of developing GD P0005 OR CI “ Table Genotype frequenciesfurther showed that individuals carrying the GG genotype at rs2569103 in the CD74 had a reduced risk of developing GD P3390 — ˆ’ OR CI ˆ’ which was consistent with results from allelefrequencies however the patients with GD carrying the AG genotype at rs2569103 in the CD74 had an increasedrisk of developing GO P0009 OR CI ˆ’ Table The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Linkage disequilibrium LD values between the two polymorphisms rs13175409 and rs2569103 in the CD74region in a TaiwaneseChinese populationThe color scale reflects the strength of LD between the two single nucleotide polymorphisms SNPs A Healthy individuals BPatients with Graves disease GD with and without Graves ophthalmopathy GO C Patients with GD without GO D Patientswith GD with GOTable Allele distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDPaControl vs GDOR 95CINonGO vsGO PaNonGO vsGO OR95CIMIF rs476240AGMIF rs507715ACCD74 rs13175409CTCD74 rs2569103AG ˆ’0929bAbbreviations CI confidence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using — or — contingency tablesbOdds ratios and CI per genotype were estimated by applying unconditional logistic regressionP005 with Bonferroni correction OR with significanceKnockdown of the expression of CD74 inhibits 3T3L1 adipocytedifferentiationThe swelling of extraocular orbital fat is one reason that the development of GO is triggered [] To understand thepossible regulation between CD74 and adipocyte differentiation 3T3L1 cells were chosen as an experimental modelThe expression of CD74 in CD74 knockdown CD74KD cells by shRNA was confirmed as compared with those withcontrol of shRNA Figure 2A Cell numbers of CD74KD and control cells were counted every day The knockdownof CD74 decreased cell proliferation from “ days after induction Figure 2B In addition the degree of Oil Red The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Genotype distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDP aControl vs GDOR 95CINonGO vsGO P aNonGO vsGO OR95CIMIF rs476240AAAGGGMIF rs507715AAACCCCD74 rs13175409CCCTTTCD74 rs2569103AAAGˆ’2495cGG — ˆ’ bˆ’0154bˆ’2467bˆ’Abbreviations CI confidence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using — or — contingency tablesbOR and CI per genotype were estimated by applying unconditional logistic regressioncOR and CI per genotype were estimated by adjusting with gender age and myxedemaP005 with Bonferroni correctionOR with significanceFigure Changes in adipocyte differentiation and proliferation after knockdown of CD74A Endogenous expression of CD74 protein in 3T3L1 cells was examined and knockdown of CD74 was examined by Westernblotting Actin was used as an internal control B The downregulation of CD74 inhibits cell growth 3T3L1 cells were detachedfrom sixwell plates and counted P001 P0001 CD74 knockdown vs control cells C Cells were stained with Oil Red Oafter inducing differentiation Quantitative analyses were performed by measurement of optical density OD at nm in extractsfrom Oil Red Ostained cells transfected with CD74 short hairpin RNA shRNA and control shRNA P0001 CD74 knockdownvs control cellsO staining was weaker in CD74KD cells than in control cells on day and on day respectively forCD74 shRNA vs control cells Figure 2C The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Different binding affinities of NR3C1 and FOXP3 for CD74 promoterdepends on SNP rs2569103The CD74 SNP rs2569103 was located within the upstream region of CD74 and showed the strongest associationwith the disease making it a possible target for transcription factors Indeed the putative transcription factorbindingsites were predicted using PROMO [] At SNP rs2569103 the A allele generates motifs for nuclear receptorsubfamily group C member NR3C1 TCAGG whereas the G allele generates a motif for forkhead box P3FOXP3 GTTTCG Bulk RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasetsPRJEB4337 were demonstrated Figure 3A To interpret the possible regulatory mechanisms of these moleculespublished mRNA expression results were explored The mRNA expression of NR3C1 only showed a negative correlation with that of CD74 in thymoma samples Pearson™s correlation ˆ’ Spearman™s correlation ˆ’ Figure3B whereas the mRNA expression of FOXP3 showed a positive correlation with that of CD74 Pearson™s correlation Spearman™s correlation in thymoma samples welldifferentiated papillary thyroidcarcinoma and welldifferentiated thyroid cancer respectively Figure 3C“E The qDNAIP results supported thatNR3C1 tends to bind to probes with promoter sequence containing AA at rs2569103 whereas FOXP3 tends to bindto probes with promoter sequence containing GG at rs2569103 Figure 3F These results suggested that the CD74expression may be orchestrated by complex transcription factor networks The AA genotype may play a role in response to NR3C1induced CD74 downregulation whereas the GG genotype on rs2569103 on the CD74 promotermay play an additional role in response to FOXP3induced CD74 upregulationDiscussionEnvironmental factors and genetic loci have been thought to be associated with immune regulation [] Here weidentified new candidates CD74 alleles and genotypes for the susceptibility of GD and GO in a TaiwaneseChinesepopulation CD74 is involved in adipocyte differentiation through its differential promoter binding affinity for transcription factors To the best of our knowledge this is the first study to demonstrate novel CD74 polymorphisms inassociation with the development of GD and GO Our results support wholegenome screening studies in that thechromosome 5q32 may play a role in generating GD and GO in humansThe thyroid gland of patients with GD revealed marked enlargement of the gland due to autoantibodies Patientswith accompanying GO exhibited enlargement of the retroorbital connective tissue and extraocular muscles inpart due to the inflammatory deposition of glycosaminoglycans collagen and fat [] Indeed genes involved inthe regulation of cell survival DNA transcription and protein synthesis have been considered risk factors for GDand GO [] Overexpression of CD74 plays a crucial role in preventing hyperreactivity between immature antigens and major histocompatibility complex class II as well as cell growth and survival whereas downregulation ofCD74 is often correlated with autoimmunity and cell apoptosis [] Upon expression of surface CD74 the cellsmay transduce survival signaling through extracellular signalregulated kinase or cJun Nterminal kinase JNKmitogenactivated protein kinase MAPK pathways or AKT pathways in a MIFdependent manner thereby improving cell survival and proliferation [] Due to the limitation to find identical cells expressed GG or AA genotypeon rs2569103 current results we did not show the direct impact of these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients withor without GO although loss the protective GG genotype most of them hold AG heterogenous genotype insteadsuggested the lossofprotect effect on the disease In the present study cellbased experiments showed that CD74 isinvolved in adipocyte differentiation but the link toward GO development remained to be investigated On the otherhand the GG genotype on rs2569103 with a higher frequency in healthy individuals Table increased the bindingof FOXP3 to the CD74 promoter Figure 3F thereby increasing CD74 upregulation and protecting autoimmuneresponses Conversely the AA genotype on rs2569103 increases the binding of NR3C1 to the CD74 promoter whichdownregulates CD74 and increases autoimmune response and manifestations of GDGO Due to the limitation tofind identical cells expressed GG or AA genotype on rs2569103 current results we did not show the direct impactof these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients with or without GO although they lost the protective genotype mostof them hold the AG heterogenous genotype instead suggesting the lossofprotection effect of the disease Furtherstudies on the detailed mechanisms through CD74derived adipocyte differentiation are warrantedConversely the ligand of CD74 MIF has previously been reported to be counterregulatory to glucocorticoid secretion [“] The glucocorticoidinduced MIF secretion was noted at min after dexamethasone administration[] In addition nonsteroidal antiinflammatory drugs such as aspirin ibuprofen and naproxen have been used torelieve the pain and inflammation of GO This evidence further supports the crucial role of CD74 in the transduction The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Different binding affinities of NR3C1 and FOXP3 for CD74 promoter depends on singlenucleotide polymorphismSNP rs2569103A RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasets PRJEB4337 B“E Bioinformaticanalysis of mRNA expression correlation between NR3C1 and CD74 or FOXP3 and CD74 The mRNA expression of NR3C1 andCD74 in thymoma samples B and the mRNA expression of FOXP3 and CD74 in thymoma samples C welldifferentiated papillarythyroid carcinoma D and welldifferentiated thyroid cancer E F Probe with promoter sequence containing rs2569103 probe Ahas a higher affinity for NR3C1 whereas G at rs2569103 probe G has a higher affinity for FOXP3 as shown by quantitative DNAimmunoprecipitation qDNAIP assay P001 P0001 probe A vs probe G The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072of MIF signaling However due to the limited population of the minor polymorphism the present study is unable toreach the interactions among cells and molecules in the orbital microenvironment and their association toward thetarget polymorphism due to the inaccessibility of the orbital tissues The current finding may have further implications for understanding the link between the polymorphismexpression of CD74 and current treatments for GO”atherapeutic effect issue that might be of value for future treatment strategies targeting MIF or CD74In conclusion the current study identified new SNPs in the CD74 that were found to be associated with GD and GOin a TaiwaneseChinese population Biological studies provide insights into the genetic information that influencesthe development of GD and GO via adipocyte proliferation and differentiationPerspectives¢The impact of genetic factors on the orbital microenvironment cannot be closely monitored due tothe inaccessibility of the orbital tissue Studies on feasible cellbased models may help elucidate howgenetic factors such as CD74 SNPs modulate the target gene expression¢¢The present study combined clinical observations and cell models to investigate how CD74 polymorphisms affect adipocyte proliferation and differentiationThe present clinical observations suggest that the genetic factors of CD74 should be considered inclinical practiceCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThis work is supported by Ministry of Science and Technology Taiwan [grant numbers MOST 1042815C039002B and MOST1072320B039032MY3] the peak project and thematic project of Academia Sinica Taiwan the higher education sproutproject by the Ministry of Education MOE Taiwan via œDrug Development Center of China Medical University from The FeaturedAreas Research Center Program and China Medical University [grant numbers CMU105S33 and CMU106S46] TaichungTaiwanAuthor ContributionYHL proposed the concept designed the experiment anized the study wrote and reviewed the manuscript CYS performed the experiments FJT coordinated patient enrollment collected the clinical samples and applied official applicationAcknowledgementsWe thank Taiwan Biobank for providing related data all anonymous for our research The sponsorfunding anization had norole in the design or conduct of this researchAbbreviationsCD74 cluster of differentiation CI confidence interval FOXP3 forkhead box P3 GD graves disease GO graves ophthalmopathy HLA human leukocyte antigen HWE Hardy“Weinberg equilibrium JNK cJun Nterminal kinase LD linkagedisequilibrium MAPK mitogenactivated protein kinase MIF macrophage migration inhibitory factor NR3C1 nuclear receptorsubfamily group C member OR odds ratio PCR polymerase chain reaction qDNAIP quantitative DNA immunoprecipitation SNP singlenucleotide polymorphism T3 triiodothyronine T4 free thyroxine TRAb thyrotropin receptor antibody TSHthyroid stimulating hormoneReferences Smith TJ and Hegedus L Graves™ Disease N Engl J Med “ 101056NEJMra1510030 Brent GA Clinical practice Graves™ disease N Engl J Med “ 101056NEJMcp0801880 Ginsberg J Diagnosis and management of Graves™ disease CMAJ Canadian Med Assoc J J de l™Assoc Med Canadienne “ The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BY 0cBioscience Reports BSR20202072101042BSR20202072 McIver B and Morris JC The pathogenesis of Graves™ disease Endocrinol Metab Clin North Am “101016S0889852905702991 Bednarczuk T Gopinath B Ploski R and Wall JR Susceptibility genes in Graves™ ophthalmopathy searching for a needle in a haystackClin Endocrinol Oxf “ 101111j13652265200702854x Anvari M Khalilzadeh O Esteghamati A Esfahani SA Rashidi A Etemadi A et al Genetic susceptibility to Graves™ ophthalmopathy therole of polymorphisms in proinflammatory cytokine genes Eye Lond “ 101038eye2009244 Bahn RS Understanding the immunology of Graves™ ophthalmopathy Is it an autoimmune disease Endocrinol Metab Clin North Am “ vi Manji N CarrSmith JD Boelaert K Allahabadia A Armitage M Chatterjee VK et al Influences of age gender smoking and familyhistory on autoimmune thyroid disease phenotype J Clin Endocrinol Metab “ 101210jc20061402 Stan MN and Bahn RS Risk factors for development or deterioration of Graves™ ophthalmopathy Thyroid Official J Am Thyroid Assoc “ 101089thy20101634 Bahn RS and Heufelder AE Pathogenesis of Graves™ ophthalmopathy N Engl J Med “ Tomer Y Barbesino G Greenberg DA Concepcion E and Davies TF Mapping the major susceptibility loci for familial Graves™ andHashimoto™s diseases evidence for genetic heterogeneity and gene interactions J Clin Endocrinol Metab “ Tomer Y Ban Y Concepcion E Barbesino G Villanueva R Greenberg DA et al Common and unique susceptibility loci in Graves andHashimoto diseases results of wholegenome screening in a data set of multiplex families Am J Hum Genet “101086378588 Gianoukakis AG and Smith TJ Recent insights into the pathogenesis and management of thyroidassociated ophthalmopathy Curr OpinEndocrinol Diabetes Obesity “ 101097MED0b013e32830eb8ab Shiina T Ota M Shimizu S Katsuyama Y Hashimoto N Takasu M et al Rapid evolution of major histocompatibility complex class I genesin primates generates new disease alleles in humans via hitchhiking diversity Genetics “101534genetics106057034 Liu YH Chen YJ Wu HH Wang TY and Tsai FJ Single nucleotide polymorphisms at the PRR3 ABCF1 and GNL1 genes in the HLA class Iregion are associated with Graves™ ophthalmopathy in a genderdependent manner Ophthalmology “101016jophtha201404027 Wang S Sun H Chen HY Zhao ZF Yang Y Zhao YJ et al Intercellular adhesion molecule gene polymorphisms do not contribute toGraves™ disease in Chinese patients Endocrine “ 101007s1202000700329 Liu YH Chen RH Chen WC Tsai Y Wan L and Tsai FJ Disease association of the CD103 polymorphisms in Taiwan Chinese Graves™ophthalmopathy patients Ophthalmology “ 101016jophtha200912037 Bednarczuk T Hiromatsu Y Seki N Ploski R Fukutani T Kurylowicz A et al Association of tumor necrosis factor and human leukocyteantigen DRB1 alleles with Graves™ ophthalmopathy Hum Immunol “ 101016jhumimm200402033 Khalilzadeh O Anvari M Esteghamati A Mahmoudi M Tahvildari M Rashidi A et al Graves™ ophthalmopathy and gene polymorphisms ininterleukin1alpha interleukin1beta interleukin1 receptor and interleukin1 receptor antagonist Clin Exp Ophthalmol “ Siegmund T Usadel KH Donner H Braun J Walfish PG and Badenhoop K Interferongamma gene microsatellite polymorphisms inpatients with Graves™ disease Thyroid Official J Am Thyroid Assoc “ 101089thy199881013 Wong KH Rong SS Chong KK Young AL Pang CP and Chen LJ Genetic Associations of Interleukinrelated Genes with Graves™Ophthalmopathy a Systematic Review and Metaanalysis Sci Rep 101038srep16672 Bucala R and Shachar I The integral role of CD74 in antigen presentation MIF signal transduction and B cell survival and homeostasis MiniRev Med Chem “ 1021741389557515666150203144111 Leng L Metz CN Fang Y Xu J Donnelly S Baugh J et al MIF signal transduction initiated by binding to CD74 J Exp Med “ 101084jem20030286 Liu YH Chen CC Yang CM Chen YJ and Tsai FJ Dual effect of a polymorphism in the macrophage migration inhibitory factor gene isassociated with newonset Graves disease in a Taiwanese Chinese population PLoS ONE e92849 101371journalpone0092849 Nakabayashi

Thyroid_Cancer

"Gastroenterol Res Primary Localized Amyloidosis of the Intestine A Pathologist ViewpointSaeed Ali Alshehria c Mahmoud Rezk Abdelwahed HusseinbAbstractBackground Localized amyloidosis of the intestine is a rare entity which can clinically masquerade several conditions such as colitis polyps and malignant tumors This study aims to evaluate the clinicopathological features of this entityMethods To evaluate the clinicopathological features of this entity a comprehensive search of the literature to was done using the following keywords œamyloidosis and œsmall intestine or œduodenum or œileum or œjejunum or œcolon We identified studies about gastrointestinal amyloidosis Data were examined for studies about localized intestinal amyloidosis The clinicopathological features were describedResults The age at presentation ranged from to years The male to female ratio was The jejunum and sigmoid colon were the most commonly involved sites Abdominal pain and intestinal obstruction small intestine or rectal bleeding sigmoid region were the most common clinical presentations Colonoscopic findings included wall thickening mucosal ulcerations small intestine and tumorlike masses colonConclusions The clinical presentations of localized intestinal amyloidosis depend on the site of the deposition of the amyloid In most cases amyloid deposits consisted of light chain proteinKeywords Amyloidosis Amyloid Colon Small intestine Rectum Gastrointestinal tractIntroductionThe term œamyloid was initially coined to describe materials Manuscript submitted June accepted July Published online August aDepartment of Pathology Armed Forces Hospitals Southern Region King Fahd Hospital Saudi ArabiabDepartment of Pathology Faculty of Medicine Assuit University Hospitals Assuit EgyptcCorresponding Author Saeed Ali Alshehri Department of Pathology Chemistry Section Armed Forces Hospital Southern Region Khamis Mushyte Saudi Arabia Email syd46118gmailcom 1014740gr1303found in plants and has been thought to represent carbohydrate Greek œamylon starch In medical literature the term œAmyloidosis has been known for more than years It was named after the deposition of the hallmark protein called crosssheet amyloid fibrils which represents either misfolded or misassembled proteins The nomenclature of the amyloid protein uses two letters The first letter œA refers to amyloid whereas the second refers to the precursor proteins [] The amyloid proteins include several types such as immunoglobulin light chains AL in primary amyloidosis transthyretin TTR beta2microglobulin in hemodialysisassociated amyloidosis and serum amyloid A SAA protein in secondary AA amyloidosis [] There are some associations between AL amyloid deposition and several hematological conditions including myeloma lymphoplasmacytic disorders and plasma cell dyscrasia Moreover AL amyloidosis and myeloma may show similar genetic abnormalities such as 14q translocations and 13q deletion []Amyloidoses are degenerative conditions associated with significant suffering not only for patients but also for their families as well [] They are characterized by the deposition of extracellular protein fibrils in the ans such as the kidneys nerves gastrointestinal tract heart and skin Amyloidoses are associated with significant morbidities such as malabsorption renal insufficiency and hematological disorders or even lifethreatening events such as intestinal obstruction renal failure and fatal arrhythmia The clinicopathological features of amyloidosis are nonspecific They depend on the type of the precursor protein site and severity of the amyloid deposition The diagnosis of amyloidosis depends on the examination of the tissue biopsies from the lesions It is established by the detection of amyloid deposits with its characteristic birefringence under polarized light following staining with Congo red or thioflavinT or immunohistochemical stains Management of amyloidosis includes reduction of the supply of amyloid fibril precursor proteins stabilization of the precursor protein formation administration of antiplasma therapies general care systemic amyloidosis or surgical resection in localized amyloidosis [ ]Amyloidosis includes both primary and secondary types In the former also known as AL for amyloid light chain there is no evidence of coexisting disease and it has an immunoglobulin light chain as a major protein component In secondary amyloidosis also known as AA for amyloidassociated there is evidence of coexisting disease resulting in the production of inherently amyloidogenic proteins or overproduction of potentially amyloidogenic normal proteins and it has serum amyloidassociated protein SAA The deposition of amys The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastroresThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cLocalized Amyloidosis of the Intestine Gastroenterol Res loid may be either widespread systemic amyloidosis [ ] or restricted to a specific an localized amyloidosis In systemic amyloidosis the amyloidogenic proteins are synthesized at sites far away from the sites of amyloid deposition The amyloid protein is deposited at several sites and therefore the patients have disparate clinical manifestations such as arrhythmia neuropathies malabsorption and proteinuria due to cardiac neural intestinal and renal involvement respectively [] Currently the classification of amyloidosis is based on the type of precursor proteins []Localized amyloidosis refers to the infiltration of the tissues by amyloid where the precursor proteins are synthesized at or close to the site of deposition It includes both anspecific amyloidoses where the amyloid is produced by the an itself and nonan specific amyloidosis where the fibrillar protein is not specific to the affected an [] Localized amyloidosis of the intestine is a rare condition that involves the duodenum jejunum and ileum appendix or colorectum The clinicopathological features are nonspecific and therefore they can masquerade several conditions such as collagenous colitis ischemic colitis bowel infarction perforation polyps and malignant tumors [] The colonoscopic features include mucosal granularity friability erosions ulcerations petechial hemorrhage and polypoid lesions [] The radiological findings include ulcerations thickening of the mucosal folds narrowing of the colonic lumen effacement of haustrations and nonspecific mucosal nodularity []To date few cases of localized intestinal amyloidosis have been reported [ ] In Tada examined patients with small intestinal amyloidosis Clinical findings included malabsorption mass lesions diarrhea and hematochezia Endoscopy of the jejunum revealed polypoid lesions wall thickening erosions ulcerations and fine granular appearance The amyloid proteins included amyloid A protein AA light chain protein AL beta 2microglobulin and prealbumin Histologically amyloid deposits were seen in the submucosa and muscle layer [] Chen reported a case of localized amyloidosis of the transverse colon in a 52yearold male patient The clinical presentations included abdominal pain bloody stool and weight loss Colonoscopy revealed a marked narrowing of the transverse colon associated with the presence of multiple polypoid growths The clinical and radiological findings supported the initial impression of carcinoma and therefore the patient underwent right hemicolectomy with lymph node dissection [] Grossly there was an ulcerative mass with marked narrowing of the lumen Histologically amyloid deposits were seen in the lamina propria []To date our knowledge about the clinicopathological features of the localized intestinal amyloidosis is limited To gain insights into these issues we conducted a comprehensive literature search and metaanalysis of the relevant published literature Also we presented a rare case of primary isolated intestinal amyloidosisMaterials and MethodsThis is a viewpoints study not an original research investigation that did not include any interaction or intervention with human subjects or any access to any identifiable private information Therefore the study did not require institutional review board review [] This study was performed in accordance with the principles of the Declaration of Helsinki []Search strategy and selection criteriaThe authors adhered to Preferred Reporting Items for Systematic Reviews and MetaAnalyses PRISMA guidelines [] The literature was reviewed by searching the PubMed electronic database using the key term œamyloidosis and the following words œintestine or œsmall intestine or œduodenum or œileum or œjejunum or œappendix or œcolon or œsigmoid or œrectum andor œcecum to identify the eligible studies The search results were considered based on their titles abstracts and publication date Their full texts were examined to confirm their eligibility and then were included in the study A summary of the search strategy and selection criteria is shown in Figure [ ]Data extraction a methodological assessment and statistical analysisThe clinicopathological findings were extracted including the family name of the first author year of publication patient age gender symptoms endoscopic features histology special stains and presence or absence of systemic involvement The authors independently read and analyzed each study They defined the final diagnosis as œthe histopathological diagnosis to ensure homogeneous evaluation of the findings Statistical computations were performed using IBMSPSS IBMSPSS Inc Chicago IL USA Descriptive statistics mean standard error of mean and ranges were calculatedResultsFlow trialOur search yielded previous studies about gastrointestinal amyloidosis of which were excluded The remaining s have undergone œabstract review These studies covered a period of years to This relatively long period allowed proper evaluation of a good number of cases in the literature Twentyseven studies with the final diagnosis of localized intestinal amyloidosis were included in the present study In all cases the workup for monoclonal gammopathy and systemic amyloidosis serum protein electrophoresis and pathological examination of the tissue specimens was performed The diagnosis of localized amyloidosis was substantiated by the use of ancillary studies including radiological examination such as abdominal ultrasound barium enema []wholebody computed tomography [] magnetic resonance imaging positron emission tomography [] skeletal surveys electrocardiography and echocardiography [ ] s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res Figure Flow chart of literature search and study selection for cases of localized intestinal amyloidosis The inclusion criteria included patients who had amyloid deposition in the intestine without evidence of systemic involvement or an associated condition leading to secondary amyloidosis [] The exclusion criteria included patients with one or more of the followings Evidence of chronic diseases such as collagen vascular disease that may have led to secondary amyloidosis [] Additional an involvement by amyloidosis Intestinal symptoms suggestive of amyloidosis but without confirmatory diagnostic tissue biopsy Plasma cell dyscrasia myeloma lymphoplasmacytic disorders or other Bcell malignancies such as Waldenstrom™s macroglobulinemia [] Systemic AL type [] Laboratory findings revealing positive detection of monoclonal light chains in serum andor urine []The reported cases did not have any evidence of generalized metabolic complications or extraintestinal complications of accumulated amyloidosis [ ] A summary of the flow chart of the literature search and study selection criteria is shown in Figure [ ]Clinical presentations and treatment modalities in localized intestinal amyloidosisThe colorectum jejunum transverse colon ileum and duodenum were the most commonly affected sites The lesions were more common in males than in females The mean age at presentation was ± years small intestine and ± years colorectum [ ] In the small intestine the main symptoms were abdominal pain [ ] and intestinal obstruction [ ] followed by diarrhea and vomiting [ ] bleeding [ ] malabsorption [] and constipation [] In the colorectum the main symptoms were rectal bleeding [ ] followed by abdominal pain [ ] anemia [] intestinal obstruction [] and weight loss [] Chen described a case of localized amyloidosis of the transverse colon in a 52yearold male patient who presented with a periumbilical pain rectal bleeding and weight loss Colonoscopic examination revealed mucosal ulcerations and luminal narrowing by multiple polypoid lesions [] Treatment modalities included in these cases included drug therapy prednisolone azathioprine melphalan and colchicines [ ] intestinal resection partial jejunectomy endoscopic submucosal resection [ ] and colonic resection [ ] with close followup of the patients [ ] A summary of these results is presented in Tables [ ]s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Table Data From Individual Studies of Primary Localized Amyloidosis of the Small IntestineCase Sex Age years SitesIleumClinical manifestationsColonoscopic findingsStudies[][][][][][][][][][][][][][]FMFMFMMMMMMFMiddle ageAgedAcute abdominal pain diarrhea and vomitingPseuntestinal obstructionMelena and anemiaJejunumJejunumSmall intestineIntestinal obstructionJejunum Chronic diarrhea and and ileummalabsorptionSmall intestine Acute abdomenDuodenum and jejunumSmall intestineAbdominal pain nausea and constipationSmall bowel obstructionSmall intestine Abdominal pains and hematemesisThickening of the wall of the ileum and ascending colonThickening of the mucosal folds and multiple polypoid protrusionsDiffuse thickening of the mucosa with friability nodularity erosions and polypoid protrusionsDiffuse thickening of the wall of the small intestineThickening of the jejunal and proximal ileal mucosal foldsPerforation of the wallMultiple shallow ulcers and several erosionsBand of amyloid and connective tissue surrounding the small intestineMucosal ulcerations with discrete nodulesSmall intestine Nonspecific digestive symptoms Multiple mucosal polypsJejunumSmall intestine Nonspecific digestive symptoms Thickening of the wall with rough and Nonspecific digestive symptoms Polypoid pseudotumoral mucosal formationsHeartburn and constipationpolypoid intestinal mucosal foldsF M MSmall intestine three casesDuodenumScreening for cancer stomachA localized depressed lesionEndoscopic and histological findings in localized intestinal amyloidosisIn the small intestine the most common endoscopic findings were thickening of the intestinal wall [ ] and polypoid mucosal protrusions [] followed by mucosal ulceration [ ] friability and nodularity of the mucosa [ ] and wall perforations [] In the colorectum polypoid masses with luminal narrowing were the most common endoscopic finding [ ] followed by mucosal ulcerations solitary or multiple ulcers [ ] or and thickening of the wall [ ] nodularity and friability of the mucosa [ ] and perforation [] In all cases deposition of pale eosinophilic amorphous amyloid material with the prominent cracking artifact in the mucosa submucosa muscle layer or walls of the blood vessels represented the main histological feature [ ] The further Congo red staining revealed the characteristic salmonpink color confirming the presence of amyloid deposits The deposits showed the characteristic applegreen birefringence under polarized light Immunohistochemical stains were also performed in some cases to substantiate the diagnosis Other histological features included mucosal ulceration amyloid angiopathy necrotizing angiitis [] foreign body giant cell reaction [] ischemic changes focal active colitis and cryptitis a personal observation The amyloid type was AL light chain protein in the majority of the cases of the small intestine [ ] and colorectum [ ] Other amyloid protein types included AAamyloid [] and high serum amyloid SAA in some cases [] Summary of clinicopathological findings in patients with localized intestinal amyloidosis is presented in Tables [ ]Case reportPrimary localized amyloidosis of the intestineA 55yearold female patient presented with anorexia diarrhea rectal bleeding and weight loss Colonoscopy revealed picture reminiscent of active pancolitis friable mucosa with mucosal erosions ulcerations and hemorrhage and six biopsies from the ileum cecum and colon ascending transverse descending colons and rectum were obtained On histology all biopsies showed deposition of pale eosinophilic amorphous material with the prominent cracking artifact characteristic of amyloid in the lamina propria and submucosa In some areas s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res Table Data From Individual Studies of Primary Localized Amyloidosis of the ColonCase SexSitesClinical presentationColonoscopic findingsStudiesAge yearsMMMMMFMMMFMFTransverse colonbleeding and weight lossIntestinal obstructionTransverse colon A periumbilical pain rectal Descending colon AnemiaSigmoid colonUlcerations and luminal narrowing by multiple polypoid lesionsElderlyA stenosing massMiddle age Transverse colon Nonspecific digestive symptoms Submucosal massUlcerative lesionsMucosal ulceration perforation and thickening of the bowel wallA submucosal tumorlike massMucosal ulcerA single friable rounded mucosal lesionA 15cm shallow depressed mucosal lesionAcute abdominal pain rectal bleeding and fecal peritonitisHematocheziaHemepositive stoolsRectal bleedingRoutine colonoscopyNonspecific digestive symptoms Several irregularlyshaped discrete ulcerationsSigmoid colonSigmoid colonSigmoid colonSigmoid colonSigmoidDescending colon Abdominal pain bloating flatulence and hematocheziaRectal bleedingHemorrhagic mucosa and amass lesionDiffuse nodular friable lesions ascending colon and several irregular large ulcers with nodularity descending colonThickening of the wall and mucosal ulcerationsAscending and descending colonsColon seven casesAscending colonF M Middle age ColonF M Rectal bleeding two casesHeartburn and constipationFAcute abdominal pain diarrhea and vomitingThickening of the wall of the ileum and ascending colon[][][][][][][][][][][][][][][]Table Clinical Colonoscopic and Histological Findings in the Primary Localized Amyloidosis of the IntestineAspectsAge mean ± SEMMale to female ratioSite of involvementClinical presentationsColonoscopic findingsHistological featuresSmall intestine duodenum jejunum and ilium ± years [ ]Jejunum Ileum Colorectum ± years [ ]The left colon Transverse colon Abdominal pain and intestinal obstruction [ ]Rectal bleeding [ ] followed by abdominal pain [ ] and anemia []Thickening of the wall [ ] polyps and polypoid mucosal protrusions [] ulcerations [ ] friability and nodularity of the mucosa [ ] and perforation []Mass lesions tumorlike lesion polypoid protrusions and polyps with narrowing were the most common [ ] Ulcerations with solitary or multiple ulcers [ ] thickening of the wall [ ] nodularity and friability of the mucosa [ ] and perforation []Amyloid deposits increased density of mixed inflammatory cells in the lamina propria light chain protein [ ]Amyloid deposits increased density of mixed inflammatory cells in the lamina propria foreign body giant cell reaction amyloid angiopathy necrotizing angiitis focal active colitis and immunoglobulin lightchain AL [ ]s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Figure Isolated primary amyloidosis of the intestine case report a e Colonic mucosa with deposition of pale eosinophilic amorphous material star in the submucosa with abundant deposits of pink amorphous materials with some cracking artifacts characteristic of amyloid Focal surface mucosal ulceration is noted The background mucosa shows focal active inflammation including cryptitis arrow and there is patchy crypt distortion No granulomas are seen hematoxylin and eosin a × b × c d × and e × f g Congo red stain which reveals the characteristic salmonpink color confirming the presence of amyloid deposits Congo red f × and g × h i The deposits exhibit characteristic applegreen birefringence under polarized light Congo red under polarized light h × and i × the lamina propria was entirely replaced by these deposits The background mucosa showed focal active inflammation focal ulceration cryptitis occasional crypt abscess This focal chronic active inflammatory change may be secondary to erosionulceration associated with amyloid deposits [] Congo red stain confirmed the presence of amyloid deposits with applegreen birefringence under polarized light Fig Immunohistochemical analysis revealed that the amyloid deposits were positive for amyloid lambda but negative for amyloid A These findings were consistent with amyloid lightchain AL amyloidosis We performed several laboratory and radiological investigations to rule out the possibility of systemic involvement The hematological indices red blood cells RBCs count hemoglobin mean corpuscular volume MCV mean corpuscular hemoglobin MCH platelets and white blood cells count and the biochemical tests total protein albumin globulin alkaline phosphatase aspartate aminotransferase AST total bilirubin ferritin vitamin B12 folic acid and prostatespecific antigen PSA urea creatinine electrolytes and spot urine proteincreatinine ratio protein electrophoresis and immunoelectrophoresis of serum and urine were within normal limits Thyroid function tests were within normal limits Skeletal surveys electrocardiograms and radiological findings liver spleen and kidneys were unremarkable Bone marrow biopsy showed no abnormalities Therefore the diagnosis of localized primary intestinal AL amyloidosis involving terminal ileum cecum colon and rectum associated ulceration and inflammatoryregenerative changes was establishedThe study presents a representative retrospective case in which the archival formalinfixed paraffinembedded tissues from the consultation files of the authors were used [] All the materials paraffinembedded tissue block slides and pathology report were coded The information obtained was analyzed and reported in such a way that the identity of the case presented cannot absolutely be ascertained The patient consent was obtainedDiscussionLocalized amyloidosis of the intestine is of interest to the pathologists and gastroenterologists because some cases may s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res mimic tumors Here we present a comprehensive review of the literature about this entity In agreement with previous reports [ ] our analysis indicates that the primary localized intestinal amyloidosis is a rare condition with disparate and nonspecific clinicopathological and endoscopic features Also its outcome depends on the site of the deposition of the amyloid small intestine versus colorectum [ ] In Cowan examined the clinicopathological features of patients with amyloidosis Seventysix patients had gastrointestinal amyloidosis The age range of the patients was years The patients include cases with localized gastrointestinal amyloidosis and cases with gastrointestinal involvement as a part of systemic amyloidosis In the latter mutations of the transthyretin gene were found Gastrointestinal bleeding and weight loss were the most common presenting symptoms []Threlkeld and Nguyen reported as a case of isolated colonic amyloidosis in a 46yearold male patient who presented with hematochezia and abdominal pain Colonoscopic studies revealed the presence of a tumorlike mass in the descending colon and therefore partial colectomy was performed Grossly the mucosa was necrotic but no mass lesion was identified There were light chainspositive amyloid deposits in the muscle layers submucosa and submucosal blood vessels Laboratory investigations for inflammatory condition and plasma cell disorders were negative and therefore the diagnosis of isolated gastrointestinal amyloidosis was established The patient received colchicine with marked clinical improvement [] Similarly Diaz Del Arco reported a case of globular amyloid deposits involving the left colon in a 74yearold male patient presenting with anemia and colitis Histologically there were Congo red positive deposits in the lamina propria of the colon []The duodenum is an important site for the deposition of amyloid protein Patients with primary duodenal amyloidosis may present with epigastric pain nausea constipation steatorrhea protein loss malabsorption hemorrhage and vomiting Endoscopic findings include the thickening of the duodenal wall and the presence of polypoid lesions [ ] Deguchi described a case of an isolated duodenal and jejunal amyloidosis in a 47yearold male patient who presented with features of intestinal pseudoobstruction Endoscopy revealed thickening and polypoid lesions of the duodenal mucosal folds The further histological evaluation confirmed the presence of amyloid deposits in the duodenal and jejunal walls [] Similarly Choi reported a case of localized amyloidosis of the small intestine duodenum and jejunum in a 62yearold male patient who presented with abdominal pain nausea and constipation of several years duration Endoscopy revealed mucosal erosions ulceration and tumorlike masses Immunohistological analysis revealed the presence of lambda light chain protein therefore establishing the diagnosis of amyloidosis []In the primary localized amyloidosis the isolated deposition of amyloid proteins may be due to the local tissue damage with the synthesis and deposition of amyloid substances in the individual ans [] The synthesis of amyloid results from plasma cell reaction to the inflammatory antigens The otherwise benign polyclonal plasma cells produce monoclonal immunoglobulin light chains L that are deposited as AL amyloid [] It is still possible that the deposition of amyloid results from the inability of the body to clear light chains produced by plasma cells [] Amyloid deposits in the intestine are usually irreversible but can sometimes be reversible with chemotherapy treatment especially with melphalan and steroid therapy []In primary localized intestinal amyloidosis we found variations in the site of deposition of amyloid proteins There was the deposition of the amyloid proteins in the walls of the blood vessels mucosa submucosa muscularis mucosae muscularis propria and the neural elements [] The destructive effects of amyloid usually resulted from the local replacement of these important histological compartments For instance amyloid deposition in the wall of the blood vessels usually leads to ischemia bowel infarction [] or intestinal perforation [] The involvement of the mucosa by amyloid deposits usually produces mucosal ulcerations leading to malabsorption [] hematochezia and anemia [ ] The deposition of amyloid in the submucosa and the muscle layers usually leads to the formation of the mass lesions amyloidoma or amyloid tumor resulting in intestinal obstruction [] The involvement of the neural elements results in altered bowel motility and chronic ileus [] Hemorrhage melena and bleeding per rectum is reasoned to bowel ischemia hemorrhagic necrosis of the mucosa damage of the vessel walls and abnormal platelet aggregation []The variations in the gross findings of intestinal amyloidosis may be reasoned to the type and the site of deposition of the amyloid protein When the deposition of the amyloid protein occurs predominantly in the perivascular and interstitial spaces it usually produces diffuse thickening of the intestinal wall rather than polypoid or tumorlike lesions [ ] Elevations or localized tumorlike lesions may reflect the presence of nodular aggregates of plasma cells locally producing abundant amyloid proteins which usually originate from AL protein produced by local aggregates of plasma cells involving the mucosa submucosa or the muscle layer [ ] Fine granular lesions or mucosal erosion or ulcerations are commonly associated with the deposition of AA amyloidosis [ ]To conclude the data presented here provides an indepth characterization of the clinicopathological features of primary localized intestinal amyloidosis It indicates that this entity is rare and its presenting features are generally nonspecific and therefore the diagnosis is challenging and can be easily missed Knowledge of this type of amyloidosis is warranted to avoid aggressive management such as treatment with an alkylating agent A high index of suspicion should be maintained both on the part of the gastroenterologists and the histopathologistsAcknowledgmentsNone to declareFinancial DisclosureNone to declares The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Conflict of InterestNone to declareInformed ConsentInformed consent was obtainedAuthor ContributionsSaeed Ali Alshehri conceived and designed the analyses collected the data biochemical and clinicopathologic aspects of amyloidosis performed the analyses tools including the statistical data analysis and wrote the paper Mahmoud Rezk Abdelwahed Hussein supervised the data analysis and contributed to the conception of the idea of the manuscript data collection Histological aspects of amyloidosis writing up and revising of the manuscriptData AvailabilityThe authors declare that data supporting the findings of this study are available within the References Westermark P Benson MD Buxbaum JN Cohen AS Frangione B Ikeda S Masters CL Amyloid fibril protein nomenclature Amyloid Monge M Chauveau D Cordonnier C Noel LH Presne C Makdassi R Jaureguy M Localized amyloidosis of the genitourinary tract report of new cases and review of the literature Medicine Baltimore Mathis H [Rokitansky Virchow and Heschl on the problem of amyloidosis a historical study] Zentralbl Allg Pathol Sack GH Jr Serum amyloid A a review Mol Med Gillmore JD Wechalekar A Bird J Cavenagh J Hawkins S Kazmi M Lachmann HJ Guidelines on the diagnosis and investigation of AL amyloidosis Br J Haematol Cowan AJ Skinner M Seldin DC Berk JL Lichtenstein DR O'Hara CJ Doros G Amyloidosis of the gastrointestinal tract a 13year singlecenter referral experience Haematologica Ankarcrona M Winblad B Monteiro C Fearns C Powers ET Johansson J Westermark GT Current and future treatment of amyloid diseases J Intern Med Selkoe DJ Folding proteins in fatal ways Nature Otaka Y Goda F Nakazato Y Tsutsui T Systemic heavy and lightchain amyloidosis presenting nephrotic syndrome and congestive heart failure a case presentation and literature review Amyloid 201926sup19596 Picken MM The pathology of amyloidosis in classification a review Acta Haematol Trinh TD Jones B Fishman EK Amyloidosis of the colon presenting as ischemic colitis a case report and review of the literature Gastrointest Radiol Threlkeld C Nguyen TH Isolated amyloidosis of the colon J Am Osteopath Assoc GarciaGonzalez R Fernandez FA Garijo MF Fernando ValBernal J Amyloidosis of the rectum mimicking collagenous colitis Pathol Res Pract Tada S Iida M Yao T Kawakubo K Yao T Okada M Fujishima M Endoscopic features in amyloidosis of the small intestine clinical and morphologic differences between chemical types of amyloid protein Gastrointest Endosc

Thyroid_Cancer

Breast cancer is a common malignancy in women Among breast cancer types triplenegative breast cancer TNBC tends to affect younger women is prone to axillary lymph node lung and bone metastases and has a high recurrence rate Due to a lack of classic biomarkers the currently available treatments are surgery and chemotherapy no targeted standard treatment options are available Therefore it is urgent to find a novel and effective therapeutic target As alteration of ion channels and transporters in normal mammary cells may affect cell growth resulting in the development and progression of TNBC ion channels and transporters may be promising new therapeutic targets for TNBC This review summarizes ion channels and transporters related to TNBC and may provide new tumor biomarkers and help in the development of novel targeted therapiesKeywords Triplenegative breast cancer Ion channels Ion transporters Pathological roles Targeted therapyBackgroundBreast cancer BC is the common malignancy in women its incidence is increased each year [] and it has become a significant threat to women™s health [] BC is a heterogeneous disease that can be divided into multiple molecular subtypes based on estrogen receptor ER progesterone receptor PR and human epidermal growth factor receptor HER2 expression providing important prognostic and predictive information [] There are four BC subtypes depending on receptor status luminal A luminal B HER2overexpressing and triplenegative breast cancer TNBC Among them TNBC is defined as ER PR and HER2 negative and it tends to affect younger women a0years of age it is Correspondence onlyoneliuxuemei163com 0078029sinacom Chengli Lu and Zhiyuan Ma contributed equally and share first authorship Department of Thyroid and Breast Surgery Affiliated Hospital of Zunyi Medical University Zunyi Guizhou Province China Department of Gastroenterology Affiliated Hospital of Zunyi Medical University Zunyi Guizhou Province ChinaFull list of author information is available at the end of the prone to axillary lymph node lung bone metastases and has a high recurrence rate [ ] Lehmann et a0al classified TNBC into six subtypes based on gene cluster sequence expression basallike basallike immunomodulatory mesenchymal mesenchymal stemlike and luminal androgen receptor subtypes [] After analyzing the RNA and DNA profile of TNBC tumors Matthew et a0 al classified TNBC into four subtypes including luminal androgen receptor mesenchymal basallike immunesuppressed and basallike immuneactivated subtypes [] The two classification methods have similarities and both provide theoretical bases for exploring targeted therapies for TNBCAlthough TNBC is the BC subtype that responds best to chemotherapy its recurrence and metastasis rates are higher than those of other BC subtypes [] Furthermore due to the lack of classic biomarkers TNBC lacks standard treatments guided by tumor biology and only surgery and chemotherapy are currently available as treatments [] Previous studies have shown that ion channels and transporters play important regulatory roles in mammary physiology and the initiation and progression of BC The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLu a0et a0al Cancer Cell Int Page of [] However the detailed functional role of ion channels and transporters in TNBC has not been clarified and summarized In recent studies upregulation of NaH exchanger has been shown to promote the proliferation migration and invasion of the TNBC cell line MDAMB231 [ ] In addition Ca2 channels such as mitochondrial calcium uniporter MCU can promote TNBC cell migration invasion and lung metastasis [] and Alvarez et a0al [] reported that the twopore domain potassium channel KCNK5 is associated with a poor prognosis in TNBC Therefore ion channels and transporters play important regulatory roles in the pathophysiology of TNBC but there is currently no relevant review on this topic Here we review the pathological roles of ion channels and transporters including AQPs Clˆ’ channels Ca2 channels K channels and acidbase transporters in the initiation and progression of TNBCAQP channelsAQPs which compose a family of transmembrane water channel proteins modulate the movement of water and small solutes into and out of cells and maintain suitable concentrations of water and solutes for cell survival [] At least AQP subtypes AQP012 have been identified in mammals and are divided into two families based on transfer specificity namely the classic watertransporting AQP family and the solute water and glyceroltransporting glycoprotein family [] AQP02 AQP4 to AQP68 are mainly waterselective AQP3 AQP7 AQP9 AQP10 and AQP12 also transport glycerol and possibly other small solutes AQPs also play roles in the transport of ammonia urea carbon dioxide metalloids nitric oxide and certain ions [] Expression of AQP1 AQP35 and AQP1012 has been detected in normal human mammary tissue and is closely related to milk secretion [ ] In addition deletion of œCCAATenhancer binding protein a family of transcription factors isoforms results in changes in mammary ductal morphogenesis and changes in expression of transport proteins such as AQP5 suggesting that AQP5 may be involved in mammary development [] Recent studies have shown that AQPs play carcinogenic roles by promoting angiogenesis enhancing invasive and metastatic potential and enhancing the transport of reactive oxygen species ROS [ ] In femalespecific cancers such as BC AQP1 and are the most important AQPs and they are been reported to be upregulated []AQP1 the membrane protein was the first reported mammalian AQP and plays a significant role in tumor cell migration proliferation and angiogenesis [] Clinical studies have shown that patients with TNBC have higher levels of AQP1 expression and that upregulation correlates with a poor prognosis [ ] AQP1 expression is induced by hypoxia through the EBoxChoRE transcription element which is affected by increased glucose consumption and metabolism [] AQP1 expression has been detected only in a subgroup of CK14positive basallike breast cancer BLBC cases [] CK14 has been used as a marker of basal mammary epithelial cells with in vivo regenerative ability in studies on mammary gland progenitor and stem cells [] Therefore it is speculated that expression of AQP1 is related to the stem cell characteristics of BLBC cells Hu et a0al demonstrated that AQP1 upregulation promotes extravasation and increases migration in a0vivo and in a0vitro in the mouse TNBC cell line 4T1 suggesting that this aquaporin enhances the rate of cell migration by promoting water permeability in cell protrusions [] Thus upregulation of AQP1 can promote the proliferation migration and invasion in TNBC cells Moreover in a0 vivo experiments have shown that AQP1 deficiency can reduce tumor mass volume vessel density and lung metastases in MMTVPyVT mouse mammary tumor virusdriven polyoma virus middle T oncogene mice and inhibition of AQP1 function andor expression is predicted to attenuate angiogenesis via reduced migration and invasion of endothelial cells [] Recently Irene AbreuRodriguez et a0al [] revealed that AQP1 expression is also responsive to hypoxiainducible factor HIF which may play a role in the VEGFindependent signaling mechanism inducing angiogenesis in a hypoxic environment Helen et a0al [] also reported that the triterpenoid saponins bacopaside I and bacopaside II can synergistically reduce the transcriptional expression of AQP1 and inhibit proliferation migration and invasion in MDAMB231 cells Similarly ginsenoside Rg3 a compound with anticancer activity isolated from ginseng inhibits AQP1 to attenuate cell proliferation through a mechanism that involves downregulation of AQP1 to induce cell cycle arrest in G0G1 phase by inhibiting cyclin D and E and inhibition of chemoattractantinduced cell migration and invasion by blocking AQP1mediated water flux in MDAMB231 cells [] These findings indicate that AQP1 plays an important role in the development and progression of TNBCOverexpression of AQP3 has been detected in the membranes and cytoplasm of TNBC tumor cells and is significantly associated with poor prognosis [] XuChen Cao et a0 al [] found that the presence of fibroblast growth factor2 FGF2 induced cell migration and metastasis in MDAMB231 cells by increasing AQP3 expression Moreover FGF receptor kinase FGFRK inhibitors PI3K inhibitors and MEK12 inhibitors all inhibit AQP3 expression suggesting that FGF receptor kinases increase AQP3 expression and promote FGF2induced cell migration by initiating downstream PI3K and ERK pathways In addition CuSO4 a water transport 0cLu a0et a0al Cancer Cell Int Page of inhibitor of AQP3 inhibits migration in MDAMB231 cells AQP3 downregulation reduces the proliferation invasion and migration of MDAMB231 cells while increasing sensitivity to 5fluorouracil chemotherapy The mechanism may be related to a decrease in glycerol permeability caused by AQP3 downregulation [] Overall these findings demonstrate that AQP3 plays a pivotal role in the initiation and progression of TNBC and specific inhibitors of AQP3 in clinical applications may improve the therapeutic effect of TNBC patientsSimilarly overexpression of AQP5 in the membrane and cytoplasm of TNBC cells has been detected and is significantly associated with poor prognosis [] Moreover patients with higher Ki67 expression are more likely to have abnormal AQP5 protein expression than patients with lower Ki67 expression [] Ki67 is a widely accepted proliferation marker [ ] and it is speculated that upregulation of AQP5 may promote proliferation in TNBC cellsIn summary AQP1 AQP3 and AQP5 are significantly upregulated in TNBC this upregulation is related to a poor prognosis and can promote the proliferation migration and invasion of TNBC cells These AQPs are promising new targets for the diagnosis and treatment of TNBCClˆ’ channelsCFTRCFTR is a member of the ATPbinding cassette transporter family that localizes at the apical membranes of normal epithelial cells CFTR is mainly responsible for conducting HCO3ˆ’ and Clˆ’ and promoting HCO3ˆ’ secretion in many tissues including the airway intestines and pancreas [] However when the extracellular concentration of Clˆ’ is higher than a0mmolL the permeability of CFTR to Clˆ’ is much greater than that of CFTR to HCO3ˆ’ thus CFTR mainly conducts Clˆ’ under physiological conditions [] CFTR can also transport two other anions glutathione and thiocyanate which are involved in airway inflammation and oxidative stress [ ] Interestingly Pierre et a0al [] reported that CFTR is required for the tightly connected functions of normal epithelial tissues loss of CFTR reduces epithelial resistance and epithelial integrity and this effect is not related to the anion channel function of CFTRCFTR has been reported to be associated with several cancers such as cervical cancer [] colorectal cancer [] prostate cancer [] and BC [] Significant downregulation of CFTR expression is observed in BC tissue compared to normal mammary tissue [] Zhang et a0al demonstrated that overexpression of CFTR inhibits EMT invasion and migration in MDAMB231 cells via a mechanism that involves CFTR inhibition of NFκB targeting of urokinasetype plasminogen activator [] In addition CFTR overexpression inhibits the EMT and the invasiveness of MDAMB231 cells and reduces lung metastasis in xenograft models Increasing evidence reveals that downregulation of CFTR occurs after treatment with EMTinducing factors such as TGF suggesting that as a downstream effector CFTR plays important roles in mediating various EMT effects [ ] Moreover hypermethylation of the cancer genome leads to activation of oncogenes or suppression of tumorsuppressor genes thereby resulting in tumorigenesis [] It has also been observed that the methylation level of CFTR in BC tissues is much higher than that in normal tissues and treatment with DNA methylation inhibitors in TNBC cell lines MDAMB231 and MDAMB435 can rescue CFTR mRNA indicating that CFTR methylation plays an important role in TNBC [] ΔF508 is the most common mutation in CFTR causing the protein to be retained and degraded in the endoplasmic reticulum due to misfolding [] It is worth noting that although there is no difference in the incidence of BC between ΔF508 carriers and noncarriers patients with ΔF508 CFTR mutations all have grade III cancer indicating that CFTR defects are associated with BC progression [] Therefore CFTR methylation or mutation need to be further investigated in the future which may provide novel therapeutic intervention for TNBCChloride channel The chloride channel ClC family also plays an indispensable role in the transport of Clˆ’ [] There are nine family members in humans which are divided into two categories based on their distribution and physiological function Clˆ’ channel proteins ClC1 ClC2 ClCKa and ClCKb which mainly exist in the plasma membrane and play roles in stabilizing membrane electric potential or mediating epithelial transport and Clˆ’H reverse transporter proteins ClC37 which mainly exist in the vascular intima of the endosomelysosomal pathway and are localized at the plasma membrane only to a limited extent due to protein degradation and hydrolase activity [ ] In recent years it has been discovered that ClC3 can transport one hydrogen ion in exchange for two chloride ions [] with important roles in cancers such as nasopharyngeal carcinoma [] and BCClC3 overexpression is observed in tissues and the TNBC cell line MDAMB231 [ ] Studies by Zhou et a0 al revealed that knockdown of ClC3 downregulates expression of cyclin D1 and cyclin E and increases levels of p21 indicating that knockdown of ClC3 can block the cell cycle of MDAMB231 cells at G0G1 phase inhibiting cell proliferation Moreover knockdown of ClC3 suppresses tumor growth in xenograft models and significantly reduces levels of pERK12 in MDAMB231 cells 0cLu a0et a0al Cancer Cell Int Page of This indicates that ClC3 can promote the progression of TNBC by acting on the ERK12 signaling pathway [] Nevertheless relative research on ClC3 in TNBC is still very limited and extensive work is needed in the furtherCa2 channelsCa2 is a key nutrient in milk that plays a vital role in the mineralization of bones and teeth and as a second messenger ionized Ca2 is a key regulator of proliferation migration cell cycle progression and apoptosis [] The level of Ca2 is very low in the cytoplasm ˆ’ a0molL whereas it is somewhat higher in anelles ˆ’ a0 molL and highest in the extracellular level milieu ˆ’ a0 molL Hence a small amount of Ca2 can significantly change intracellular levels to activate downstream signaling molecules including calmodulin nuclear factor of activated Tcells NFAT NFκB calmodulindependent protein kinase II calpain and others [ ] In nonexcitatory mammary cells calcium channels play important roles in lactation and the maintenance of normal physiological functions [ ]Continuous increases in intracellular Ca2 levels will drive expression of oncogenes resulting in tumor growth and development especially the metastatic behavior of cancer cells and conferring tumor cells with resistance to apoptosis [] Abnormal expression of several Ca2 transporters and ion channels such as calcium releaseactivated calcium modulator Orai1 has been observed in TNBC and may lead to oncogenic Ca2 signaling [] Interestingly specific changes in the expression and function of Ca2 channels are related to hormone receptor status and differ significantly among BC subtypes []Calcium modulator Ca2 influx mainly depends on storeoperated calcium channels SOCCs When the Ca2 concentration in the endoplasmic reticulum declines to a certain level the STIM stromal interaction molecule which is located on the endoplasmic reticulum membrane moves to a position close to the highly selective calcium channel protein Orai on the cell membrane Subsequently Orai is activated to cause Ca2 influx and storeoperated calcium entry SOCE is initiated thereby replenishing the calcium store Some researchers have proposed that the canonical transient receptor potential TRPC also participates in the above process though the mechanism remains controversial There are two different claims that both Orai and TRPC form independent channels activated by the STIM protein and that Orai and TRPC subunits form heterochannels triggered by STIM [] There are three Orai1 isomers Orai1 to Orai3 and two STIM homologs STIM1 and STIM2 SOCE has been found to be primarily mediated by Orai1 and STIM1 in TNBC [] Compared with that in nonmalignant breast epithelial cells expression of Orai1 and STIM1 is significantly higher in TNBC cell lines and is associated with a poor prognosis [ ] Liu et a0 al [] reported that hypoxia can induce expression of Orai1 Notch1 and Jagged1 and Orai1 is significantly downregulated after blockade of Notch signaling suggesting that hypoxia can increase Orai1 expression in TNBC by activating Notch signaling Notch1Orai1SOCENFAT4 axis Similarly Mognol et a0al [] found that Orai1 promotes the invasion and angiogenesis of TNBC cell lines and activates NFAT4 which can regulate genes involved in the cell cycle apoptosis angiogenesis and metastasis In addition Yang et a0 al [] demonstrated that Orai1 and STIM1 promote the migration and invasion of MDAMB231 cells both in a0vivo and in a0vitro and the authors proposed that these proteins may at least partially control cell migration by regulating focal adhesion turnover Furthermore treatment with TGF can reduce expression of STIM1 whereas blockade of SOCE can impair TGFinduced G0G1 cell cycle arrest and inhibit the proliferation of MDAMB231 cells [] Based on the above research Orai1 and STIM1 may be new therapeutic targets for TNBC Indeed some selective SOCE inhibitors have shown encouraging inhibitory effects on TNBC but they are still only in the preclinical trial stage For example phemindole a diindole derivative reduces SOCE by downregulating STIM1 expression significantly inhibits the proliferation and migration of MDAMB231 cells reduces the growth of solid tumors in mouse models and produces a targeted antitumor effect in TNBC [] In addition Miroslava Didiasova et a0al [] revealed that elevated cell surfaceassociated enolase1 ENO1 expression correlates with augmented MDAMB231 cell migratory and invasive properties Pharmacological blockade a selective SOCC inhibitor NS1643 or knockdown of STIM1 or Orai1 reduces ENO1dependent migration of MDAMB231 cells These results demonstrate the pivotal role of SOCE in the regulation of ENO1 exteriorization and thus in the modulation of TNBC cell migratory and invasive properties indicated that Orai1 and STIM1 might be promising threptic targets for TNBCSecretory pathway Ca2‘ATPaseThe secretory pathway Ca2ATPase SPCA can direct Ca2 and Mn2 from the cytoplasm to the Golgi and postGolgi vesicles Two isotypes SPCA1 and SPCA2 are known and the distribution and function of the two differ SPCA1 is commonly expressed in mammalian tissues expression of SPCA2 is limited to highly absorptive and secretory epithelial cells including mammary 0cLu a0et a0al Cancer Cell Int Page of and salivary gland cells [] SPCA1 is highly expressed in TNBC cell lines and SPCA2 is highly expressed in cell lines of other subtypes [] Interestingly based on clinical samples Desma et a0al reported SPCA1 levels to be significantly elevated in the basal subtype of BC compared with all other subtypes and it is worth noting that changes in its expression affect posttranslational modification and transport of certain proteins important for tumor progression without significantly changing cytosolic calcium signaling SPCA1 inhibition also decreased MDAMB231 cell proliferation [] Moreover SPCA1 is a key regulator of insulinlike growth factor receptor IGF1R processing in TNBC cells and promotes the production of functional IGF1R IGF1R activity is associated with poor prognosis suggesting that targeting SPCA1 is an alternative IGF1Rinhibiting strategy [ ] Overall upregulation of SPCA1 may promote the initiation and progression of TNBC The main mechanism reported to date involves SPCA1mediated increase in functional IGF1R expressionMitochondrial calcium uniporterUpregulation of MCU expression on the mitochondrial membrane is closely related to a poor prognosis in BC [] miR340 correlates negatively with the metastatic potential of TNBC cells [] it may directly inhibit MCU expression to reduce glycolysis and exercise capacity and knockdown or inhibition of MCU inhibits the growth invasion and metastasis of MDAMB231 cells [] Interestingly Anna et a0 al [] demonstrated that mitochondrial Ca2 uptake is required for TNBC progression in a0vivo and that absorption of Ca2 by mitochondria promotes the production of sustained mitochondrial reactive oxygen species activating the HIF1α signaling pathway and promoting tumor growth and metastasis In addition inhibiting or silencing MCU also block seruminduced migration of MDAMB231 cells and reduce serum or thapsigargininduced SOCE suggesting that MCU promotes TNBC cell migration by regulating SOCE [] The above results indicate that overexpression of MCU may play an important oncogenic role in the growth invasion and metastasis of TNBC cells However the precise mechanism is unclearOther promising calcium channel targets in TNBC include TRPV6 [] Overall calcium channels are promising targets for TNBC treatment but most compounds targeting these channels are only in the preclinical trial stage Thus further research is neededK channelsThrough the action of NaKATPase two K molecules are transported into a cell in exchange for three sodium molecules which increases the intracellular K concentration K channels on the cell membrane are numerous and in humans more than genes encode major K channel subunits [] K channels play key roles in maintaining acidbase balance by functioning in concert with the NaH exchanger and NaKATPase [] controlling electrical excitability of nerves and muscles and participating in energy metabolism and other physiological processes In addition K channels can help regulate cell proliferation and cell cycle progression and are involved in tumorigenesis [] Many studies have reported dysregulated K channel expression in human cancers including BC astrocytictype brain cancer and prostate cancer [ ] Tumorrelated K channels can be divided into four main categories according to their domain structures and activation mechanisms voltagegated potassium channels which are controlled by changes in membrane potential calciumactivated potassium channels which are activated by intracellular calcium inwardly rectifying potassium channels and twoporedomain potassium channels K2P KCNK [] However the carcinogenic mechanism of K channels remains rather clear Nuria et a0 al [] proposed that K channels may participate in and regulate tumor progression through permeationrelated mechanisms including changes in membrane potential Ca2 driving forces and cell volume regulation and nonconductive mechanisms dependent on proteinprotein interactionsThe Kv111 channel also known as human etheragogorelated gene hERG1 is not expressed in normal breast cells but is expressed in BC with a relationship with subtype Indeed TNBC exhibits lower expression of Kv111 compared with other subtypes [] Olivia Crociani et a0al [] showed that the mRNA levels of Kv111 change throughout the cell cycle peaking in G0G1 phase Moreover Lansu et a0al [] reported that stimulation of Kv111 led to inhibition of proliferation in MDAMB231 cells and that an agonist the diphenylurea derivative NS1643 caused a significant inhibition of cell proliferation This phenomenon can be linked to a rapid decrease in the cyclin E2 protein level which causes accumulation of cells in G0G1 phase and an increase in tumor suppressor proteins and markers for cellular senescence including p21 p16INK4a and galactosidase activity Therefore Kv111 inhibits TNBC cell proliferation by activating a cellular senescence program [] Breuer et a0 al confirmed that NS1643 reprograms the EMT by attenuating the Wntcatenin signaling pathway inhibits cancer cell stemness and significantly reduces the metastatic spread of breast tumors in a MDAMB231 mouse model [] Regardless cardiotoxicity is an important limiting factor for potential therapeutic molecules acting on Kv111 Although the activator is well tolerated 0cLu a0et a0al Cancer Cell Int Page of in BC potential effects include tachycardia [] Overall the potential benefits of Kv111 activators as anticancer drugs outweigh their side effectsIn addition many other channels are altered in TNBC For example some K2P channels with differential expression may serve as novel molecular markers associated with TNBC RNASeq analysis of K2P channels has shown that overexpression of KCNK5 KCNK9 and KCNK12 and low expression of KCNK6 and KCNK15 are related to TNBC [] The above findings indicate that K channels play an important role in TNBC and are expected to be diagnosis markersAcid‘base transportersThe pH of milk is significantly lower than that of plasma indicating that there may be some acidbase transporters in the mammary gland that regulate the pH between the extracellular fluid and milk [] A uniform feature among solid tumors with high metabolic and proliferative rates is a significantly different pH from that of normal tissue [] Cancer cells can maintain a weakly acidic intracellular pH that is even more alkaline than that of normal cells suggesting that tumor cells have a powerful pH regulation system a0[]The NaH exchanger NHE a membrane transporter mainly catalyzes the exchange of intracellular H for extracellular Na in mammals thereby maintaining the pH balance inside and outside the cell [ ] There are subtypes of NHE with tissue and membranespecific expression patterns NHE15 are located on the plasma membrane and NHE69 are on intracellular anelle membranes NHE10 is only expressed in osteoclasts [] In addition NHE plays indispensable roles in maintaining normal mammary structure and physiological functions [] NHE1 SLC9A1 is universally expressed in epithelial cells and upregulated in BC tissues compared to normal tissues [] Studies have shown that hypoxia various growth factors and hormones among others can activate NHE1 and enhanced NHE1 activity can reduce extracellular pH and promote metastasis of MDAMB231 cells [] Furthermore it has been proposed that NHE1 promotes metastasis and remodeling of the extracellular matrix by acidifying the extracellular microenvironment [] In addition NHE1 knockdown reduces the migration invasion and growth of xenograft tumors of MDAMB231 cells increasing the susceptibility of these cells to paclitaxel [ ] Moreover knockdown of NHE1 or NBCn1 SLC4A7 in the MDAMB231 cell line significantly reduced the steadystate intracellular pH value after acid load the ability to restore pHi and the primary tumor growth of xenografts in a0 vivo but NBCn1 knockdown prolonged tumorfree survival and reduced cell proliferation [ ] It has been confirmed that NHE1 and NBCn1 promote the development of TNBC through different mechanisms There are two main NHE1 inhibitors amiloride and cariporide which are more effective than amiloride and highly selective [] Amiloride is a potassiumsparing diuretic and has blocking effects on a variety of ion channels such as NHE and the NaCa2 exchanger Cariporide is a highly specific and powerful NHE1 inhibitor that is relatively well tolerated in humans with heart disease [] Moreover a study has suggested that KR33028 a novel small molecule inhibitor of NHE1 produces a cellular phenotype comparable to that of NHE1 knockout cells and significantly decreases rates of migration invasion and colony growth in TNBC cell lines MDAMB231 MDAMB468 and Hs578T [] The above findings suggest that NHE1 may play an important role in the progression TNBCAdditionally other acidbase transporters are also altered in TNBC and are expected to emerge as new targets for TNBC treatment For instance NBCe1 SLC4A4 knockdown reduces cell proliferation invasion and migration in TNBC cells expressing high levels of NBCe1 [] The above findings all suggest that the acidbase transporters have essential functions in the occurrence and development of TNBC but further research is neededConclusionsDysfunction of ion channels and transporters in the mammary resulted in development and progression of TNBC Despite extensive work has been performed to investigate the expression pattern functional diversity regulatory mechanism and pathophysiology of different ion transporters in TNBC the systematic review is rare in this field Therefore this review focuses on different pathological function of multiple families in the development and progression of TBNC including the AQPs Clˆ’ channels Ca2 channels K channels and acidbase transporters Fig a0 Table a0 We hope that we can provide a basic systemics and summarised knowledge to this field advocating researchers play more attention on the pathophysiological role of ion channels and transporters in the development and progression of TNBC which may provide novel targets for the clinical diagnosis and treatment of TNBC 0cLu a0et a0al Cancer Cell Int Page of Fig Pathological roles of ion channels and transporters in triplenegative breast cancer cells Alteration and dysfunction of AQPs Clˆ’ channels Ca2 channels K channels NaHCO3ˆ’ transporter and NaH exchanger results in abnormality of ion transport and disorder of multiple signaling pathway including WNT PI3K TGF Notch and VEGF etc eventually promoting TNBC cell proliferation migration and invasion but inhibiting apoptosis

Thyroid_Cancer

Gastric cancer GC persists as a worldwide public health crisis According to the American Cancer Society the 5yearsurvival rate of GC remains at worldwide and withinthe United States1 These survival statistics have increasedoverall since the 1980s when the 5year survival rate for stageII disease was below and near for stage IIIB andhigher1 With the development of chemotherapies such as platinums and taxanes survival beyond stage II increased steadilyto Although chemotherapies improved overall survivalthis is not as dramatic as that in other solid malignancies suchas prostate or breast Furthermore even with the identificationof molecular targets such as BRCA mutations and HER2amplifications clinical success with available therapies hasbeen minimal23 A recent clinical trial with olaparib a polyADP ribose polymerase inhibitor showed little efficacy compared to standard of care4 Although a subset of gastric diseasehas HER2 amplification monoclonal antibodies against HER2have demonstrated very limited success in GC unlike theresponse seen in HER2 positive breast cancer5 It is clear that Department of Oncology Wayne State University School of MedicineDetroit MI USACorresponding AuthorAsfar S Azmi PhD Department of Oncology Wayne State University Schoolof Medicine Karmanos Cancer Institute John R HWCRC DetroitMI USAEmail azmiakarmanosCreative Commons CC BY This is distributed under the terms of the Creative Commons Attribution License creativecommonslicensesby40which permits any use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cTechnology in Cancer Research Treatmentmore work is needed to elucidate the underlying moleculardrivers and resistance mechanisms in GCGastric cancer is classified mainly using either the Laurenclassification or the World Health anization WHO criteria The Lauren classification compares tumors based ongrowth invasion pattern with subtypes intestinal well differentiated diffuse poorly differentiated and intermediatemixed67 The majority of patients outside US with GC areyounger 60years old and have the poorly differentiated diffuse subtype which is located within the distal portion of thestomach characterized by poor cellular differentiation and highintratumor heterogeneity This subtype has poorer outcomesdue to its widespread infiltration and invasive nature of thedisease78 Conversely within the United States the pathologyof GC is similar to that of malignancies found within the gastroesophageal junction8 Older patients are primarily impactedand the disease is commonly well differentiated intestinalThe welldifferentiated subtype is found in the cardia or lowerregion of the stomach with welldefined glandular structuresand growth pattern The WHO designation for GC was createdin and expands vastly on the Lauren classification67There are subtypes tubular adenocarcinoma papillary adenocarcinoma mucinous adenocarcinoma poorly cohesiveSignet ring cell carcinoma and mixed carcinoma67 Similarities exist between the Lauren and WHO classificationsSignetring cell carcinoma comparable to poorly differentiatedGC is steadily increasing in incidence within the United Statesand around the world9 This increase is attributed to eradication efforts of Helicobacter pylori a pathogen known toinduce intestinal type GC increases in genetic predisposition to genes such as Ecadherin CDH1 hypermethylationand less screening and detection due to the œlow riskpopulation within the United States compared to other regionssuch as Japan10Here we aim to analyze the molecular signatures as well asdifferences between Lauren classified GCs We also aim tounderstand the molecular differences between male and femalepatients with GC We chose to look solely at Lauren classifiedcancers within this due to its established use within themedical community as well as its availability and relevancewithin publicly available data sets Our overarching goal is toidentify and dissect some of the heterogeneous aspects of GCthat are commonly overlooked within the literatureMethodsOncomine Database SearchOncomine Compendia Bioscience was used for analysis andvisualization Three separate data sets were used to explore theup and downregulation of Lauren subtypes of GC Chen Gastric Mol Biol Cell mRNA DErrico Gastric EuropeanJournal Dataset2 mRNA and Cho Gastric ClinicalCancer Research mRNA For the nonsubtyped GC analysis we have used separate data sets Cui Gastric NucleicAcids Research mRNA Wang Gastric MedicalOncology mRNA and Cho Gastric Clinical CancerResearch mRNA To find highly ranked genes weselected our subtype of interest or GC compared to normaland assessed upregulated or downregulated genes We averaged the fold changes for genes in the individual analyses andhave used the computed P values provided by the OncominesoftwareKyoto Encyclopedia of Genes and Genomes PathwayAnalysisTo identify pathways involved in the genes found to be upregulated or downregulated from our Oncomine analysis weutilized the Kyoto Encyclopedia of Genes and GenomesMiRWalk Database AnalysisMiRWalk Database University of Heidelberg was used foranalysis of gene“microRNAs miRNA interactions11Drug“Gene Interaction AnalysisDGIdb database was used to identify druggable targets withinour genes found to be differentially expressed12Protein DatabaseThe Human Protein Atlas available from httpwwwproteinatlas was used to identify survival curves in stomach cancerwith the following proteins CWD43 Stage IIV Survivalcurves wwwproteinatlasENSG00000109182CWH43pathologystomach¾cancer METLL7A Stage IIVwwwproteinatlasENSG00000185432METTL7Apathologystomach¾cancer SLC2A12 Stage IIV wwwproteinatlasENSG00000146411SLC2A12pathologystomach¾cancer MAL Stage IIV wwwproteinatlasENSG00000172005MALpathologystomach¾cancer DMRT1 Stage IIV wwwproteinatlasENSG00000137090DMRT1pathologystomach¾cancerAll are available from v19proteinatlasProtein“Protein Interaction NetworksSTRING Database was used to identify protein“proteininteractions for the following genes CWH43 METLL7ASLC2A12 MAL BTD CAPN9 ADAM17 EPB41 TOM1L1and DMRT113GEO Database AnalysisThe data discussed within this publication have been previously deposited in NCBI™s Gene Expression Omnibus andare accessible through GEO Series accession numberGSE118916 wwwncbinlmnihgovgeoqueryacccgiacc¼GSE118916 0cSexton et alTable Top Upregulated Genes Found in Gastric Cancer Cohort viaOncomine DatabaseaGene nameFold change diffusevs normal averagePublicationsP valuefoundINHBACOL1A2CLDN1CDH11COL3A1COL5A2COL1A1TIMP1SULF1SPON2549E7949E12664E6117E10241E6289E6299E6383E6465E6644E10aP values were calculated using Oncomine softwareStatisticsOncomine software and Human Protein Atlas providedStatisticsEthical ApprovalThe data are not obtained from patients and does not requireinstitutional review board approvalResultsGenetic Analysis of Upregulated GC GenesWithin the literature various genetic aberrations have beenproposed that can serve as prognostic or therapeutic markersincluding SOX17 hypermethylation BCL2 transforminggrowth factor beta TGFb vascular endothelial growth factorVEGFR and HER21418 Many of these proposed markersare studied extensively and do not serve as ideal targets dueto their limited clinical utility as either drug targets or predictors of therapeutic response Some examples of this include lesssuccessful attempts to target HER2 with monoclonal antibodiesand the use of TGFb inhibitors which although promisinghave proven to be highly toxic1920 Additionally these targetshave demonstrated limited clinical utility due to the crosstalkbetween TGFb and other signaling pathways such as RAS aknown nontargetable protein2122 While VEGF inhibitors areused as a therapeutic modality in GC they do not improveoverall survival23 An indepth investigation of the molecularmechanisms are urgently and investigations need to be distinctfrom the commonly studied and clinically intractable targetsAlthough this is the case discrepancies exist within the literature as some groups look at the molecular composition of GC asa whole while others focus on differences within the Laurenclassification systemUsing the Oncomine database we have found significantupregulation in several understudied genes in all GCs including COL3A1 COL5A2 SPON2 and CDH11 Table Wealso have confirmed the upregulated status of many of thegenes found within the literature that are somewhat well knownsuch as INHBA a gene associated with poor overall outcomes24 but are still understudied Claudin CLDN1 hasbeen found to be highly expressed in GC and is a poor predictive disease marker by mediating tumor necrosis factorainduced cell migration enhancement of proliferation andmetastasis while SULF1 has been found to be significantlyhypomethylated causing significant downregulated proteinexpression2528 This SULF1 downregulation may be indicativeof a posttranslational modification feedback loop or degradation event via protein“protein interactions but is still unclearNot surprisingly a significant underrepresentation was notedwhen comparing publications related to these genes over publications to the commonly studied genes such as MAPKPI3K and TP53 over total publicationsGenetic Analysis of Upregulated GC Genes Using LaurenType Classified GCsWe stratified the data sets based on the respective Lauren distinguished subtype and have highlighted the vast heterogeneticmolecular landscape within the poorly differentiated diffusewell differentiated intestinal and mixed GC subtypesTable Poorly differentiated GC shares many similaritieswith GC overall including perturbations in various collagentranscribing genes stimulation of PI3KAKT signaling andperturbations in cellular structural components This is a dominant subtype throughout the world for reasons we have previously mentioned Due to the overabundance of collagentranscribing genes we wanted to explore whether a potentialgenetic link exists Literature search identified a study correlating EhlersDanlos syndrome EDS a disease caused bycollagen gene perturbations to the development of GC29EhlersDanlos syndrome also presents with gastrointestinalinvolvement such as increased rates of heartburn which is arisk factor for developing esophageal cancer3031 Based on thelocation of these gastric tumors within the stomach that is inthe proximal stomach near the esophagus and the connectionbetween gastric and esophageal cancers it is quite possiblethere may be a much stronger correlation between EDS anddiffuse GC than previously thoughtWe have found GC overall does not share many molecularsimilarities with the welldifferentiated subtype of GC withinthe scope of our analysis We have found only a similarityCLDN1 expression Claudin is a gene involved in coding forthe protein involved in epithelial barrier functions and is part ofthe claudin family Within GC CLDN1 has found to be differentially expressed in GC and has been found to be upregulatedin a small patient population being linked to poor survival outcomes indicative of an oncogenic function32 Other groupshave found claudin1 has tumor suppressive activities and canreverse the epithelialtomesenchymal transition in GC cellsand was found to be downregulated in intestinal type GC in aof patients cohort3334 It is clear that work needs to be donein order to elucidate the role CLDN1 plays within intestinaltype gastric tumors as it has differing functions based on the 0cTechnology in Cancer Research TreatmentTable Top Significantly Upregulated Genes Based on Molecular Subtype of Gastric Cancer Well Differentiated Poorly DifferentiatedMixed Subtype Based on Oncomine DatabaseaFold change diffusevs normal averageP valueKEGG pathway analysisGastric cancersubtypeGene nameTHY1TIMP1BGNCOL1A2SULF1COL6A3OLFML2BRAB31THBS2COL1A1TTYH3THY1CADUBE2CCLDN1PRC1DAZAP1ATP11ADCAF13MTHFD1LCOL6A3FBN1RCC2AHCYTGIF1FN1MYO9BVCANLUMMCM4PI3KAKT focal adhesion ECM receptor proteoglycansPI3KAKT focal adhesion ECM receptor““Immune component161E12124E11 HIF signaling238E11223E10139E9 Metabolism585E9404E8361E9 Membrane trafficking118E8165E7232E23 Transporter346E21202E8Phagosome PI3KAKT focal adhesion ECM“receptor interactionPI3KAKT focal adhesion ECM receptor proteoglycansImmune componentPhenylpropanoid biosynthesis metabolic pathways biosynthesis ofsecondary metabolitesmRNA surveillance262E20 Ubiquitinmediated proteolysis650E15 Cell adhesion tight junction134E14 Tubulin binding protein680E8768E19 Metabolism translocase971E8893E9109E7191E7161E9213E6733E9943E9Ribosome biogenesisOne carbon metabolismPI3KAKT signaling focal adhesion ECM“receptor interactionTGFb signaling“Cysteine and methionine metabolismTGFb signalingPI3KAKT signaling focal adhesion ECM“receptor interactionregulation of actin cytoskeleton proteoglycans and pathways in cancer224E6 Membrane trafficking260E6380E6833E6Cell adhesion molecules CAMsProteoglycans in cancerDNA replication cell cycleDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalMixedMixedMixedMixedMixedMixedMixedMixedMixedMixedAbbreviations ECM extracellular matrix KEGG Kyoto Encyclopedia of Genes and Genomes TGFb transforming growth factor betaaP values were calculated via Oncomine software and KEGG pathway analysis was used to analyze gene functionliterature Many of the processes underlying intestinal GCinvolve alterations in metabolism and cellular crosstalkTable It is not surprising that the intestinal and diffuse GCsare distinctly different but we did find similarity with THY1expression both having similar fold changes Although thisgene has not been investigated in GC it is overexpressed inthe pancreatic cancer microenvironment35 Further investigation may be needed as this gene may have importance in GCdevelopmentWe finally investigated the mixed subtype of GC a subtypethat is commonly overlooked within the literature Table Interestingly mixed GC has some similarities to the diffusesubtype including PI3KAKT signaling a collagen transcribinggene and upregulation of cellular anizational componentsInterestingly we have found the genes perturbed within thissubtype are involved in driving a number of genetic diseasessuch as Marfan syndrome FBN1 and hypermethioninemiaAHCY Research has shown Marfan syndrome due to aberrant TGFb signaling can induce GC development in a murinemodel36 Hypermethioninemia which can go undetected foryears was found to induce aggressive cancers by protectingtumors from 5flurouracil 5FUinduced death a chemotherapy commonly used to treat GC3738 It is likely the diffusesubtype is not the only subtype with a strong genetic link butthe mixed subtype may have a stronger genetic component thanpreviously thought We hypothesize some of the genetic diversity within GC is masked when analyzed as a whole whichfurther supports the notion of this disease being highlyheterogeneousGenetic Analysis of Downregulated GC GenesThere are about twice as many published studies looking atupregulated GC genes compared to downregulated vs The most common downregulated GC genes are influenced in part by aberrant DNA methylation3940 Other thanthis much less is studied pertaining to highly significant downregulated genes in GC Using the Oncomine database we have 0cSexton et alTable Top Significantly Downregulated Genes According to Oncomine Database in Gastric CanceraGene nameLIFRCWH43RDH12MFSD4METTL7AATP4BSLC2A12GHRLMALADH7Fold change diffusevs normal averageP valueKEGG pathway analysis 251E6Cytokine“cytokine receptor interaction signaling for pluripotency in stem cells279E9136E8220E5227E5165E10365E10617E8119E9947E8JAKSTAT signaling“Retinol metabolism metabolic pathways““Oxidative phosphorylation metabolic pathways gastric acid secretionTransportercAMP signaling neuroactive ligand“receptor interaction growth hormonesynthesis secretion and action“Glycolysisgluconeogenesis fatty acid degradation tyrosine metabolism retinolmetabolism chemical carcinogenesisAbbreviation KEGG Kyoto Encyclopedia of Genes and GenomesaP values were calculated via Oncomine software and KEGG pathway analysis was used to analyze gene functionfound the most significant downregulated genes were LIFRRDH12 MSFD4 ATP4B GHRL and ADH7 All of these arepoorly represented within the literature Table We haveinvestigated the survival outcomes of select genes from table that have not been investigated in gastric cancer to the best ofour knowledge These genes include METTL7A MALSLC2A12 and CWH43 Figure 1A We found a trend towardimproved survival with upregulated CWH43 and downregulated METLL7AWe have included protein interaction networks for the genes we have obtained using the STRING databaseFigure 1BE SLC2A12 interacts with AKT1 a commonly studied gene of interest within GC known to contribute to chemoresistance41 Although many of the interacting proteins are not aswell studied as AKT1 various genes such as MTUS1 PGAP3ALDOA and PMP22 have been shown within the literature toonly influence GC but pancreatic cancer as well4246 It is clearthat further investigation into these understudied specific geneticinteraction networks are needed We then wanted to look intowhether any of these genetic aberrations or their interactor proteins were targetable To do this we utilized the DGIdb METTL7A is a methyltransferase that is located primarily in lipiddroplets and is silenced via DNA methylation in thyroid cancer47 There is a variety of drug interactions within the networkof METTL7A including CDA gemcitabine cytaribine deoxycytidine LTA4 H Kelatophan Ubenimex and a variety of preliminary drug compounds B2 M pembrolizumab QPCTpramipexole ALDOA a variety of preliminary compoundsand HP Estradiol pyridoxine Pembrolizumab has been FDAapproved for the treatment of advanced staged GC with positivePDL1 expression B2M acquired mutations were found to conferresistance to pembrolizumab in other malignancies48 but little isknown in GC Downregulation of these genes may partiallyexplain why there is some efficacy issues with pembrolizumabor other chemotherapies MAL encodes a membrane proteinwithin the endoplasmic reticulum ER of Tcells and is involvedin myelin biogenesis49 Drug interactions within the networkinclude ACTA1 kabiramide c latrunculin ab aplyronine a anda variety of preclinical compounds LIMK1 dabrafenibPMP22 progesterone and MAG GSK249320 CWH43 isinvolved in cell wall biogenesis and involved in lipid remodeling50 Drugs that interact with the protein network include UPP2fluorouracil brivudine Understanding the genetic landscape ofGC gene interaction networks and how those genes respond totherapies may explain partially why this disease is highly resistant to conventional chemotherapies However more work isneeded to understand the possible underlying resistance mechanisms within subsets of GC that would bring forward the idealpopulations that benefit from conventional and commonly usedtherapiesIncreasing interest has been placed around small RNAsincluding miRNAs involvement within GC development5152We wanted to investigate the interaction networks betweenthese uncharacterized genes of interest bold and miRNAsUsing the miRWalk database we found miRNA to interactwith our genes of interest Figure 1FI Many of the miRNAsare uncharacterized in GC but we did find that miRNA612miR612 a METTL7A interacting miRNA induces PAX8 atumorsuppressor and represses FOXM1 to inhibit angiogenesis and metastasis of GC53 Our lab™s work in part involves studying the role of nuclear export and miRNA expression and uncovering ways in which tumor suppressive miRNAs canbe upregulated within the nucleus by manipulating nuclearexport Nuclear export via XPO1 has a limited role in exportingmiRNA from the nucleus to the cytosol rather than its nuclearexport family member XPO5 which exports the majority ofcellular miRNAs54 XPO1 overexpression was found to be atherapeutic target in GC and we have found blocking the protein with the small FDA approved molecule selinexor XPOVIO influences the expression of a subset of tumorassociatedmiRNAs55 Furthermore we have found via small RNAsequencing that after XPO1 inhibition with selinexor as well 0cTechnology in Cancer Research TreatmentFigure Gastric cancer is a highly heterogeneous disease A Survival curves taken from the human protein atlas for CWH43 METTL7ASLC2A12 and MAL BD Protein interaction networks for CWH43 METTL7A SLC2A12 and MAL taken from the STRING Database EHmiRNA interaction networks found from top interactions with CWH43 METTL7A SLC2A12 and MAL in the miRDb as the second generation inhibitor KPT8602 miR7977CWH43 interacting miRNA is significantly upregulated foldchange P ¼ 392E23 and fold change P ¼ 546E in the early stage diffuse gastric cell line SNU1 suggestiveof the tumor suppressive role of this miRNA The connectionbetween nuclear export and cancerspecific miRNAs in GC has 0cSexton et alTable Top Significantly Downregulated Genes Based on Molecular Subtype of Gastric Cancer Well Differentiated Poorly DifferentiatedMixed Subtype Based on Oncomine DatabaseaGenenameMT1GMT1FGCNT2SLC9A1PPFIBP2DBTMT1MPXMP2MT1HGSTA2MALPGA3SIDT2ADRB2BRP44 LSSTGCNT2CKMT2RAB27ASTK32BFGAPXMP2NRXN1GSTA2PKIBPOU2AF1SLC22A23AQP4MLXCXCL14Fold change diffuse vsnormal averageP value KEGG pathway analysisGastric cancersubtype 143E4 Mineral absorption213E10 Mineral absorption597E7 Glycosphingolipid biosynthesis metabolism762E7 Transporter150E9654E4 Valine leucine isoleucine degradation propionate metabolism metabolicpathwayPeroxisome903E7172E9113E6 Mineral absorption231E9 Glutathione metabolism drug metabolism platinum drug resistancepathways in cancer chemical carcinogenesis881e11 Ribosome biogenesis454e12 Protein digestion and absorption199E10 103E12 cAMP signaling neuroactive ligand“receptor interaction188E12 Mitochondrial biogenesis422E8cAMP signaling neuroactive ligand“receptor interaction gastric acidsecretion growth hormone synthesis secretion and action206E12 Glycosphingolipid biosynthesis metabolic pathways537E8 Arginine and proline metabolism metabolic pathways258E12 Membrane trafficking156E9 Metabolism318E10 Membrane trafficking175E8190E7 Cell adhesion molecules CAMs155E6 Glutathione metabolism drug metabolism platinum drug resistancePeroxisomepathways in cancer chemical carcinogenesis184E6890E7111E5 anic acid transporters384E6 Bile secretion vasopressinregulated water absorption139E5139E5 Cytokine“cytokine receptor interaction viral protein interaction chemokineInsulin resistance nonalcoholic fatty liver disease NAFLDDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalMixedMixedMixedMixedMixedMixedMixedMixedMixedMixedAbbreviation KEGG Kyoto Encyclopedia of Genes and GenomesaP Values were calculated via Oncomine software and KEGG pathway analysis was used to analyze gene functionsignaling pathwaynot been investigated in depth We are working toward not onlycharacterizing this novel interaction but also using this information to uncover novel genes pertinent to GC growth anddevelopmentGenetic Analysis of Downregulated GC Genes UsingLauren Type Classified GCsWe stratified the data sets based on the respective Lauren distinguished subtype as we did previously and have highlightedthe vast heterogenetic molecular landscape within the diffuseintestinal and mixed Table GC subtypes All subtypes areexpectedly distinct from one another within our molecular analysis The diffuse and intestinal type GCs seem to have moreprominent downregulation of metabolism related genes suchas GSTA2 and DBT GSTA2 is involved with chemoresistancedue to the action of glutathione metabolism an antioxidant andthis observation suggests that this subtype may be more sensitiveto platinum drugs56 This overall downregulation of metabolicpathways may also point to an increase in the Warburg effectThis alternative metabolic pathway has been suggested to contribute phenotypically to high rates of invasion and aggressiveGCs57 We also observed downregulation of ADRB2 in theintestinal type GC Table Zhang et al described ADRB2signaling as essential in GC and is likely related to stressinduced tumor induction58 They suggest treating with antagonists of ARDB2 likely will provide survival benefit This may beimportant to note and be beneficial for nonintestinal like GCsbecause there is a clear trend of significant downregulation ofthis gene fold differenceWe next assessed the molecular aberrations in the downregulated genes of mixed subtype GC Table Interestingly 0cTechnology in Cancer Research Treatmentwe found various genes that are significantly downregulatedwith no pathway analysis and no real evidence of a mechanismat the protein level Table PKIB function has not beenexplored within the literature in regard to GC but has beenshown to promote proliferation through PI3KAKT pathwayin breast cancer59 POU2AF1 is another gene that has not beencharacterized within the GC literature but has been found to bea highrisk gene in gastrointestinal stromal tumors a type ofsoft tissue sarcoma and rheumatoid arthritis6061 Again themixed subtype is molecularly different from the intestinal anddiffuse gastric subtypes based on this genetic pathway analysiswith notably less involvement of metabolism related genesAlthough this is expected due to its difference in subtypingthe mixed gastric subtype has a much smaller representationwithin the literature than the intestinal and diffuse types and itis clear that further investigation is needed A better understanding of the diverse nature of downregulated genes in allaspects of GC is needed as a first step to identify new therapeutic options that will benefit patients with GCGastric Cancer Exhibits High Molecular DifferencesBetween GendersWithin the United States men and women older than are athigher risk for developing GC while the male population ishigher in risk for welldifferentiated GC development than thefemale population mainly due to the protective effect of estrogen against developing H pylori induced gastric carcinogenesis62 Females have higher incidence of poorly differentiatedGCs compared to their male counterparts for reasons largelyunknown Various environmental factors play a role in diseasedevelopment as a whole including obesity smoking drinkingand a poor diet6366 A retrospective study by Kim et al hasshown that women not only have a higher incidence of diffusetype GC but have a worse overall prognosis as well as geneticdifferences compared to men including ERb expression67 suggesting a hormonal component may also be a contributing factor to this subset of disease Due to the evident genderdisparities in GC we investigated the underlying moleculardifferences between male and female patients by preformingGEO2R analysis on the GSE118916 data set Our results showstriking differences in differentially expressed genes betweenmales and femalesOverall both male and female patients with GC showed anabundance of upregulated genes Figure 2A After stratifyingbased on gender the female patients with GC have a higherabundance of upregulated genes oncogenic like genes genes greater than 5fold upregulation compared to downregulated genes Figure 2B while male patients with GC have agreater abundance of downregulated genes tumor suppressorlike genes Figure 2B This trend can also be seen from just thetop differentially expressed genes in the provided tables Current treatment options for GC are somewhat limited in achieving a longterm survival benefit and we wanted to use ourcohorts to identify whether there are differences in actionabletargets between gendersFemale Patients With GC Are Vastly UnderrepresentedWithin Clinical StudiesWe found no direct druggable targets according to the DGIdbdatabase with the top differentially expressed genes Therefore we looked further into the individual protein“proteininteraction networks using STRING database Figure 2CFBroadening the scope of our search allowed us to find manypotential druggable targets Table We narrowed the scopeof our search to inhibitorsantagonist type compounds due tothe substantial genes found to be upregulated Many of thedruggable targets such as estimated glomerular filtration rateEGFR tyrosine kinase inhibitors TKIs are currently beingexplored in a variety of malignancies including GC Erlotinibwas investigated in a phase II clinical trial in combination withoxaliplatinleucovorin5FU in metastatic GC68 Lapatinib aTKI responsible for inhibiting HER2neu and EGFR wastested in a phase III clinical trial TyTAN Trial in Asianpatients with GC69 There was no statistically significant difference in overall survival for Paclitaxel plus Lapatinib overPaclitaxel alone70 We looked further into the patient demographics of the TyTAN trial and noticed a large underrepresentation of female patients within all arms of the study total female patients Another example of this is a trial withBortezomib which interacts with the ADAM17 pathway andhas been tried unsuccessfully in Phase II clinical trials in combination with paclitaxel and carboplatin in metastatic patientswith GC71 As with the Lapatinib trial this one had an overrepresentation of male patients compared to femalepatients A common occurrence within many of theGC clinical trials is combination of new therapies with paclitaxel or some type of Taxol We have found the female cohortto have an abundance of druggable targets interact with paclitaxel including EPB41L4B and CAPN9 Table but largelythis demographic is underrepresented within clinical trial studies It is clear that based on the molecular profile of femalepatients with GC this issue demands further investigationMale Patients With GC May Benefit From HormoneInhibiting TherapiesAs we have previously mentioned the male cohort has anopposite molecular profile compared to the female cohort withWhen screening for actionable drug targets we limited thescope of our analysis to agonists due to the substantial geneticdownregulation already occurring naturally and notion thatmale patients with GC have an abundance of tumor suppressorlike genes In doing so we have found direct druggable targetssuch as SSTR1 and GPT Table GPT is a gene that encodesthe alanine aminotransaminase protein and catalyzes thereversible transamination between alanine and 2oxoglutaratewithin the tricarboxylic acid TCA cycle to generate pyruvatea TCA intermediate and glutamate72 Glucagon and tacrolimus interact with GPT but the stimulation of this gene wouldlikely enhance glucose metabolism through the TCA cyclelikely being nonbeneficial as a treatment option Furthermore 0cSexton et alFigure Male and female patients with gastric cancer have different molecular signatures A Density plots of differentially expressedgenes in the GSE118916 data set for all gastric cancer cases within the cohort B Male and female cohort density plots of the differentiallyexpressed genes in the GSE118916 data set CG STRING Database interaction networks for protein networks from genes found to bedifferentially expressed in female gastric cancer cases within the cohort BTD CAPNS9 EPB41L4B ADAM17 TOMIL1Tacrolimus can influence the development of lymphomas73Although targeting GPT would not be beneficial targetingSSTR1 may have more benefit Hypermethylation of SSTR1was found to contribute to the pathogenesis of GC by actingin a tumor suppressive manner This hypermethylation wasfound to be caused by EpsteinBarr virus infection74 a positive 0cTechnology in Cancer Research TreatmentTable Genes Found to Be Significantly Differentially Expressed Within the Female Cohort From the GEO Database GSE118916aFold change diffuse vsGene namenormal averageP value DrugFBX13DMRTA1BTDPFDN2GRAMD1CCAPN9PBLDEPB41L4BADAM17 109E9 201E8 Testosterone Tretinoin LY294002201E8 Biotin Hydrocortisone Aspartic Acid Celiponase alfa319E9 533E8 620E8 Emricasan Paclitaxel Rizatriptan Celecoxib Idronoxil956E8 961E8 Paclitaxel Vindesine Colchicine Docetaxel Cabzitaxel Erbulin mesylate IxabepiloneLexibulin Tamoxifen Ornithine144E7 Cetuximab Nimotuzumab Tesevatinib Infliximab Etanercept Adalimumab GolimumabHydrocortisone Everolimus Methotrexate Mercaptopurine Bortezomib PrednisoloneDexamethasone Ribociclib Nitrogacestat Dacomitinib Lapatinib Erlotinib PoziotinibIbrutinib PelitinibTOM1L1155E7 Erlotinib Afatinib Gefitinib Cetuximab Lapatinib Panitumumab Rociletinib IcotinibLacomi

Thyroid_Cancer

obesity and ethnicity alter gene expression in skinJeanne M Walker18 Sandra Garcet28 Jose O Aleman34 Christopher E Mason5 David Danko5 Simone Zuffa6 Jonathan R Swann67 James Krueger2 Jan L Breslow3 peter R Holt3Obesity is accompanied by dysfunction of many ans but effects on the skin have received little attention We studied differences in epithelial thickness by histology and gene expression by Affymetrix gene arrays and PCR in the skin of obese BMI “ and normal weight BMI “ postmenopausal women paired by age and ethnicity Epidermal thickness did not differ with obesity but the expression of genes encoding proteins associated with skin blood supply and wound healing were altered In the obese many gene expression pathways were broadly downregulated and subdermal fat showed pronounced inflammation There were no changes in skin microbiota or metabolites African American subjects differed from European Americans with a trend to increased epidermal thickening In obese African Americans compared to obese European Americans we observed altered gene expression that may explain known differences in water content and stress response African Americans showed markedly lower expression of the gene encoding the cystic fibrosis transmembrane regulator characteristic of the disease cystic fibrosis The results from this preliminary study may explain the functional changes found in the skin of obese subjects and African AmericansObesity defined as a body mass index BMI greater than a0kgm21 has become a major epidemic in industrial and emerging countries The prevalence of obesity has doubled since the 1980s and it is now estimated that million adults worldwide are obese2 Obesity affects many ans of the body and it is this an dysfunction that leads to excess mortality and morbidity3 Much attention has focused on the consequences of obesity in the heart liver and pancreas and other ans in which increased inflammation and oncogenesis become apparent4 Less attention has been paid to the effects of obesity on the skinObesity increases psoriasis5 which can be ameliorated with weight loss and cutaneous infections6 Since diabetes is common in obesity disorders such as fibroepithelial polyps and acanthosis nigricans also occur in the skin of obese subjects78 Moreover physiologic changes found in obese skin include increased transepidermal water loss with lower capacitance dry rough textured skin with pronounced erythema and reduced microvascular reactivity Altered collagen formation and increased delayedtype hypersensitivity have also been reported9Adipocyte depots that exist adjacent to the epidermis have distinct morphology and physiologic characteristics and are termed dermal or subdermal adipose tissue In addition to the principal role for dermal adipocytes in lipid storage and thermal insulation10 they also promote skin immunity11 wound healing and hair follicle cycling12 Obesity is accompanied by inflammatory immune changes in subcutaneous and visceral adipose tissues13 but the role of inflammatory changes within the adipose layer of the skin has received little attention Furthermore obesity is associated with increased circulating leptin levels which appear to independently affect dermal cell proliferation and hair growth14 In addition the microanisms that live on the skin surface also affect skin immunity11 so that it is important to analyse the skin microbiome comparing obese and normal individuals1The Rockefeller University Hospital New York NY USA 2Laboratory of Investigational Dermatology The Rockefeller University New York NY USA 3Laboratory of Biochemical Genetics and Metabolism The Rockefeller University New York NY USA 4Laboratory of Translational Obesity Research New York University Langone Health New York NY USA 5Weill Cornell Medical College New York NY USA 6Department of Metabolism Digestion and Reproduction Imperial College London London UK 7School of Human Development and Health Faculty of Medicine University of Southampton Southampton UK 8These authors contributed equally Jeanne M Walker and Sandra Garcet email walkerjrockefelleredu holtprockefellereduScientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cIn view of the profound clinical and physiologic changes described in the skin in obesity it would not be surprising also to find biologically important molecular changes The present study was designed to compare gene expression in skin of healthy obese and nonobese subjects and to evaluate the potential importance of parallel changes in the microbiome and metabolites found on the adjacent skin surface and in the adipose tissue immediately below the skinMaterials and methodsSubjects Participants were recruited from the surrounding community through advertisements and from the Rockefeller University subject repository Eligible were healthy obese BMI “ a0kgm2 and nonobese BMI “ a0kgm2 postmenopausal women between the ages of and a0years The two groups were matched by age ± a0years and defined by selfreported ethnicity and by skin colour Exclusions were unstable weight change within the past three months HIV infection weight loss surgery inflammatory bowel disease history of malignancy other than nonmelanoma skin cancer in the previous a0years generalizable or systemic skin diseases history of a bleeding disorder current anticoagulant therapy or regular NSAID use current weight control medication or hypoglycaemic therapy individuals taking oestrogenprogesterone hormones and current tobacco smokers Also excluded were candidates with fasting blood glucose a0mgdl liver function tests ALT AST alkaline phosphatase greater than times the upper limit of normal ULN abnormal thyroid function test or serum creatinine — ULNFourteen obese subjects were screened two refused skin biopsies one was withdrawn due to an intercurrent inflammatory illness and one was not postmenopausal by our criteria Ten obese subjects met our inclusion criteria and underwent skin swab collections and punch biopsy Twenty nonobese subjects were screened Two subjects refused to undergo punch biopsy one was withdrawn due to an intercurrent illness two with a BMI outside the required range one withdrew consent one was excluded with a history of keloid formation one with a low platelet count one with uncontrolled hypertension One nonobese subject who underwent skin punch biopsy was not included in the analysis because we were unable to find an age and ethnicitymatched obese subject These obese and agematched ethnicitymatched nonobese postmenopausal women completed all aspects of the study Fig a0 Six participants were European American and four were African Americans in each group Postmenopausal women were chosen to exclude effects of the menstrual cycle upon study end points and to exclude gender effectsBased on preliminary data from a previous study comparing skin from seven obese and six nonobese postmenopausal women there was a variation of in a set of RTPCR genes unpublished data Assuming the same variation and proportion of differentially expressed genes to be we calculated that a sample size of n subjects per group matched by age and ethnicity would provide power at a falsediscovery rate to detect the expected number of differentially expressed genes based on a threshold of twofold changesDesign and setting This was an label comparison of a group of postmenopausal obese women and postmenopausal nonobese women who were agematched ± a0years and racematched Screening comprised a complete history and physical examination and fasting blood testing for complete blood count sedimentation rate comprehensive chemistry panel lipid panel thyroid function tests hepatitis C antibody uric acid and haemoglobin A1C Observing Good Clinical Practice guidelines all participants read and signed an informed consent document approved by the Institutional Review Board and the Advisory Committee for Clinical and Translational Science at The Rockefeller University Protocol JWA0921Procedure methods Anthropometric measurements Body weight was measured daily to the nearest a0kg using a ScaleTronix scale Welch Allyn Skaneateles Falls NY with precision of ± a0kg Subjects were weighed in a hospital gown after an overnight fast and postvoiding Height was measured to the nearest a0cm at baseline with a Seca216 stadiometer Hamburg Germany in a0cm increments Body mass index BMI was calculated as kgm2 using the NIH Standard Metric BMI calculatorBlood collection and analysis Fasting blood samples were analysed in the Clinical Pathology Laboratory of the Memorial SloanKettering Cancer Center for complete blood count electrolytes glucose creatinine blood urea nitrogen liver function Creactive protein sedimentation rate and uric acid Research serum samples were drawn pre and post intervention aliquoted and stored at ˆ’ a0°C for subsequent analysisSkin swabbing Subjects were permitted to shower but did not wash the planned biopsy area over the midlower abdomen with soap for a0days before the biopsyFor microbiome analysis two areas of skin approximately — a0cm were swabbed using the eSwab collection and preservation system for aerobic anaerobic and fastidious bacteria Copan Diagnostics Marietta CA Swabs were labelled sealed separately in the provided tubes and immediately stored at ˆ’ a0°C For metabolome analysis two different areas of skin approximately — a0cm were swabbed using salinemoistened sterile cottontipped applicators The tips were cut sealed in separate sterile collection tubes and immediately stored at ˆ’ a0°CSkin microbiome The DNA extraction protocol was adapted from the Maxwell RSC Buccal Swab DNA kit Catalogue number AS1640 Promega Corporation Madison WI Briefly a0μl of lysis buffer and a0μl of Proteinase K was mixed and added to each swab tube Swab tubes were then incubated for a0min at C using a Thermo Fisher water bath removed from the tubes and fluid was transferred to well of the Maxwell RSC Cartridge The swab head was centrifuged using a ClickFit Microtube Cat V4741 and extracted fluid was added to the corresponding well of Maxwell Cartridge and eluted in a0μl of provided elution bufferScientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Consort flow chart of eligible subjectsExtracted DNA was taken forward to the Nextera Flex protocol by Illumina Briefly a0μl of extracted DNA was taken into library prep protocol and run with cycles of PCR Libraries were cleaned up with a left sided size selection using a bead ratio of 08x The right sided size selection was omitted Libraries were then quantified using a Thermo Fisher Qubit Fluorometer and an Advanced Analytical Fragment Analyzer Libraries were sequenced on an Illumina HiSeqPE — at the Weill Cornell Epigenomics CoreAll bioinformatic analysis was performed on Weill Cornell Medicine™s Athena compute cluster a highperformance grid compute system Secondary analysis was performed on a Linux and MacOS systems Unless otherwise noted programs were run with default settingsRaw sequence data were processed with AdapterRemoval v217 to remove low quality reads and reads with ambiguous bases15 Subsequently reads were aligned to the human genome hg38 including alternate contigs using Bowtie2 v230 fast preset16 Read pairs where one or both ends mapped to the human genome were separated from read pairs where neither mate mapped Read pairs where only one mate mapped were discarded Hereafter we refer to the read sets as human reads and nonhuman readsTaxonomic profiles were generated by processing nonhuman reads with KrakenUniq v032 with a database based on all draft and reference genomes in RefSeq Microbial bacteria fungi virus and archaea ca March KrakenUniq identifies kmers that are unique to taxa in a database Reads are broken into kmers and searched against this database Finally the taxonomic makeup of each sample was given by taking the proportion reads which were assigned to each clade KrakenUniq counts the number of unique marker kmers assigned to each taxon and we filtered taxa with fewer than unique markers17We performed differential abundance testing over microbial species using the ALDEx2 R package ALDEx2 performs variance stabilization read counts using a centred log ratio transformation that models samples as Scientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cDirichletMultinomial distributions over taxa then compares taxonomic abundances across groups18 Comparison of abundances across groups was done with a Wilcoxon rank sum test and Benjamini Hochberg Correction for multiple hypothesis testingDimensionality reduction of taxonomic profiles was performed with Principal Coordinates Analysis PCA based on a matrix of JensenShannon Divergences JSD between samples Analysis of intersample beta diversity was performed using the same matrix of JSD Intrasample alpha diversity was measured by finding Shannon™s Entropy of the taxonomic profile and by counting the total number of species identified in each sample richness Shannon™s entropy accounts for the relative size of each group in diversity estimation and is defined as H ˆ’ cid31 ai log2ai where ai is the relative abundance of taxa i in the sampleWe generated profiles of antimicrobial resistance genes using MegaRes v10119 To generate profiles from MegaRes we mapped nonhuman reads to the database using Bowtie2 v230 very sensitive presets Subsequently alignments were analysed using Resistome Analyzer commit 15a52dd and normalized by total reads per sample and gene length to give RPKMs MegaRes includes an ontology grouping resistance genes into gene classes AMR mechanisms and gene groupsSkin metabolome The skin was swabbed using two sterile salinemoistened culture swabs and immediately frozen at ˆ’ a0°C Swab heads were removed and placed in a0ml methanol water Following sonication a0min a0ml of isopropanol was added and the solution was spun at a0g for a0min The swab was removed and the samples were dried using a vacuum concentrator operating at a0°C Prior to UPLCMS analysis samples were reconstituted in a0μl of HPLCgrade water sonicated for a0min and transferred to vials for analysisA Waters 2777C sample manager Waters Corp Milford MA USA was used for sample handling This was equipped with a a0μl Hamilton syringe a a0μl loop used for fullloop injections of prepared sample and a 3drawer sample chamber maintained at a0°C with a constant flow of dry nitrogen gas to prevent the buildup of condensation The LC component was an ACQUITY UPLC Waters Corp Milford MA USA composed of a binary solvent manager and column heatercooler module Metabolic profiles were acquired using reversedphase chromatography Water and acetonitrile each supplemented with formic acid mobile phases A and B respectively were selected for the mobile phase A — a0mm HSS T3 column was used at a0°C with a mobile phase flow rate of a0mlmin This generated a maximum pressure of psi in a wateracetonitrile gradient After a a0min isocratic separation at initial conditions A a linear gradient elution A to A in a0min proceeded followed by a quicker gradient A to A in a0min to final conditions The mobile phase flow rate was simultaneously increased to a0mlmin in the latter stage to facilitate faster column washing The MS component comprised a Xevo G2S QToF MS Waters Corp Manchester UK coupled to the UPLC via a Zspray electrospray ionization ESI source The cone gas flow was set to a0lh to protect the cone from residue accumulation during operation Both positive and negative ion modes RPC and RPCˆ’ respectively were used Raw spectra were converted into mzML files using MSConvert20 and processed with XCMS in R21 Peak picking and peak grouping were performed using inhouse scripts in R and matrices were normalized using a median fold change approach Log transformation scaling and data analysis was performed in SIMCA Umetrics Umea SwedenSkin biopsy After the skin swabbing the abdominal site was cleansed with Chloraprep swabs chlorhexidine and isopropyl alcohol Becton Dickinson Canaan CT Using sterile technique local anaesthesia was induced by infiltration of the area with a0ml of lidocaine Hospira Inc Lake Forest IL mixed with a0ml sodium bicarbonate The skin biopsy was performed using a a0mm punch Miltex Instruments York PA Fat tissue was carefully removed from the skin core of the biopsy The dermis and epidermis were divided into two halves one half placed in a cryomold for OTC flash freezing Agar Scientific Essex UK and stored at ˆ’ a0°C and the other half was placed in RNAlater Stabilization Solution Thermo Fisher Scientific Fair Lawn NJ refrigerated for a0h and then frozen at ˆ’ a0°C The fat tissue was removed from the biopsy divided between RNAlater refrigerated for a0h then frozen at ˆ’ a0°C and a dry Sarstedt tube that was flash frozen in liquid nitrogen and placed in ˆ’ a0°C The biopsy site was sutured closed and a dry sterile dressing was applied Subjects were discharged and scheduled to return for suture removalGene‘array and quantitative real‘time PCR analysis RNA was extracted followed by hybridization to Affymetrix Human U133 Plus gene arrays Santa Clara CA or quantitative RTPCR as previously described2223All statistical analyses were carried out in R Limma Log 2transformed qRTPCR measurements hARP normalized and microarray expression values were assessed with a mixedeffect The fixed factors were condition obese vs nonobese race African American vs Caucasian with random intercept for each subject Quality control of microarray chips was carried out using standard QC metrics and R package microarray quality control Images were scrutinized for spatial artefacts using Harshlight24 Expression measures were obtained using the GCRMA algorithm25 A batch effect corresponding to the hybridization date was detected by PCA and adjusted using the ComBat function from the SVA package Probe sets with at least samples with expression values were kept for further analysis Fold changes for the comparisons of interest were estimated and hypothesis testing was conducted with contrasts under the general framework for linear models with the limma package P values from the moderated paired ttests were adjusted for multiple hypotheses using the Benjamini“Hochberg procedure Hierarchical clustering was performed with Euclidean distance and a McQuitty agglomeration scheme26Data was deposited into Gene Expression Omnibus GEO repository GSE151839All study methods and procedures were carried out in accordance with Good Clinical Practice Guidelines by trained practitioners The protocol and informed consent were evaluated and approved by the Institutional Scientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression between the skin of obese and nonobese subjects A Heat map of the most differentially expressed genes in the skin of obese and nonobese subjects with an FCH fold change fdr false discovery rate B PCA principal component analysis plot of differentially expressed genes in the skin of obese and nonobese subjects with an FCH fdr Review Board and the Advisory Committee for Clinical and Translational Science at Rockefeller University prior to initiation of the study and annually thereafter Protocol JWA0921ResultsThis study was performed in ten healthy obese and ten healthy nonobese postmenopausal women matched for age and ethnicity Obese subjects had a mean weight of a0kg BMI of a0kgm2 and waist circumference of a0cm Nonobese subjects had a mean weight of a0kg BMI of a0kgm2 and waist circumference of a0cm Supplemental Table a0S1 The skin thickness for subjects with obesity was not significantly different from that of nonobese subjects Supplemental Fig a0S1Gene expression analysis of the skin The most differentially expressed genes in the skin between obese and nonobese subjects are displayed in the heat map in Fig a02a Comparing gene expression in obese versus nonobese skin showed greater gene expression of S100A7A encoding a calcium binding protein involved in psoriasis and CORIN encoding a natriuretic peptide converting enzyme which is expressed in the dermis and is involved in specifying skin colour However the expression of CREB3LA encoding a cyclic AMP response element was lower in the skin of obese subjectsA PCA model constructed on of the most differentially expressed genes between obese and nonobese subjects showed partial separation of the groups This difference was seen in PC1 which accounted for of the variation in the included genes Fig a02bThe complete list of skin genes whose expression significantly differed between the two groups are shown with fold changes in Supplemental Tables a0S2 and S3 Again the gene expression of S100A7A was 344fold higher in the obese skin compared to the nonobese skin Similarly the expression of DEFB4A Defensin B4A which encodes an antimicrobial peptide part of the betadefensive system and SPRR2C which encodes a proline rich protein strongly induced during differentiation of human epidermal keratinocytes was also significantly higher in the obese skin being and 17fold higher respectively Genes with lower expression profiles in obese subjects than nonobese included AOP that encodes aquaporin involved in water channels present in the skin PROM1 prominin involved in cell differentiation and proliferation and Keratin and important for fibrogenesis in the epidermis Also of interest was the significantly higher expression 282fold of CFTR the cystic fibrosis transmembrane conductance receptor in nonobese subjects compared to the obese groupQTPCR analysis of genes selected from the total list of significantly differentially expressed genes in skin confirmed increased expression of the S100A 373fold DEFB4A defensin B4A 329fold and CORIN fold in the skin of obese subjects Fig a0 Significantly lower gene expression in the skin of obese subjects was found with CFTR 36fold PROM1 556fold and GABRP gamma aminobutyric acid receptor 29foldScientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cGene expression pathway analysis showed a broad downregulation of many pathways in obesity with only out of of the most highly differentially expressed pathways higher in the obese Supplemental Fig a0S2 The pathways most downregulated included cardiac beta adrenergic signalling which appears to function in skin cyclin dependent Kinase CDK5 a mutation which is important in melanoma formation and functions in skin healing and gonadotrophic releasing hormone GNRH signalling which has many extra pituitary functionsGene expression analysis in skin fat We next examined differences in gene expression between the groups of subjects in subdermal fat removed from immediately below the skin portion of the biopsy A heat map of gene expression shows a markedly different pattern between the two groups Fig a04a The expression of many of the genes upregulated in obese subdermal fat are involved with inflammation and immune function including platelet activating factor PLA2G7 ILIRN involved in IL1 activation SPPI a cytokine that can increase interferon gamma and IL12 activity and several serpins mediators involved in inflammation and immune functionThe PCA plot of genes whose expression differed significantly in subdermal fat of obese and nonobese subjects Fig a04b clearly shows separation between the two groups Most of the difference was seen in PC1 which includes of the genes whose expression was determinedSupplemental Tables a0S4 and S5 show a list of genes whose expression was relatively greater in the subdermal fat of obese subjects The expression of SPPI that encodes osteopontin which can act as a cytokine augmenting the action of interferon gamma and interleukin was approximately tenfold higher in the obese subjects EGFL6 expression which encodes an epidermal growth factor found to be enhanced in obesity and alters insulin action was increased by 85fold MMP9 which encodes metalloproteinase and ILTRN was increased by sevenfold in obesity Genes significantly downregulated in obese subdermal fat included SLC27A2 acetylCoAsynthase tenfold and C6complement fivefoldBy QTPCR in subdermal fat from obese subjects the increased expression of genes encoding proteins important in inflammation and immune function was confirmed Fig a0 This includes genes encoding proteins that determine accumulation of immune cells in adipose tissues such as CD52 the high affinity immunoglobulin gamma FC receptor FCGR1β CCL3 CZXCL8 interleukin and CLEC7A a pattern recognition receptor found in monocytes and other myeloid cells IL17F a member of the IL17 family also was specifically increased in subdermal fat from the obese as compared to nonobese individualsExpression pathway analysis Supplemental Fig a0S3 showed upregulation of several inflammatory immune pathways including the Thelper dendritic cell maturation and inflammatory signalling pathways further indicating profound effects of obesity on inflammation in subdermal fat Dramatically lower in the obese subdermal fat was the LXRRXR activation pathwayGene expression analysis by race Two subgroups were observed in the gene expression profiles of the skin based on the subject™s race Using selfreported data and skin colour as criteria the data from African Americans was analysed separately from the data from European Americans This analysis showed striking differences in this very small group of subjects A heatmap of the most differentially expressed genes in skin from obese subjects divided by ethincity is shown in Fig a06a No clear differences in gene expression in the skin by ethnicity were found in nonobese subjects In contrast gene expression clearly differed between obese African American and obese European American subjects Fig a06a and also is illustrated in the PCA plot Fig a06b with PC1 responsible for the greatest variation The pattern of differences between obese and non “obese skin is further illustrated in Fig a06cA list of genes whose expression significantly differed between the obese African Americans and the obese European Americans is shown in Supplemental Tables a0S6 and S7 The expression of SLC6A4 a serotonin transporter CORIN and COL8AI a collagen gene encoding a protein that is dysregulated in atopic eczema was higher in African Americans while the expression of SCCB2A2 the secretoglobin expressed in skin sweat glands and CFTR was expressed higher in European AmericansComparing the skin of obese African Americans to obese European Americans by QTPCR the former showed significantly lower expression of MYBCPI 516fold and PROM1 43fold and CFTR Fig a0 In contrast there was a small increase in the expression of CORIN 22fold a gene encoding the atrial naturalistic peptide converting enzyme and BMP2 fold also present in the skin compared to obese European AmericansPathway analysis found no racerelated differences in the nonobese samples However in the obese Supplemental Fig a0S4 there was markedly reduced expression of oestrogen mediated sphase entry pathway aryl hydrolase receptor signalling pathway and cell cycle regulation through cyclins pathways in the African Americans compared to the skin of the European American groupSubdermal fat in African Americans exhibited few differences from that found in European Americans Fig a08A Figure a08bc show the differences by weight and by ethnicity respectively illustrating the impact of obesity in the two racial groups The expression of numerous inflammatoryimmune genes was upregulated in the fat of both groups of obese subjects Fig a0 Tables a0S5 and S6 Microbiota analysis We next examined whether the microbiota collected from skin swabs around the biopsy site differed between the obese and nonobese subjects We generated taxonomic profiles for each sample using KrakenUniq and a database built from all available microbial species in RefSeq We measured the total number of AMR genes detected in each sample by aligning reads to MegaRESOverall differences between groups were minor No significant differences were noted in average taxonomic alpha diversity as measured by either Shannon™s entropy or richness between the groups A PCA plot of the taxonomic profiles showed slight separation between obese and lean samples and slightly higher beta diversity for obese samples however these differences were minor Fig a010a The number of antimicrobial resistant AMR Scientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RT PCR between the skin of obese and nonobese subjects LS means of gene expression by RTPCR showing significant differences as p p p Figure a0 Differences in gene expression between the subdermal fat of obese and nonobese subjects A Heat map of the most differentially expressed genes in subdermal fat of obese and nonobese subjects with an FCH fdr B PCA plot of differentially expressed genes in subdermal fat of obese and nonobese subjects with an FCH fdr Scientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RTPCR between the subdermal fat of obese and nonobese subjects LS means of gene expression by RTPCR showing significant differences as p p p Figure a0 Differences in gene expression between the skin of African American and European American subjects A Heat map of the most differentially expressed genes in skin of African American and European American subjects with an FCH fdr B PCA plot of differentially expressed genes in skin of obese and nonobese African American and European American subjects with an FCH Left side of plot indicates differences in gene expression by ethnicity in nonobese subjects Right side of plot indicates differences in gene expression by ethnicity in obese subjects C PCA plot of differentially expressed genes in skin of obese and nonobese subjects by ethnicity with an FCH Left side of plot indicates differences in gene expression between obese and nonobese African American subjects Right side of plot indicates differences in gene expression between obese and nonobese European American subjectsScientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RT PCR between the skin of obese and nonobese African American and European American subjects LS means of gene expression by RTPCR showing significant differences as p p p genes identified was higher on average from obese subjects but this difference did not reach significance p Wilcox test Fig a010bDifferentially abundant taxa At the given sample size n no taxa were identified as significantly differentially abundant after Benjamini“Hochberg correction Before correction five taxa were significantly differentially abundant at p Wilcox test These five taxa were Corynebacterium aurimucosum Corynebac‘terium jeikeium Corynebacterium urealyticum Streptococcus salivarius and Streptococcus sp A12 All five taxa were more abundant in samples from obese subjects on averageMetabolome analysis As highlighted by the PCA analysis no variation in the metabolites analysed by liquid chromatographymass spectrometry from the skin swabs was observed between the obese and nonobese individuals PCA Supplemental Fig a0S6 Similarly no ethnicityrelated metabolic variation was observed These results were further confirmed by the poor predictive ability of the orthogonal projection to latent structuresdiscriminant analysis OPLSDA models comparing the two different ethnic groupsDiscussionFew comprehensive reviews of skin changes occurring with obesity have been conducted despite over of the US population being obese27 A broad review of the physiologic and clinical consequences and associations was published by Hirt et a0al in The authors include a discussion of circulatory and lymphatic changes which may enhance the frequency and severity of skin ulceration and provide a comprehensive review of skin disorders that can be associated with obesity expanding on previous reviews29 and studies in rodents30In our study the thickness of

Thyroid_Cancer

emergence of promising targeted therapies for the treatment of hepatocellular carcinoma HCCSorafenib has been the mainstay of treatment for a decade and newer modalities were ineffective and did not confer any increasedtherapeutic benefit until the introduction of lenvatinib which was approved based on its noninferiority to sorafenib Thesubsequent success of regorafenib in HCC patients who progress on sorafenib treatment heralded a new era of secondlinetreatment and was quickly followed by ramucirumab cabozantinib and the most ‚uential immune checkpoint inhibitors ICIsOver the same period combination therapies including antiangiogenesis agents with ICIs dual ICIs and targeted agents inconjunction with surgery or other locoregional therapies have been extensively investigated and have shown promise andprovided the basis for exciting clinical trials Work continues to develop additional novel therapeutic agents which could potentiallyaugment the presently available options and understand the underlying mechanisms responsible for drug resistance with the goalof improving the survival of patients with HCCSignal Transduction and Targeted Therapy 101038s4139202000264xINTRODUCTIONPrimary liver cancer remains a major problem for all health caresystems worldwide and is associated with a significant clinicaleconomic and psychological burden Hepatocellular carcinomaHCC accounts for of cases of nonmetastatic tumors of theliver1 During the past decades research has shed light on theepidemiology risk factors and molecular and genetic profiles ofHCCš contributing to the evolution of strategies for preventionsurveillance early diagnosis and treatment23 Liver resectionablation and liver transplantation are potentially curative butrequire diagnosis at a sufficiently early stage Unfortunately asignificant proportion of HCC patients present with intermediateand advanced stage disease often despite diligent surveillanceand curative treatments are frequently not possible4 In thesepatients systemic therapy remains essential and its pivotal roleand potential have stimulated considerable research over the pastdecade In this review we examine recent advances in targetedtherapy and discuss the impact this has had on the managementof HCC We also provide an overview of the most important areasof HCC research including novel clinical trials and technicalplatforms which promise to facilitate substantial progress withinthe next decadeAPPROVED FIRSTLINE AGENTS FOR HCCSorafenibThe success of SHARP and AsiaPacific trial promoted the approvalof sorafenib as firstline targeted therapy for advanced HCC5“ushering in the era of systemic treatment Subsequently virtuallyall trials were centered around sorafenib and it was used as acontrol with which novel firstline agents were compared andevaluated in an attempt to improve the prognosis of patients withHCC Unfortunately despite a number of trials which comparedthese novel agents including sunitinib10 brivanib11 cediranib12linifanib13 dovotinib14 and immunecheckpoint inhibitors ICIs tosorafenib none achieved the predefined primary end points Fig In addition during the decade when these agents were investigated the median overall survival OS of sorafenib monotherapy asfirstline treatment for advanced HCC increased from monthsSHARP to months CheckMate459 further consolidating itsposition Meanwhile the antitumor activity and safety of sorafenibhave been validated in realworld setting Subanalyses of the SHARPand AsiaPacific trials found sorafenib was effective and safeirrespective of disease etiology disease burden ECOG EasternCooperative Oncology Group performance status status etc15“The safety of sorafenib was consistent across ChildPugh A and Bpatients in clinical practice18 and the occurrence of side effects suchas handfoot syndrome and diarrhea were associated with animproved OS19 Baseline hepatic function clinicopathologicalfactors and etiology also affect the prognosis in HCC patientstreated with sorafenib20 In addition sorafenib exerts antitumoreffects with recurrenttransplantationconferring a survival advantage when compared with bestsupportive care BSC21“ Noticeably the application of sorafenibin clinical practice displays significantregional variations andincompliance with guidelines besides its usage as firstline therapyIt is common that initially unresectable HCCs got downstaged aftersorafenib treatment and underwent curativeintent surgery24“ andlocoregional therapies before sorafenib were commonly encountered in realworld settings2930tumors following liver1Department of Liver Surgery and Transplantation Liver Cancer Institute Zhongshan Hospital Fudan University Shanghai China 2Key Laboratory of Carcinogenesis and CancerInvasion Fudan University Ministry of Education Shanghai China 3Shanghai Key Laboratory of an Transplantation Zhongshan Hospital Fudan University Shanghai China4Department of Hepatobiliary and Pancreatic Surgery University Hospitals of Leicester NHS Trust Leicester UK 5Institute of Biomedical Sciences Fudan University ShanghaiChina and 6State Key Laboratory of Genetic Engineering Fudan University Shanghai ChinaCorrespondence Jian Zhou zhoujianzshospitalshcnThese authors contributed equally Ao Huang XinRong YangReceived April Revised July Accepted July The Authors 0cTargeted therapy for hepatocellular carcinomaHuang et alFirstlineSorafenibSharpAsiaPacificCediranibNCT00427973SunitinibNCT0069937BrivanibBRISKFLLinifanibNCT01009593NintedanibNCT01004003DovitinibNCT01232296ATEZOBEVGO30140IMbrave150LenvatinibREFLECTPEMLENKEYNOTE524NivolumabCheckMate459DonofenibChina SecondlineBrivanibBRISKPSEverolimusEVOLVE1AxitinibNCT01210495RamucirumabREACHRegorafenibRESORCENivolumabCheckMate040PEMKEYNOTE224TivantinibMETIVHCCS1SCUBEPEMKEYNOTE240CabozantinibCELESTIALRamucirumabREACH2NIVIPICheckMate040CAMChinaApatinibChinaFig Overview of the targeted agents approved for HCC ATEZO atezolizumab BEV bevacizumab CAM camrelizumab LEN lenvatinib PEMpembrolizumab NIV nivolumab IPI ipilimumabThe clinical benefit of sorafenib however remains modest andthe complex molecular pathogenesis of HCC stimulated theinvestigation of combinations of sorafenib with other moleculartargeting drugs Sorafenib has been combined with antiangiogenic agents MEKERK pathway inhibitors mTOR pathwayinhibitors histone deacetylase inhibitors EGFEGFR pathwayinhibitors and HGFcMet pathway inhibitors31 Other agentssuch as interferon32 selumetinib33 capecitabine34 tegafururacil35 gemcitabine and oxaliplatin GEMOX3637 and gemcitabinealone38 have also been evaluated but to date no treatmentsinvolving combinations containing sorafenib have succeeded inphase III trialsSince sorafenib and TACE are both recommended therapies foradvanced HCC it is reasonable to expect that their combined usewould confer benefits when compared with monotherapy Resultshowever highlighted regional differences and the heterogeneityof the trial protocolsIn TACE the multicenter randomizedplacebocontrolled phase European trial when compared withTACE alone the addition of concurrent sorafenib unlike the SPACEtrial39 did not improve progression free survival PFS40 It was alsoshown that the addition of sorafenib did not confer any survivalbenefit in patients with unresectable HCCs who had alreadyresponded to TACE41 Contrasting with these findings retrospective studies from China have shown that combination therapywith sorafenib and TACE increased OS by more than compared with TACE alone42“ which was supported by thefindings from a number of other groups5455 RecentlytheTACTICS trial a randomized multicenter prospective trial fromJapan reported an improved PFS for TACE plus sorafenibcompared with TACE alone vs months p although this trial used a redefined PFS not conventional PFS buttime until œunTACEable progression The TACTICS trial also usedtime to any cause of death plus OS as primary endpoints resultsnot reported and compared with sorafenib monotherapy TACEplus sorafenib was only superior in controlling tumor progressionand did not prolong OS5758The acceptance of sorafenib as the standard to which othernewer agents and nonsurgical interventions are compared hasresulted in studies comparing its use as monotherapy with TACEplus external beam radiotherapy59 and TACE plus intensitymodulated radiotherapy combined with sorafenib60 In the SARAHstudy selective internal radiotherapy with yttrium90 resin microspheres did not produce any survival benefit compared withsorafenib in locally advanced and inoperable HCC median OS vs p and did not meet the primary endpoint of OS61Similarly the addition of selective internal radiation therapy tosorafenib did not result in a significant improvement in OScompared with sorafenib alone62 Bettinger et al63 however diddemonstrate that stereotactic body external beam radiotherapyemployed as monotherapy SBRT was able to improve OScompared with sorafenib and SBRT with TACE also providedimproved OS and PFS when compared with sorafenib and TACE incombination64 In a recentinfusionchemotherapy HAIC NCT02774187 He et al65 reported thatsorafenib plus HAIC with FOLFOX improved OS compared withsorafenib alone in advanced HCC when portal vein invasion waspresent which was supported by other studies6667 Although theSCOOP2 trial found sequential HAIC with cisplatin and sorafenibdid not improve the survival benefit compared with sorafenibalone this is likely to have resulted from the study beingunderpowered for the primary and secondary endpoints68trial of hepatic arterialDue to the high recurrence rates following hepatectomy fortherapy has been extensivelyHCC approaches to adjuvantinvestigated although previous attemptsincluding the use ofantiviral agents have been largely unsuccessful Based on thepalliative use and success of sorafenib its potential in the adjuvantsetting was investigated and improved survivals following surgeryanticipated Unfortunately this has not been demonstrated and itfailed to reduce postoperative tumor recurrence in the STORMtrial69 and other western studies70 Explanations for the negativeoutcome in the STORM trial include highdose modification ratesshort treatment durations and the enrollment of patients whowere not at high risk of tumor recurrence with no evidenceof tumor satellites with one lesion and with nomicroscopic vascular invasion71 Consistent with this viewpointWang et al72 reported no case of recurrence during the sorafenibdosing period whereas patients suffered recurrence of theirtumor within months of discontinuation of sorafenib72 andpersistent sorafenib intake following postoperative recurrenceimproved OS73 Considering that patients who respond tosorafenib may belong to limited clinical or biological subsetsthe effectiveness of sorafenib in an unselected population cohortsupports its use in the adjuvant setting A number of studies fromthe Far East including China Japan and Korea include patientswith HCCs who are treated with hepatectomy despite their tumorsbeing outside Barcelona Clinic Liver Cancer Classification BCLCguidelines and although the results are difficult to compare dueto heterogeneity ofthe protocols the results are positiveSorafenib significantly reduces tumor recurrence in BCLC stage Cpatients7475 and increases diseasefree survivalDFS76 andSignal Transduction and Targeted Therapy 0cZhuang et al77 demonstrated that adjuvant therapy increaseddisease free survival DFS and OS Sorafenib treatment followinghepatectomy significantly prolonged the OS of advanced HCCthan intermediate HCC78 In addition to BCLC stageratherpatients who underwent hepatectomy and were pathologicallydiagnosed with microvascular invasion MVI also benefited fromadjuvant sorafenib treatment79 In line with these results a largerecent study with propensity score matching analysis alsodemonstrated that sorafenib significantly improved overall andrecurrencefree survival following resection80 The results fromthese studies which include all eligible patients suggest that moreprecise stratification would enable the identification of thosepatients who will benefit most from the use of adjuvant sorafeniband those in where additional treatment is not appropriateOngoing trials are attempting to evaluate the role of sorafenib inpatients with MVI following radical resection NCT02867280 andNCT02537158LenvatinibFollowing the approval of sorafenib for use in the treatment ofHCC it takes more than a decade before the second firstlinetargeted agent for HCC emerged Lenvatinib was approved for thefirstline therapy in advanced HCC following the results of theREFLECT trial a randomized phase III noninferiority trial publishedby Kudo et al81 Although not approved for long further multicenter data from œrealworld conditions confirmed the efficacy oflenvatinib regardless of previous tyrosine kinase inhibitor TKItherapies8283 and lenvatinib monotherapy demonstrated antitumor activity for more than years in unresectable HCC whenportal vein invasion was present84 In intermediatestage HCCpatients with tumors exceeding the uptoseven criteria for whomTACE is not helpful lenvatinib could provide significant longer OS vs months and PFS vs months85 Lenvatinibpharmacokinetics in HCC is affected by body weight8687 and asufficient dose relative dose intensity RDI is required to achieve agood therapeutic effect and consequently improved outcomesand prognosis are associated with the preservation ofliverfunction which reduces the number of patients who need todiscontinue their treatment88“ With lenvatinib unlike other TKIsthere are issues with thyroid toxicity and surveillance for thyroidabnormalities during treatment is important92 Hypothyroidism isnot unusual and there are also fewer common reports ofthyrotoxicosis and destructive thyroiditis93 From a healtheconomics standpoint however lenvatinib is more cost effectivethan sorafenib9495Secondline targeted agents for HCCStill sorafenib displays limited antitumor activity and someinitially sorafenibsensitive would eventually succumb to thedisease indicating the acquired resistance to sorafenib reducesits beneficial effects and an urgent need for secondline therapyRegorafenibInitial attempts to discover effective secondline agents wereunsuccessful and mirrored attempts to develop firstline agentswhich were superior to sorafenib96 The RESORCE trial was arandomized double blind placebocontrolled and phase III trialdemonstrating the effectiveness of regorafenib in patients whohad progressed on sorafenib treatment Thisstudy finallyconfirmed the potential of secondline agents and ushered inthe era of secondline and sequential therapy97 Regorafenibprovided survival benefitthe rate of diseaseprogression during prior sorafenib treatment or since the lastsorafenib dose98 This was confirmed by Yoo et al99 in aretrospective study of safety and efficacy in Korean patientswhere data were consistent with those from the RESORCE trialRegorafenib was even shown to be effective in patients with HCCrecurrence following liver transplantation with a median OS ofregardless ofSignal Transduction and Targeted Therapy Targeted therapy for hepatocellular carcinomaHuang et al months following regorafenib initiation and monthsfollowing sorafenib initiation CI “ for the sorafenibfollowed by regorafenib sequential therapy100However not all patients who progress on sorafenib arecandidates for secondline therapy101 In clinical practice only of patients are eligible forsecondline regorafenibtreatment102 Good liver functional reserve and ECOG performance status during sorafenib treatment contributed to theefficacy and better outcomes of subsequent treatment103104including lenvatinib105 This may in part be due to the RDIrequired to achieve a clinically significantinprognosis106 This is supported by the demonstration that thenew liver reserve function biomarker albuminbilirubin gradeALBI107 successfully identified regorafenib candidates and thatin the selected cohort a median OS of months was achievedcompared with months for noncandidates108 Even in patientsnot eligible for regorafenib the ones with an ECOGPS score of the absence of MVI and TTP time to progression ‰¥ monthscould still have acceptable postprogression survival109 Longtermtreatment with regorafenib has also been shown to reduceangiogenesis and improve portal hypertension PHT and acuteadministration ameliorates portal haemodynamics suggestingthat it may be especially suitable for patients with PHT andpreserved liver function110improvementCabozantinibCabozantinib is another small molecule inhibitor of the tyrosinekinases which are implicated in the progression of HCC and theacquired resistance to sorafenib Cabozantinib blocks the receptors involved in oncogenesis and angiogenesis including VEGFR hepatocyte growth factor receptor MET AXL and theangiopoietin receptors TIE2 RET cKit and FLT3 in vitro andin vivoIn CELESTIAL trial cabozantinib achieved the primaryendpoint with median OS of months compared with months for the placebo group111 and was consequentlyapproved in the EU and USA There remains a paucity of datahowever from realworld clinical practice examining the sequentialtreatment utilizing cabozantinib as the secondline agent it is acostly option associated with frequent highgrade adverse eventsConsequently several studies have addressed the costeffectivenessof cabozantinib using the cost and utility data extracted from theCELESTIAL trial The conclusion from these studies is consistent andconfirms that at its current cost point the gain of qualityadjustedlifeyears for cabozantinib QALYs “ and the incrementalcosteffectiveness ratio ICER “ mean that it isnot a costeffective treatment option for patients with sorafenibrefractory HCC112“ compared with regorafenib QALY “and ICER “RamucirumabRamucirumab is a fully human recombinant IgG1 monoclonalantibody targeting the VEGF2 receptor Although ramucirumabfailed to meet its primary endpoint as secondline treatment in theREACH trial117 subgroup analysis found survival benefit in patientswith AFP of ngml or higher118“ This was later confirmed inthe REACH2 trial122 which led to the approval of ramucirumab assecondline treatment for advanced HCC REACH2 is the firstpositive phase trialin patients with HCC performed in abiomarkerselected patient cohort and more recent findingsdemonstrated that AFPenriched HCCs displayed significantactivation of VEGF which suggests the underlying mechanism ofaction and confirms the potential value of biomarkerdrivenclinical trials123Immune checkpoint therapy and TKI inhibitorsICIs stand as the mainstream of immunotherapy The CheckMate and KEYNOTE224125 studies evaluated the safety andefficacy of nivolumab and pembrolizumab in patients with 0cTargeted therapy for hepatocellular carcinomaHuang et alphaseIIrandomizedparallelgrouptislelizumab sintilimabrealworld cohort studyadvanced HCC refractory to previous sorafenib treatment whichestablished the basis for accelerated approval by the FDA assecondline treatment Subanalysis of CheckMate040 data validated the safety and efficacy of nivolumab in Asian cohort126 Inan internationalICIs have showedpromising efficacy and safety in advanced HCCs as systemicfirstsecondthirdfourthline treatment with median OS andPFS of and months respectively127 and an excellentresponse to antiPD1 therapy has also been described in casereport128 Although the subsequent phase III KEYNOTE240 trialdid not meet its prespecified statistical significance in respect ofimproved PFS and OS the results were consistent with previousKEYNOTE224129 The KEYNOTE394 presently underway in Asianpatients may clarify the role of pembrolizumab in cases ofadvanced HCC with a viral background NCT03062358 RecentlyCheckMate459 the multicenter phase III randomized sorafenibcontrolled trial evaluating nivolumab as firstline treatment foradvanced HCC failed to achieve its endpoints ESMO butnivolumab did prolong OS vs months and achievelongtime disease control less adverse events AEs and survivalbenefit regardless of the level of PDL1 expression Furthermorenivolumab improved the survival of HCC patients whose etiologywas HBVHCV and did not reactivate hepatitis CamrelizumabSHR1210 Hengrui Pharmaceutical is an antiPD1 inhibitor fromChina investigated for the treatment of Hodgkin lymphoma andHCC It has been shown to have antitumor activity in previouslytreated Chinese patients with advanced HCC in a multicenteropenlabeltrialNCT02989922130 providing evidence for the effectiveness ofPD1 therapy for HBV related HCC in Chinese patients The resultsICIs including durvalumabfrom other trials investigating novelavelumabtremelimumabipilimumabspartalizumab and toripalimab will hopefully yield positive resultsand provide further options for the treatment of patients withHCC particularly those who have relapsed on firstline treatmentsFurther efforts to enhance the treatment effect of ICIs includedual ICIs treatment and combination therapy of ICIs with otherkinds of targeted agents For dual ICIs treatment the initial resultsfrom CheckMate 9DW were astonishing the objective responserate was higher than monotherapy of any ICIs alone FDA hasapproved nivolumab in combination with ipilimumab for patientswith HCC previously treated with sorafenib Treatment modalitiessuch as radiotherapy and antiangiogenesis agents which affectantigen release or modulate the tumor microenvironments havethe potential to increase the efficacy of immunotherapy and thecombination oftargeted agents with ICIs are attracting theattention of a number of research groups and in vitro studies andearlyphase clinical trials assessing combination treatments haveshown promising antitumor effects in patients with advancedIn vitro evidence by Qui et al131 demonstrated thatHCClenvatinib and regorafenib could affect the expression of PDL1and realtime PCR results suggested that the mRNA expression ofPDL1 in the lenvatinib group was significantly higher than that inthe control group while its expression in the regorafenib groupwas significantly lower When combined with antiPD1 lenvatinibcan modulate cancer immunity in the tumor microenvironmentand enhance antitumor activity132133 In July the FDAannounced its approval of the first combination therapy employing the TKI lenvatinib with the ICI pembrolizumab based on theresults from the KEYNOTE524Study NCT03006926 for thetreatment of HCC Recently results from Study Phase IbNCT03418922 showed marginally better results for lenvatinibwith nivolumab than lenvatinib with pembrolizumab METmediated phosphorylation leads to a decreased expression ofPDL1 using the combination of MET inhibitors tivantinib andcapmatinib antiPD1 and antiPDL1 produced an additive effectwhich slows the growth of HCCs in mice134 Clinically based onthe results from the experimental arm A of the GO studyNCT02715531 the FDA approved atezolizumab plus bevacizumab as breakthrough therapy for untreated advanced ormetastatic HCC135 Individual case studies also reported promisingresults for the use of combined TKI and antiPD1PDL1 agents foradvanced HCC136“ Such results were confirmed in the phase IIItrialIMbrave study NCT03434379 which reported thatatezolizumab combined with bevacizumab resulted in better OSand PFS than sorafenib in patients with unresectable HCC139Other combination therapies include Galunisertib with nivolumabNCT02423343 spartalizumab with and without capmatinibNCT02795429 FGF401 with spartalizumab NCT02325739regorafenib with pembrolizumab NCT03347292 cabozantinibwithaxitinibNCT03289533 ramucirumab with durvalumab NCT02572687and XL888 with pembrolizumab NCT03095781 Table avelumab withNCT03299946nivolumabImmunerelated adverse events IRAEs occur frequently duringtreatment with ICIs and the clinical consequences can besignificant140 Activation of the immune system leads to damageof normal healthy tissues and IRAEs can have myriad effects andinvolve a number of different ans and have been reported toproduce colitis hepatitis pneumonitis dermatitis myocarditisendocrine glands ‚ammation and rheumatic and musculoskeletal phenotypesincluding ‚ammatory arthritis arthralgiamyositis and sicca syndrome141 Although the precise pathophysiology underlying the IRAEs side effects during treatment withICIs remains unknown discontinuing administration and the useof steroids is generally effectiveIn severe cases howeveradditional immunosuppressants may be required but based oncurrent available evidence immunosuppression for IRAEs does notappearresponse to the ICItreatment142143to compromise the antitumorPromising agents and treatment regimensDespite abovementioned targeted drugs novel agents have beencontinuously under development Table Of note apatinib anovel inhibitor of VEGFR2 tyrosine kinase has attracted considerable attention and there is now a significant body of workdescribing clinical experience with its use Although less effectivethan sorafenib as a firstline treatment in a retrospective study144apatinib still displayed promising antitumor effects in sorafenibresistant HCC145“ where portal vein invasion was present148when metastases have occured149150 and for unresectable andrelapsed HCCs151152 Combination therapy in studies utilisingapatinib with TACE have achieved better clinical effectivenessthan TACE alone with tolerable AEs153“ Recentlythecombination of apatinib with the antiPD1 monoclonal antibodycamrelizumab achieved partial response rates of Theresults of other ongoing trials including the phase IIItrialcomparing TACE and apatinib with sorafenib as firstline treatmentfor locally advanced or metastatic and unresectable HCC NCT and the adjuvant apatinib after hepatectomy for theprevention of tumor recurrence NCT03722875 and NCT03261791will hopefully prove effective and add to the presently availabletherapeutic optionsThese promising results have stimulated the investigation ofother new agents the combinations of agents and regimenswhich have been thoroughly discussed in a recent review fromZhu and Sun154 The combination of bevacizumab and erlotinibhas been extensively evaluated as first155 or secondline inadvanced HCCs156“ but unfortunately the heterogeneousnature of the results precludes firm conclusions and recommendations Recently a singlearm metaanalysis of prospectivestudies found that combination therapy with bevacizumab anderlotinib used as secondline treatment was associated with afavorable PFS weeks P and OS months P suggesting that future welldesigned and sufficiently poweredlargescale RCTsshould be able to identify the potentialcontribution of these agents163Signal Transduction and Targeted Therapy 0cTable Trials investigating the combination therapy of ICIs and TKIs in HCCTrial nameidentifierPatient No Study type LineInterventionsPrimaryendpointStudy statusTargeted therapy for hepatocellular carcinomaHuang et alLEAP002NCT03713593Phase IIIFirstLenvatinib pembrolizumab vs lenvatinib PFSOSPhase IIIFirstPhase IIIPhase IIIPhase IIIFirstFirstFirstPhase IIIFirstFirstPhase IIFirstPhase IIPhase Ib II FirstFirstPhase IIPhase IbFirstNivolumab ipilimumab vs lenvatinib orsorafenibCabozantinib atezolizumab vs sorafenib PFSOSSintilimab IBI305 vs sorafenibOS ORRCamrelizumab apatinib vs sorafenibOSPFSOSCamrelizumab apatinib vs FOLFOX orsorafenibNivolumab lenvatinibNivolumab sorafenibSorafenib pembrolizumabAnlotinib sintilimabAvelumab axitinibOSORR AEMTD ORRORRORR AEAEActive notrecruitingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingCompletedCheckMate 9DWNCT04039607COSMIC312NCT03755791ORIENT32NCT03794440SHR1210III310NCT03764293SHR1210III305NCT03605706IMMUNIBNCT03841201NCT03439891NCT03211416KEEPG 04NCT04052152VEGF Liver NCT03289533KN743NCT03347292GOINGNCT04170556REGOMUNENCT03475953NCT02423343aaRegorafenib pembrolizumabFirstPhase ISecond Regorafenib nivolumabPhase IISecond Regorafenib avelumabPhase IIIPhase Ib II Second Galunisertib nivolumabAEAECR PRPhase Ib MTDOngoingOngoingOngoingOngoingPFS progressionfree survival OS overall survival ORR objective response rate AE adverse events MTD maximum tolerated dose CR complete response PRpartial responseaTrials enroll not only HCC patientsTable Trials investigating targeted therapy in advanced HCCTrial nameidentifierPatient noStudy typeLineInterventionsPrimary endpointStudy statusIMbrave150 NCT03434379ZGDH3NCT02645981HIMALYYA NCT03298451RATIONALE301 NCT03412773PHOCUSNCT04344158ALTER0802 NCT02809534AHELPNCT02329860KEYNOTE394 NCT03062358NCT04080154Phase IIIPhase IIIIIPhase IIIPhase IIIPhase IIIPhase IIIPhase IIPhase IIIPhase IIIPhase IIFirstFirstFirstFirstFirstFirstFirstSecond Apatinib vs placeboSecond Pembrolizumab BSC vs placebo BSCSecond AnlotinibAtezolizumab bevacizumab vs Sorafenib OS PFSDonafenib vs sorafenibOSDurvalumab tremelimumab vs sorafenib OSOSTislelizumab vs sorafenibPexaVec sorafenib vs sorafenibOSAK105 anlotinib vs sorafenibOSPFS 12WAnlotinibOSOSPFSOS overall survival PFS progressionfree survival BSC best supportive careCompletedCompletedOngoingOngoingOngoingOngoingOngoingCompletedOngoingOngoingundergoingevaluationandPreclinical evidence for cyclindependent kinase CDK targetingtherapies in HCC has showed promise and supports theirinvestigation164“ especially with the potential ability toabrogate the emergence of sorafenib resistance167 and sensitizeHCC to regorafenib treatment168 A number of CKD inhibitors arepalbociclibpresentlyNCT01356628 milciclibribociclibNCT02524119 The antiMET monoclonal antibody emibetuzumab exhibited the greatest antitumor activity in HCC whencombined with ramucirumab and had an excellentsafetyprofile169 and for HCC with high MET expression there was analmost 3fold increase in PFS vs months relative to thosewith low MET expression suggesting the potential for furtherbiomarkerdriven clinical trials Rigosertib is a synthetic benzylstyryl sulfone small molecule inhibitor which has been used in theNCT03109886includingtreatment of monomyelocytic leukemia and due to its activity as aRAS and PLK1signaling inhibitorit was investigated in HCCpatients who demonstrate upregulation of PLK1 during tumordevelopment and HRAS expression in advanced HCC Highexpression levels of PLK1 are also significantly correlated withpoor patient survival and the multiple effects of rigosertib couldbe beneficially employed to produce a therapeutic œdualhitapproach in selected patients170 Donafenib is a novel multikinaseinhibitor which is similar to sorafenib displaying comparable orbetter safety and efficacy when treating advanced HCC in phase1b trial and phase studies using sorafenib as the controlNCT02645981171 There are ongoing trials evaluating novelagents such as anlotinib another multikinase inhibitor which isorally administered and targets VEGFR fibroblast growth factorreceptor FGFR plateletderived growth factor receptors PDGFRSignal Transduction and Targeted Therapy 0cTargeted therapy for hepatocellular carcinomaHuang et alTable Molecular classification of HCCResearcherBoyault et alHoshida et alSchulze et alSia et alKurebayashi et alShinata et alJiang et alYearClassificationG1“G6S1“S3Msig “iC1“iC3 HCCs with adaptive or exhausted immune responsesImmunehigh mid and “lowMS1 ˆ’ ˆ’SI SII and SIIITypeTranscriptomeTranscriptomeExome sequencingMultiomocisImmune cell profilingImmunomicroenvironmentTranscriptome and gonomeProteomicsCase no and ckit NCT02809534 Tivozanib is another oralinhibitor ofVEGFR123 with promising activity against HCC in vivoNCT01835223 and TRC105 which despite demonstrating clinicaltolerated in HCC patients followingactivity and being wellsorafenib has notto date met prespecified criteria and itsdevelopment in HCC continues as combination therapy withsorafenib NCT02560779Biomarkerdriven targeted therapyDespite extensive research investigating potential biomarkers to aidthe development of protocols for the treatment of HCC none haveso far been identified to be able to predict the effect of or responseto treatment with sorafenib172“ Although the molecularclassification of HCC has been widely reported Table to date itremains unclear whether this basic genomic and proteomic datawill prove valuable in guiding targeted therapies183“The continued belief that the future lies with personalizedtreatment which will be made possible through the rapiddevelopments in next generation sequencing and the precisionmedicine that it underpins have encouraged the development ofnovel trial designs191 These novel trials designs offer new hopethat biomarkerdriven targeted therapies can be modul

Thyroid_Cancer

Neurologic Manifestations of Systemic Disease D Lapides Section EditorNeurologic Manifestationsof Systemic Disease SleepDisordersEric M Davis MD1Chintan Ramani MBBS1Mark Quigg MD MSc2Address1Division of Pulmonary and Critical Care Department of Medicine University ofVirginia Charlottesville VA USAEmail emd9bvirginiaedu2Department of Neurology University of Virginia Charlottesville VA USA Springer ScienceBusiness Media LLC part of Springer Nature This is part of the Topical Collection on Neurologic Manifestations of Systemic DiseaseKeywords Sleep disorders I Sleep manifestations of systemic diseases I Sleep impacts on health I Sleep apnea IInsomniaAbstractPurpose of review Sleep is intimately involved in overall health and wellbeing We provide acomprehensive report on the interplay between systemic diseases and sleep to optimizethe outcomes of systemic disordersRecent findings Spanning the categories of endocrinologic disorders metabolictoxicdisturbances renal cardiovascular pulmonary gastrointestinal infectious diseases autoimmune disorders malignancy and critical illness the review highlights the prevalentcoexisting pathology of sleep across the spectrum of systemic disorders Although it is rarethat treating a sleep symptom can cure disease attention to sleep may improve quality oflife and may mitigate or improve the underlying disorder Recent controversies inassessing the cardiovascular relationship with sleep have called into question some ofthe benefits of treating comorbid sleep disorders thereby highlighting the need for anongoing rigorous investigation into how sleep interplays with systemic diseasesSummary Systemic diseases often have sleep manifestations and this report will help theclinician identify key risk factors linking sleep disorders to systemic diseases so as tooptimize the overall care of the patient 0c Page of IntroductionCurr Treat Options Neurol All Earth™s species maintain a solar 24h cycle of rest andactivity and disrupting the cycle affects adaptation andhomeostasis Sleep™s quotidian œnormalness meansthat analogous to fish not knowing about water until itis dry sleep is not commonly thought about until it isdisruptedFor example about of the adult populationcomplain of transient insomnia and about experience chronic insomnia that disrupts daytime function[] Patients with chronic insomnia experience less workproductivity more absenteeism more accidents andmore hospitalizations leading to direct treatment costsof approximately 60B annually [] Considering thepotential widespread reach of comorbid sleep disordersevaluating sleep in the neurological patient is importantThis review will introduce the accepted anizationof sleep disorders review important features in historytaking and evaluation and survey the systemic diseasesthat have important comorbidities with particular sleepdisordersGeneral considerationsClassification of sleep disordersAn abridged listing of sleep disorders from the American Academy of SleepMedicine Table provides an overview of the current classification []Insomnia is a chronic dissatisfaction with sleep duration and quality that isassociated with daytime dysfunction Although pharmacologic treatment isoften pursued for chronic insomnia management outcomes are often betteraddressing underlying factors with the early use of cognitivebehavioral therapyfor insomnia CBTi []Sleeprelated breathing disorders involve dysfunction of the respiratory systemduring sleep usually resulting in daytime hypersomnia Obstructive sleepapnea OSA central sleep apnea CSA and respiratory effort related arousalsare classified under this category Treatment options including continuouspositive airway pressure CPAP positional therapy mandibular advancementdevices healthy weight loss and even a novel cranial nerve stimulator whichprotrudes the tongue forward during sleep [4cid129cid129]Central hypersomnias are defined as a primary dysregulation of sleep resultingfrom dysfunction of the central nervous system that causes daytimehypersomnia Often treatment addresses the underlying cause and may includeuse of strategic napping and wakepromoting medicationsCircadian disorders consist of various lesions or external disruptions of thecircadian timing system that desynchronize the brain™s clock from the externalsolar lightdark cycle resulting in hypersomnia or insomnia in a clockdependent fashion Treatment of circadian rhythm disorders involves adjustinglife around the patient™s desired sleep time or augmenting factors that entrainthe body™s clockParasomnias represent disorders of faulty inhibition of waking behaviors thatarise inappropriately during sleep and are divided into those that occur duringnonREM sleep REM sleep or state transitions REM sleep behavior disorder is aparasomnia characterized by loss of muscle atonia during REM sleep thatusually occurs in patients with neurodegenerative disorders It is often treatedeffectively addressing other sleep disturbances and treating with clonazepam ormelatonin [] 0cCurr Treat Options Neurol Page of Table Abridged classification of the AASM sleep disordersInsomniaChronic insomnia disorderShortterm insomnia disorderExcessive time in bedShort sleeperSleeprelated breathing disordersObstructive sleep apneaCentral sleep apneaSleeprelated hypoventilation disordersSleeprelated hypoxemia disordersCentral disorders of hypersomnolenceNarcolepsy types and Idiopathic hypersomniaKleineLevin syndromeHypersomnia due to medical disorder medication substance psychiatric disorderInsufficient sleep syndromeCircadian rhythm sleepwake disordersDelayedAdvancedIrregularNon hShift workJet lagParasomniasNREM relatedArousal disordersConfusional arousalsSleepwalkingSleep terrorsSleeprelated eating disorderREM relatedREM sleep behavior disorderRecurrent isolated sleep paralysisNightmare disorderOtherExploding head syndromeSleeprelated hallucinationsEnuresisSleep talkingSleeprelated movement disorders 0cCurr Treat Options Neurol Page of Table ContinuedRestless legs syndromePeriodic limb movement disorderLeg crampsBruxismRhythmic movement disorderBenign sleep myoclonus of infancyPropriospinal myoclonus at sleep onsetNormal variantsSleep historySleeprelated movement disorders consist of fragmentary often repetitive bodymovements that can disrupt sleep or sometimes worse disturb the sleep of bedpartners Periodic limb movement disorder PLMD and restless legs syndromeRLS both fall under this category and are treated with repletion of iron storesand consideration of dopaminergic agonists []A sleep history helps a patient disclose sleep findings and helps the physiciananize it into categories of hypersomnia sleep habits and scheduling sleepcharacteristics environmental issues and sleep interrupters Table The Epworth Sleepiness Scale quantifies the degree of hypersomnia []Most adults require “ h of daily sleep [] and prefer it anized into eithera monophasic nocturnal schedule or in a biphasic pattern augmented with anafternoon œsiesta The sleep pattern characterizes the presence and severity ofsleeponset insomnia sleep maintenance insomnia or terminal insomnia insomnia distributed within the last half of the sleep period œCatchup sleep aphenomenon of prolonged sleep on a free day is a classic sign of sleepdeprivation Habitual earlyphase advances œmorning larks latephase delays œnight owls or a chaotic irregular schedule can be a sign of circadiandisorders One also must inquire about common sleep disruptors including legmovements snoring witnessed apneas and environmental factorsDiagnostic testing modalitiesSleep diaryPolysomnographyThe sleep diary often available through standardized forms or evenwebsites or smartphone apps consists of “ weeks of selfreported sleeptimesThe overnight polysomnography PSG is the goldstandard measurementof sleep architecture respiratory disorders such as OSA and parasomniasIn the case of OSA the unattended home sleep study has had an 0cCurr Treat Options Neurol Page of Table A categorical sleep historyHypersomniaEpworth Sleepiness Scale Considering the last weeks how likely would you fall asleep while doing each task not at all points slight moderate severe Normal ‰¤ pointsSitting and readingWatching TVSitting inactive in public lecture church ¦Car passenger for an hourLying down to rest in the afternoonSitting conversationSitting quietly alone after lunchDriving stopped in trafficSchedulesleep timeWorkday bedtime and out of bedtimeWeekday bedtime and out of bedtimeWhat is your estimated sleep latency If min what are you doing in bed before you fall asleepHow often do you awaken at night and whyDo you need an alarm clock to awaken in the morningHow many days of the week do you nap and for how longEnvironmentDo you have a bedroomDo you have a bedpartner TV Mobile phone or other electronicsWhat are you doing right before bedtimeHow much caffeine coffeeteasoda popenergy drinks and alcohol do you consume and when is the latest intakeInterruptersDo you have leg pain or restlessnessDo you have chronic pain that prevents or interrupts sleepDo you have daytime hallucinations or dreams severe or lucid nightmares sleep paralysis or cataplexyDo you snore or have witnessed apneasMultiple sleep latency testincreasing role as a diagnostic testing alternative to the traditional inlabPSG Concerns of other sleep disorders or those that may be presentcomorbidly with probable OSA require inlab PSG that can measure sleeparchitecture and sleepassociated movementsThe multiple sleep latency test MSLT consists of a series of daytime napsfrom which sleep onset is calculated The test in combination with PSGperformed the night before is the gold standard in measuringhypersomnia especially in the evaluation of narcolepsy 0c Page of ActigraphyPersonal devicesCurr Treat Options Neurol Wrist actigraphy provides measurements of longterm patterns of rest andactivity as proxies for sleep and wakefulness Such patterns can help tocorroborate histories of sleep duration and timingPopular smartphones and other ambulatory devices with physiologicalmonitoring capabilities may transform the evaluation of sleep However arecent comparison of different brands of activity trackers found that sleepwake measurements varied widely in comparison with sleep diaries orstandard PSG [] The overall conclusion is that at the beginning of wearable devices are not ready for reliable quantification of sleep acrossindividuals Although serial recordings confined to a single individual mayhold some value these measurements have yet to be validatedSleep comorbidities with systemic diseasesEndocrine disordersThyroid diseaseConsidering the various sleep disorders and diagnostic tools afforded by a goodsleep history and sleep testing understanding the relationship between sleepdisorders and systemic diseases has farreaching implications in optimizing thecare of the patient The following sections will address sleep manifestations ofvarious neurological disorders arising from systemic disease based on an systemAlmost half of the patients with hypothyroidism report at least one sleep complaint such as restless sleep choking hypersomnia or fatigue [] OSA is presentin approximately [] A unique mechanism of airway restriction in hypothyroidism is myxedematous mucoprotein deposition in the airway™s soft tissuesand dilator muscles even though myxedema can be absent [] Larger goiters canalso cause OSA by external compression of the airway []On the other side of the thyroid spectrum hyperthyroidism is most closelyassociated with insomnia occurring in of patients [] Arousaldisorders”specifically sleep walking”also occur especially in the setting ofthyrotoxicosis [] proposed to arise from frequent arousals and impairmentof attaining slowwave sleep as the direct result of thyroid hormoneBeyond the treatment of the specific sleep disorder sleep problems usuallyremit following appropriate treatment of the underlying thyroid disorder []Type diabetes mellitusSleep disorders affect high proportions of those with type diabetes mellitusDM surveys of patients with DM compared with those of controls show a 0cCurr Treat Options Neurol Page of nearly 2fold propensity for insomnia fourfold higher use of sedativehypnoticsand a 10fold higher rate of hypersomnolence [] OSA is highly prevalent inDM and many are undiagnosed [] Contributors to a multifactorial series ofsleep disruptors include periodic limb movements and restless legs syndromeRLS diabetic neuropathy and fluctuations in blood glucose []DM presents an excellent model by which to demonstrate the reciprocaleffects of sleep disruption on the primary disease First sleep disturbances affectthe regulation of the neuroendocrine control of appetite Sleep deprivationpromotes overeating through hyperactivity of orexin system [] and activatesthe hypothalamicpituitaryadrenal system to increase cortisol secretionresulting in impaired glucose tolerance [ ] These multiple mechanismssupport clinical observations that untreated OSA may be reason for the ineffective treatment of DM and that accordingly treatment with CPAP leads toimprovements in glycemic control in some patients []Sex hormones and gender affect the distribution and susceptibility to a varietyof sleep disorders Men on the basis of relative airway collapsibility haveapproximately a twofold increased risk of OSA compared with women “ in males and “ in females [] A potential side effect in thetreatment of hypoandrogenism is the facilitation of OSA given the impacttestosterone has on upper airway collapsibility []Testosterone levels may affect the propensity for chronic insomnia Menwith hypoandrogenism demonstrate reduced sleep efficiency increased nighttime awakenings and reduced deep sleep compared with the normaltestosteronelevel controls although it is not clear whether these features improve with testosterone therapy [] Women experience higher rates of chronicinsomnia risk ratio of for women versus men which becomes even morepronounced in the elderly [] Despite sleeping longer overall sleep quality isoften lower in women than men []The distribution of sleep disorders in women varies with reproductivelifespan Younger women are more susceptible to restless legs syndromeRLS mainly on the basis of mensesassociated irondeficiency During pregnancy women are at significantly increased risk for the development of RLSwith an overall prevalence exceeding of all pregnant patients [] Treatment of RLS in pregnancy involves iron supplementation with a goal ferritinlevel mcgl Often oral iron repletion is adequate although there arereports of intravenous iron therapy in severe cases of pregnancyrelated RLSand irondeficiency [] Pregnancy is also associated with an increased prevalence of OSA up to of pregnant patients during the third trimester whichis associated with increased risks of complications including gestational hypertension gestational DM and preeclampsia []Although not a particular systemic neurological disease pharmacological effectson sleep form an important aspect of neurological sleep medicine since manymedications that are used by neurologists may affect sleep Table showscommon medications that provoke insomnia hypersomnolence respiratorysuppression parasomnias and RLSperiodic limb movement disorderSex hormonesMedications 0c Page of Curr Treat Options Neurol Table Medication classes and specific examples that can cause sleep disturbancesInsomniaCentral nervous system stimulants methylphenidate amphetamines modafinilCaffeineAntidepressantsSelective serotonergic reuptake inhibitors fluoxetine sertralineSelective norepinephrine reuptake inhibitors venlafaxine duloxetineSecondary tricyclic antidepressants desipramine nortriptylineCardiovascularBeta2 agonists albuterolVasopressors epinephrine dopamineCorticosteroidsSympathetic amines phentermineHypersomniaBenzodiazepines alprazolam diazepamNonbenzodiazepine receptor agonists zolpidem eszopicloneOpioidsH1 antihistamines diphenhydramineAntiepileptic agents phenytoin levetiracetamAntidepressantsSelective serotonergic reuptake inhibitors paroxetine sertralineTertiary tricyclic antidepressants amitriptylineTypical and atypical antipsychotics haloperidol olanzapineDopaminergic agonists ropinirole carbidopalevodopaAnticholinergic medicationsCentrally acting α agonists clonidine dexmedetomidineRespiratory suppressionOpioids oxycodone morphineBenzodiazepines diazepam clonazepamAlcoholPhenobarbitalParasomniasAntidepressants clomipramine fluoxetine citalopramNonbenzodiazepine receptor agonists zolpidemCaffeineAlcohol withdrawalRestless legs syndrome and periodic limb movementsSelective serotonergic reuptake inhibitors fluoxetine mirtazapineAntipsychotics haloperidol risperidoneTricyclic antidepressants amitriptyline clomipramine 0cCurr Treat Options Neurol Page of Renal diseaseInfectious diseasesSleep disturbances are highly prevalent in patients with chronic kidney diseaseCKD spanning the broad spectrum of sleep disorders including hypersomniainsomnia sleeprelated breathing and RLSThe prevalence of OSA in CKD ranges from to rates that are notexplained solely by overlapping comorbidities common to both OSA and CKD[] The cooccurrence of both CKD and OSA is associated with increasedcardiovascular events and allcause mortality [“] Usually OSA develops inpatients with CKD independent of underlying renal dysfunction but someevidence shows that CKD can cause or exacerbate OSA and central sleep apneaProposed mechanisms for this causal relationship include uremic neuropathyaltered chemosensitivity and hypervolemia [] Accordingly renal replacement therapy and fluid removal [] may improve obstructive or central sleepapnea Conversely treatment of sleep apnea with PAP may improve renalfunction in those with borderline renal impairment []RLS is a common and debilitating symptom in patients with CKD occurringin up to of patients on hemodialysis compared with that in approximately of the general population [] Although RLS symptoms generally follow acircadian rhythmicity with increased symptoms occurring at night RLS symptoms can occur during the long periods of daytime inactivity during hemodialysis [] Treatment is primarily focused on ensuring adequate iron stores thenconsidering medical therapy as per routine care of RLSSleep disorders and infectious diseases have few specific associations In general acute infection is associated with mild encephalopathy that masquerades ashypersomnolence and fatigue Proinflammatory cytokines are implicated inthe development of these constitutional symptoms Some infections howeverdirectly affect regulatory centers of the sleepwake systemEncephalitis lethargica is a historical pandemic cause of hypersomnolence ofrenewed interest since this review is being written in the middle of the COVID pandemic Also known as Von Economo™s encephalitis it occurred inassociation with the Spanish flu pandemic of [] An estimated millionwere affected worldwide The most common subtype the somnolentophthalmoplegic form developed after flulike symptoms of fever and malaiseand consisted of subsequent ophthalmoplegia accompanied by long periods ofhypersomnia Despite the appearance of deep sleep patients could be easilyawoken and sometimes maintained memories of activities that had transpiredaround them while œasleep This state of acute akinetic psuedosomnulencecould be followed by the development of chronic postencephaliticparkinsonismThe pandemic associated with the severe acute respiratory syndrome coronavirus SARSCoV2 ie COVID19 occurring during the writing of thisreview features evolving literature The first reports centered on respiratorysymptoms Although the involvement of the nervous system now appearsprevalent [] sleep disorders have yet to be specifically reported Howeverthe psychological responses to social distancing change in schedules and otherfeatures of an active pandemic have caused a wave of anxiety and depressionwhich in turn have been associated with poor sleep quality For example a 0c Page of Curr Treat Options Neurol survey of Chinese health care workers showed prevalences of depressionat anxiety at and insomnia at []Postinfectious or postvaccination narcolepsy is rare but is important in developing overall hypotheses in the etiology of idiopathic narcolepsy In certainvaccinations in Europe for the H1N1 pandemic caused narcolepsy at a risk of in pediatric patients [] Fortunately the risk of postvaccinationnarcolepsy appeared confined to specific vaccine formulations The incidenthowever has led to ongoing research in the immunological etiology ofnarcolepsyAfrican trypanosomiasis or sleeping sickness remains important in the developing world It is a parasitic infection spread by the tsetse fly that is endemic insubSaharan Africa The first symptoms include fever headaches and lymphadenopathy Once the parasite enters the central nervous system disorderedfragmented sleep ensues often with inversion of the circadian sleepwake cycleThe World Health anization outlines treatment with a regimen of antiparasitic medications once symptoms have started []Nonalcoholic fatty liver disease NAFLD consists of idiopathic hepatic steatosiswith a prevalence of to of the general population with increasedfrequency in individuals with obesity or DM [] Given these coassociationsOSA is common Untreated OSA may exacerbate liver injury because of oxidative stress and systemic inflammation [] and is a risk in conversion fromNAFLD to liver fibrosis [] Trials with CPAP have shown inconsistent resultsin markers of liver injury following treatment of OSA []The symptoms of gastroesophageal reflux disease GERD worsen during sleepparticularly if sleep occurs soon after a meal [] The lower esophageal sphincter that normally prevents reflux may be compromised by the increase inthoracic pressure in the setting of the upper airway obstruction [] Patientswith symptoms of GERD should be screened for OSA and conversely interruption of sleep in absence of OSA may improve with treatment with a protonpump inhibitor PPI [] or by simply elevating the head of the bedInflammatory bowel disease IBD has bilateral interactions with sleep []Given the relationship between sleep deprivationfragmentation on cytokineregulation and immune dysfunction it is hypothesized that poor sleep qualityworsens overall symptoms of IBD [ ] Additionally the proinflammatorystate disrupts the circadian rhythm [] Subjective and objective measurementsof sleep quality and timing should be considered in patients with IBD particularly in those who have frequent inflammatory flares despite otherwise adequate management An algorithmic approach to sleep assessment in IBD patients has been proposed by Canakis et al []Systemic lupus erythematosus and rheumatoid arthritis serve as the prototypical diseases of this group of disorders with a prevalence of sleep disturbancesof greater than [] The mechanisms of sleep disturbances as well as thereciprocal relationship in the contribution of poor sleep to worse autoimmunestatus are thought to be similar to those described above with IBD [ ] Thespecific sleep disorders prevalent in this group are OSA and periodic limbGastrointestinal systemAutoimmune disorders 0cCurr Treat Options Neurol Page of Pulmonarymovement disorder PLMD both with greater than prevalence [ ]As seen above hypersomnolence and activitylimiting fatigue arise from specificsleep disorders pain and medication side effects well as the primary effects ofthe primary proinflammatory status [ ] Often treating the underlyingautoimmune disorder improves associated fatigue However if sleepiness persists then evaluating for a comorbid sleep disorder such as obstructive sleepapnea is indicatedOne syndrome with possible autoimmune origins is chronic fatigue syndromeSleep disturbances insomnia and unrefreshing sleep are common symptoms yetpatients rarely report relief despite appropriate identification and treatment ofcomorbid sleep disorders [] Cognitivebehavioral therapy CBT and gradedexercise therapy are commonly pursued treatment approaches []Obstructive lung diseases most commonly asthma chronic obstructive pulmonary disease COPD and less common disorders such as cystic fibrosisCF or bronchiolitis obliterans may affect nocturnal ventilation OSA andCOPD often overlap given shared body habitus and other mutual risk factorsestimates of comorbid OSA and COPD range from to [] Patients withsevere COPD treated with nocturnal noninvasive ventilation NIPPV a moreadvanced form of positive airway pressure experience an absolute risk reduction of of the risk of hospital readmission or death at months compared with those treated with standard care and without NIPPV [64cid129]Insomnia is another common complaint among patients with COPD Circadian bronchial constriction may cause nocturnal wheezing dyspnea or othersymptoms of asthma prompting the patient to awaken [] In addition thehyperadrenergic response to beta agonist inhalers used in treatment for acutedyspnea impairs sleep onset see Table The growing success in treatments for CF patients means that sleep disordersarising from their intrinsic obstructive lung disease are now coming to theattention of caregivers Many factors contribute to sleep disruption includingchronic cough frequent infections abdominal discomfort reflux frequentstools medication side effects and psychological disease [] In addition tosleep disruption patients with CF are susceptible to hypoventilation thatworsens with disease progression Use of NIPPV in highrisk patients withhypercapnia has been shown to improve physiologic parameters and at timescan positively impact symptoms particularly in patients who have severedisease while awaiting lung transplant []Restrictive lung diseases defined by a reduced total lung capacity includethose with parenchymal damage such as idiopathic fibrosis hypersensitivitypneumonitis or other interstitial pneumonias Alternatively lung parenchymais normal in restrictive diseases such as obesity hypoventilation syndromehemidiaphragm paresis or neuromuscular disorders muscular dystrophiesamyotrophic lateral sclerosis Restrictive lung disease patients as seen abovewith obstructive disease patients are susceptible to nocturnal hypoventilationsubsequent CO2 retention and compensatory sleep fragmentation Use ofNIPPV in patients with severe restrictive lung disease spanning obesityhypoventilation syndrome to muscular dystrophies and ALS has had positiveimpacts on survival and quality of life [ ] 0c Page of CardiacCurr Treat Options Neurol Over of patients with congestive heart failure CHF have comorbid OSAmainly on the basis of mutual risk factors of DM hypertension obesity andolder age [ ] In addition insomnia in those with CHF may arise from avariety of factors including diuretic medications and subsequent nocturiapositional heart failure symptoms increased adrenergic status or psychosocialfactors [] Treatments addressing comorbid OSA and insomnia improve sleepquality but demonstrate mixed results in terms of longterm cardiovascularoutcomes [ ]Patients with acute myocardial infarction AMI experience both acute andchronic sleep disorders Due to the circadian variability of adrenergic hormonesand cardiac and systemic vasculature [] the timings of AMI sudden cardiacdeath and arrhythmia occur with increased frequency at night [] Cardiacischemia may present a series of nocturnal symptoms including paroxysmaldyspnea chest pain agitation or insomnia Surviving patients are at risk forchronic sleep disorders such as insomnia and sleepdisordered breathing withor without the cooccurrence of anxiety or depression []Retrospective longitudinal data demonstrate that those with OSA and whoare adherent with CPAP experience improved cardiovascular morbidity andmortality over nonadherent patients [] However these findings have notbeen clearly supported by prospective randomized trials The Sleep ApneacardioVascular Endpoints Trial SAVE Trial has called into question the causallink between the treatment of OSA and cardiovascular outcomes With a meanfollowup of years those randomized to PAP experienced no significantimprovements in study endpoints of death from cardiovascular causes AMIstroke and hospitalization for unstable angina CHF or transient ischemicattack compared with controls [78cid129cid129] Because of possible insufficient CPAPuse and because of the lack of main indications for CPAP treatment such assevere sleepiness interpretation of the findings of this large trial remainscontroversial In practice these authors often pursue CPAP treatment for patients with OSA and cardiovascular risk factors even in the absence of sleepiness at least for a trial period to assess adherence to treatment and to determineif there are subjective and objective improvements to sleep qualityWith a prevalence range of “ OSA is common in patients with atrialfibrillation and other arrhythmias [] Accordingly the Sleep Heart HealthStudy showed a two to fivefold higher risk of arrhythmia in patients with severeOSA compared with that in controls [] Retrospective series show that inpatients with atrial fibrillation and untreated OSA the risk of atrial fibrillationrecurrence following cardioversion is compared with in patients whoare adherent to CPAP [] However a prospective randomized control trialcalled retrospective findings into question [] Similar in design to the SAVETrial patients with atrial fibrillation were randomized to CPAP versus usualtherapy from a cohort in which sleepiness was specifically excluded This smalltrial total assessed the primary outcome of time to arrhythmia recurrenceBoth arms had recurrence rates of Although the trial showed that CPAPitself provides no specific benefit to those with atrial fibrillation the outcomesfor treatment of those with both disorders remain unclearAlthough the above studies centered on associations between cardiac diseaseand OSA patients with CHF AMI and atrial fibrillation experience high rates of 0cCurr Treat Options Neurol Page of CancerCritical illnesscentral sleep apnea CSA as well exceeding in patients with mild symptomatic CHF as an example [] CheyneStokes respiration a cyclical form ofCSA results when circulatory impairment perturbs the normal responsivenessin respiratory control resulting in alterations in œthe loop gain in modulatingchanges in carbon dioxide and oxygen levels in the bloodstream [] analogous to overly aggressive adjustments to a thermostat in response to changingtemperature The presence of CSA has been considered a marker of increasedmortality in patients with CHF although aims to resolve the treatment of CSAwith CPAP or more advanced modalities have not clearly demonstrated animprovement in cardiovascular outcomes []Estimates of the prevalence of sleep disturbances across cancer patients range widelyfrom to [ ] Insomnia is the most common disorder with prevalencelevels ranging from to [ ] Patients with cancer who undergo PSGhave shorter total sleep times longer times in bed low sleep efficiency andproportionately less deep sleep than controls [] Insomnia in patients with canceris driven by a multitude of factors including preexisting socioeconomic andpsychiatric disorders fatigue age RLS pain and medication effects [ ]Treatment follows that for the general population Although sedativehypnoticsare most commonly prescribed no evidence exists for specific pharmacologicinterventions for sleep disturbances in this population [] Cognitivebehavioraltherapy is currently the recommended firstline treatment for chronic insomnia[] Because the rarity of trained psychologists makes finding a provider difficult insome circumstances the electronic delivery of cognitivebehavioral therapy hasbeen sought as an alternative to facetoface therapy [ ]The bilateral interactions between sleep and critical illness form a rapidlychanging area of investigation which is made particularly challenging giventhe difficulties in measuring sleep in critically ill patients [ ] Lack ofsleep”or its encephalopathic analog”may affect outcomes in critical illnessesFor example a lack of scorable REM sleep correlates with longer ventilatorweaning time compared with controls with intact REM [] Failure rates onnoninvasive ventilation are impacted by sleep continuity [] Delirium acommon neurobehavioral syndrome seen in upwards of of patients inthe ICU [ ] is associated with significantly worse outcomes i

Thyroid_Cancer

This study was performed to explore the effective management of bleeding associated with radiofrequency ablation RFA of benign thyroid nodulesMethods Thirtyfive patients with benign thyroid nodules who were treated with ultrasoundguided RFA from July to December at the Third Affiliated Hospital of Sun YatsenUniversity were retrospectively reviewed The technique efficacy bleeding and other complications were assessed during the followup periodResults The mean technique efficacy was 06 at month and 06 at months after the procedure One case of an intranodular haematoma and two cases of voicechange month were observed All patients recovered with corresponding treatmentConclusion Although the incidence of haemorrhage is low serious haematomas are lifethreatening Therefore having a comprehensive understanding of the potential complicationsan accurate clinical strategy and adequate technical skills may prevent or help to properly managethese complicationsKeywordsRadiofrequency ablation benign thyroid nodules haemorrhage management haematomaultrasoundDate received January accepted June 1Department of Medical Ultrasound The Third AffiliatedHospital of Sun Yatsen University Guangzhou China2General Surgery Department The Third AffiliatedHospital of Sun Yatsen University Guangzhou ChinaThese authors contributed equally to this workCorresponding authorsBo Liu General Surgery Department The Third AffiliatedHospital of Sun Yatsen University Tianhe RoadGuangzhou City Guangdong Province China Jie RenDepartment of Medical Ultrasound The Third AffiliatedHospital of Sun Yatsen University Tianhe RoadGuangzhou City Guangdong Province ChinaEmails renjieguangzhou126com liubojake126comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cIntroductionAssociationfor AdultThyroid nodules are extremely commonand the associated morbidity rate rangesfrom to according to highresolution ultrasound US findings12Mostthyroid nodules are benign andrequire no intervention other than clinicalfollowup According to the AmericanThyroidManagementGuidelinesPatients withThyroid Nodulesand DifferentiatedThyroid Cancer thyroidstimulating hormone suppression therapy for benign thyroid nodules BTNs is not recommendedbecause the potential harm outweighs thebenefit3 Radioiodine therapy was historically an effective treatment for thyroid hotnodules and a possible alternative to surgery Howeverthis technique has beenproven to have uncertain efficacy andsome adverse effects such as hypothyroidrecurrence4“ Surgery may beism orconsideredgrowing BTNs withpressurerelated symptoms neck discomfort cosmetic concerns or decreased quality of life3 At present partialtotal thyroidsurgery is considered the gold standardtreatment Surgeryassociated withnumerous complications such as nerveinjury anaesthesiarelated problemslonghospital stays conspicuous scars haemorrhage and lesions ofthe parathyroidglands78 In addition hypothyroidism isinevitable after totalthyroidectomy andrequires lifelong hormone supplementationHenceincreasingly minimally invasivetherapeutic strategies are currently used totreat BTNs In most cases several thermalablation techniques such as laser ablationmicrowave ablation radiofrequency ablation RFA and highintensity focused UShave been shown to be effective in BTNsAmong these thermal ablation techniquesRFA is the most widely applied910forisRFA of thyroid diseases first reported in“ is considered efficacious and safeJournal of International Medical Researchfor treatment of BTNs1415 To date no lifethreatening complications related to RFAhave been reported Howeverseveralcases of haemorrhagerelated to fineneedle aspiration FNA or core needlebiopsy CNB have been reported16“Although a microinvasive procedure suchas FNA can result in massive uncontrolledbleeding resulting in upper airway respiratoryuncontrolledbleeding is a rare but lifethreatening complication of RFA Thus management ofbleeding associated with RFA of BTNs isof vitalimportance This study was performed to explore the effective managementof bleeding associated with RFA of BTNsobstructionsuchMaterials and methodsofThis study was approved by the EthicsCommitteethe Third AffiliatedHospital of Sun Yatsen University andwritten informed consent was obtainedfrom all patients prior to the performanceof USguided FNA or CNB and RFA Therequirement to obtain informed consent forpublication was waived because of the retrospective nature of the studyPatientsAll consecutive patients who underwentRFA of BTNs at our institution from July to December were analysed Thefollowing inclusion criteria were appliedconfirmation of benignancyBethesdaClass II by FNA cytology or CNB complaints of pressure symptoms compressivesymptoms neck discomfort orforeignbody sensation or cosmetic problems a2cm maximum diameter of the indexnodule anxiety about a malignancy unsuitability for surgery or unwillingness toundergo surgery and a normal serum thyrotropin concentration normal completeblood counts and normal blood coagulation test results The exclusion criteria 0cHu et alwere nodules showing malignant featuresie taller than wide spiculated marginmarked hypoechoic appearance or microcalcifications on US imaging19 abnormalthyroid function performance of othertreatments for the thyroid nodules within months before the procedure pregnancyand age of years For the presentstudy only patients with 15 months offollowup after the procedure were included Thirtyfive patients met the inclusioncriteriaPretreatment assessmentBefore the procedure conventional USfindings USguidedFNA findingscontrastenhanced US CEUS findingsand laboratory and clinical results wereevaluated Two radiologists TW and JR with and years of thyroid US experiencerespectively performed the USUSguided FNA and CEUS examinationsusing a Logiq E9 US device GE MedicalSystems Milwaukee WI USA equippedwithtransducerwith a MHzfrequency range “ MHz The USexamination included characterisation ofthe location shape size margins solidcystic proportions echogenicity calcification status and internal vascularity ofeach nodulefrequency ofan ML615centrelinearLaboratory tests included the levels ofthyroidstimulating hormone free triiodothyronine free thyroxin and thyrotropina complete blood cell count and a coagulation test prothrombin time and activatedpartial thromboplastin time The nodulevolume was calculated using the followingvolume¼ length 02 width 02equationdepth 02 In addition all patientsunderwent vocal cord function assessmentsby an experienced laryngologist before theablation procedure Atenrolment allpatients were asked to rate their pressuresymptoms on a 10cm visual analoguescale grade “ cm and the cosmeticgrading score was assessed by the physicianas described in the consensus statement20Procedures and equipmentpreventsignificantAll RFA procedures were performed by oneradiologist JR with years of experienceperforming RFA in an outpatient clinic Weused an RF generator VIVA RF SystemVR STARmed Gyeonggisi South Korea andan internally cooled 18gauge 70mmlength or 10mm activetip electrodeStar RF ElectrodeVR STARmed Localanaesthesia with lidocaine was appliedto the puncture site The hydrodissectiontechnique was used under US guidance glucose and norepinephrine weremixed and injected into the surroundingthyroid capsule which provided a safe distance between the needle tip and adjacentcritical structures During the procedurewe paid special attention to the preservation of surrounding important structurestocomplicationsTherefore two essential techniques werethe transisthmic approach andappliedtechnique2122 Ablationthe movingshotwas suspended when the index nodule wascovered by hyperechoic zones The technique efficacy TE was then evaluated byCEUS at to minutes after RFA untilthedisappearedTechnicalthechange of an entire nodule to a noenhancement zone on realtime CEUSFor nodules with an enhancement zonean additional ablation was performed todestroy the nodule as much as possibleComplications were monitored immediatelyafter the procedure and during the followup period Major and minor complicationsand adverse effects were defined accordingto the criteria established by the Society ofInterventional Radiology2324success was defined ashyperechoiczones 0cJournal of International Medical ResearchFollowup evaluationatandperformedserum thyroidAny specific complaints or concerns wererecorded for month Postproceduralfollowup wasand months after treatment At each followup visit a US examination CEUS examinationhormonemeasurements were performed pressuresymptoms and the cosmetic grading scorewere evaluated and the volume ofthetreated nodule was calculated The TE wascalculated using the following equationTE¼ final nodule volumeinitial nodule volume 02 Statistical analysisstatistical analyses were performedAllusing SPSS software version IBMCorp Armonk NY USA Continuousvariables are expressed as mean 06 standarddeviation Quantitative data for volumeand TE were analysed using a pairedttest A P value of 14 was consideredstatistically significantResultsThe patients™ characteristics are summarised in Table Thirtyfive patients underwent RFA including male and femalepatients mean age years The meanlargest BTN dimension was 06 mmrange “ mm and the mean BTNvolume was 06 mL Twentytwototal complications minor and majorcomplications were observed among thetreated patients None of these complications was lifethreatening and all occurredwithout sequelaeNodule volumeAfter treatment the overall volume of thesignificantly decreased 06nodules 06 mL at mL at month and 06 mL at monthsbaselineTable Patients™ baseline characteristics n¼ Characteristics 06 06 06 06 06 06 06 06 Age at treatment yearsMalefemale ratioBody weight kgBody height cmBody mass index kgm2Symptom score “Cosmetic score “Cosmetic score of Cosmetic score of Cosmetic score of Preablation serum FT4 level pmolLPreablation serum TSH level mIULIndex nodule on ultrasoundRight sideLeft sideLargest dimension mm to to 15Data are presented as mean 06 standard deviation ornumber of patientsFT4 free thyroxin TSH thyroidstimulating hormoneP and the TE was 06 at month and 06 at months P Table Figure shows the shrinkage of the nodules at and months after the procedure comparedwith baseline no hypoechoic blood supplywas observed within the area ofthenodulesBleeding complicationsTwelve patients developed bleeding complicationsincluding a perithyroidal haematoma minor complication in patientsand an intranodular haematoma majorcomplication in patient as shown inTable The haematomas were detectedby US scans which revealed gradualenlargement of a hyperechoic mass in oraround the nodules Figure For thepatient with intranodular haemorrhage 0cHu et alTable Changes in volume before RFA and at each followup visitParameterInitial month laterLargest diameter mm 06 “ 06 “Volume mLTechnique efficacy ”Data are presented as mean 06 standard deviation range 06 “ 06 “ 06 “ months laterP value 06 “ 06 “ 06 “ Figure a c e Ultrasound examination and b d f contrastenhanced ultrasound examination of a39yearold woman treated with radiofrequency ablation a b Ultrasound and contrastenhanced ultrasound revealed a cysticsolid nodule before ablation c d One month after ablation ultrasound showed ahypoechoic nodule with a decreased volume d e Six months after ablation the volume of the nodule haddecreased further and no blood supply was observed within the area of the nodule 0cJournal of International Medical ResearchTable Complications and adverse effects in patients who underwent RFA of thyroid nodulesComplication or adverse effectAdverse effectsFeverPainDizzinessSensation of heatMinorPerithyroidal haematomaVomitingnauseaOedemaswellingVoice change for monthMajorVoice change for monthIntranodular haemorrhageData are presented as n the haematoma was controlled throughtimely use of the ablation needle to coagulate the injured blood vessel and by injecting lyophilising thrombin powder into thehaematoma Figure Most of the perithyroidalseriesrequired only observation with or withoutcompression and disappeared within to weeks after the procedure None of the patientssubscapularhaematomahaematomasdevelopedthisinaOther complications and adverse effectsThe adverse effects of RFA included fevern¼ pain n¼ dizziness n¼ and a sensation of heatn¼ Minor complications includedoedemaswelling n¼ and a voicechange for month n¼ vomitingnausean¼ DiscussionImageguided thermal ablation techniquessuch as laser ablation ethanol ablationmicrowave ablation highintensity focusedUS and particularly RFA have recentlybecome more widely used to treat thyroidthe creation ofnodules Briefly the basic mechanism ofRFA involvesthermaldamage by friction and heat conductionwhich is generated from an oscillatinghighfrequency alternating electric currentproduced by the RFA generator and thentransferred through the electrode tip Theenergy of RFA is powerful and accurate2526 RFA is considered an effectiveand safe treatment for control of BTNsIn most cases the incidence of haemorrhage and other complications is low20However haemorrhage is sometimes lifethreatening because serious haematomasmay compress the upper airways Manyreports have described active bleedingduring FNA of thyroid nodules and RFAof hepatocellular carcinomas14““ andsome reports have described fatalities14ThusimportantcomplicationhaemorrhageanisThree types of haemorrhage may occurperithyroidal subcapsular and intranodular121427“ The mechanism of haemorrhage is thoughtto be related to themechanical or thermal injuries induced bythe RFA electrode tip3031 Thyroid nodulesreportedly have abundant capsular vesselsthat are usually anastomosed with vesselspenetrating into the core32 These numerousvessels are abnormal thinwalled and susceptible to rupture Large thyroid nodulesare another cause of haemorrhage becausemultiple insertions are often required totreat such nodules In additionthepatient cannot coordinate with the physician during the RFA procedure the perithyroidal orintrathyroidal vessels mayeasily be damaged by movement of theneedle tip or production of heat energyifIt is important to manage bleeding associated with RFA of BTNs Based on ourexperience we suggest several steps to preventshouldobtain a thorough medical history of eachpatient before the procedure All risk factorsdrugssuch bleeding Physiciansincludingbleedingfor 0cHu et alFigure Ultrasound examination and contrastenhanced ultrasound examination of patients with intranodular haemorrhage and perithyroidal haemorrhage a Ultrasound and contrastenhanced ultrasoundrevealed a hyperechoic mass lesion in the nodule b Ultrasound and contrastenhanced ultrasound revealedperithyroidal haemorrhagedrugsnonsteroidalantiplateletantiinflammatory drugs and anticoagulantsand diseases affecting coagulation shouldbe recorded33 In addition the patient™scoagulation function should be thoroughlyevaluated All patients with clinical coagulation disorders should be excluded Evenwhen coagulation indices are normalinpatients with high risk factors for bleedingsuch as liver cirrhosis endstage renal disease anticoagulant use or hypertension34sufficient preoperative preparation shouldbe emphasised A patient with active bleedingthesein the presentstudy metconditions Although his coagulation indices were normal he had a subclinical coagulation disorder due to endstage liverdisease Fresh frozen plasma or human prothrombin complex should be used inpatients with liver cirrhosis and anticoagulants should be withdrawn in these patientswhich will help to improve coagulation function before the procedure If a possibility ofbleeding exists Reptilase haemocoagulaseatrox forinjection Pentapharm BaselSwitzerland can be used preoperativelyDuring the RFA procedure an effectiveclinical strategy and adequate technical 0cJournal of International Medical ResearchFigure Ultrasound examination and contrastenhanced ultrasound examination of patients with intranodular haemorrhage or perithyroidal haemorrhage during ablation a Ultrasound revealed a hyperechoicmass lesion in the nodule b Ultrasound showed an ablation needle inserted into the nodule to coagulatebleeding vessels c After lyophilising thrombin powder was injected into the hematoma ultrasound andcontrastenhanced ultrasound showed disappearance of the hyperechoic mass lesion and microbubbleextravasation d Ultrasound showed a hyperechoic mass lesion around the thyroid and contrastenhancedultrasound showed no microbubble extravasation around the thyroid e After lyophilising thrombinpowder was injected into the haematoma no microbubble extravasation was observedskills are both essential Patient cooperationis the first requirement When the needle tipis in the patient™s body any uncooperativemotion of the patient may lead to injury ofvessels or other structures Most patientscan endure the procedure under local anaesthesia however anxious patients mayrequire general anaesthesia to achieve cooperation If possible smallbore electrodesshould be chosen to decrease the risk ofbleeding35 It is necessary to cauterise thesupplying vasculature of nodules to avoidrecurrence and residue Howeverthepuncture route should be carefully designedto avoid pericapsular vessels and the electrode tip should be closely monitoredActive bleeding during needle puncture isvisible as a rapidly expanding hypoechoicor anechoic signal Locating the haemorrhagic focus is not difficult with CEUSguidance The bleeding pointcan beblocked by RF electrode tip insertion anddirect ablation When the bleeding is toorapid to control with the RF electrode tipby increasing the power drug injection is asuitable alternative Lyophilised thrombin 0cHu et alpowder can be dissolved in normal salineand then injected at the bleeding pointthrough a syringe with US guidance Onereport also described haemorrhage treatedby local injection of hypertonic saline andepinephrine solution in a patient with hepatocarcinoma36 Mildbleeding whichappears as a hypoechoic layer can mostlybe controlled using ice and compression ofthe neck for several minutes after the procedure30 All bleeding can be controlled byconservative methodsthus no surgicalintervention is needed Ecchymosis can befound after the procedure and usually disappears in approximately to weeksPostprocedure CEUS is indispensablefor all patients regardless of whether bleeding occurs CEUS is an objective evaluationtool for active bleeding37 Close clinicalobservation for hours postoperatively isrecommended in our department becausemost bleeding occurs during the firstlobectomy38Observation of the neck can help to detecta haematoma early and may aid in preventing serious adverse effectsthyroidhoursafterConclusionAcute thyroid bleeding is one possible complication of RFA although rare it is potentially lifethreatening Proper selection ofpatients and sufficient preparation areessential During the RFA procedureboth an effective clinical strategy and adequate technical skills are indispensable Thephysician should trace the electrode tipusing realtime US and sufficiently managebleeding Mild bleeding has limited morbidity and can be easily controlled by compression Active bleeding tends to be rarehowever it may be disastrous if the operator is unaware or careless Direct ablationwith the RF electrode tip and drug injectioninto the bleeding focus are effective modalities for active bleeding CEUS and closeobservation are also recommended afterthe procedureto detect abnormalitiesearly RFA is an effective and relativelysafe alternative for selected patients withBTNs if performed by skilled physiciansAuthor contributionsI Conception and design Jie Ren and Bo LiuII Administrative support Jie RenIIIstudy materials or patientsProvision ofKunpeng Hu and Yufan Lian IV Collectionand assembly of dataJinfen Wang andWenchao Li V Data analysis and interpretation Wenchao Li and Zhicheng YaoVIManuscript writing All authors VII Finalapproval of manuscript All authorsData availabilityData regarding the patients™ characteristics usedto support the funding are shown in Table Declaration of conflicting interestThe authors declare that there is no conflict ofinterestFundingthe NaturalThis work was supported by the NationalNatural Science Foundation of China CNNoScienceFoundation of Guangdong ProvinceNo2016A030313200 the Science and TechnologyProject of Guangzhou City No the Hengrui Foundation of Hepatobiliary andPancreaticNoCXPJJH1180000120183331NaturalScience Foundation of Guangdong ProvinceNothe FundamentalResearch Funds for the Central UniversitiesSun Yatsen University No 17ykpy67 andthe Clinical Research Project of Sun Yatsen University No 2017A030313580theCancerResearchORCID iDKunpeng Huorcid00000001 0cReferences Guth S Theune U Aberle J et al Very highprevalence of thyroid nodules detected byhigh frequency MHz ultrasound examination Eur J Clin Invest “ Tan GH and Gharib H Thyroid incidentalomas management approaches to nonpalpable nodules discovered incidentally onthyroid imaging Ann Intern Med “ Haugen BR Alexander EK Bible KC et al AmericanThyroid AssociationManagement Guidelines for Adult Patientswith Thyroid Nodules and DifferentiatedThyroid Cancer The American ThyroidAssociation Guidelines Task Force onThyroid Nodulesand DifferentiatedThyroid Cancer Thyroid “ Ceccarelli C Bencivelli W Vitti P et alOutcome ofradioiodine131 therapy inhyperfunctioning thyroid nodules a years™ retrospective study Clin EndocrinolOxf “ Reiners C and Schneider P Radioiodinetherapy of thyroid autonomy Eur J NuclMed Mol Imaging S471“S478 Nieuwlaat WA Hermus AR SivroPrndeljF et al Pretreatment with recombinanthuman TSH changes the regional distribution of radioiodine on thyroid scintigramsof nodular goiters J Clin Endocrinol Metab “ LinosDEconomopoulosKPKiriakopoulos A et al Scar perceptionsafter thyroid and parathyroid surgery comparison of minimaland conventionalapproaches Surgery “ Jeannon JP Orabi AA Bruch GA et allaryngeal nervethyroidectomy a systematicDiagnosis ofpalsy afterreview Int J Clin Pract “recurrent Lang B Woo YC and Chiu KW Identifyingpredictive factors for efficacy in high intensity focused ultrasound HIFU ablationof benign thyroid nodules “ a retrospectiveInt J Hyperthermia analysis“ Mauri G Pacella CM Papini E et alImageguided thyroid ablation proposalforterminology andstandardization ofJournal of International Medical Researchreporting“criteria Thyroid Sato M Tateishi R Yasunaga H et alMortality and hemorrhagic complicationsassociated with radiofrequency ablation fortreatment of hepatocellular carcinoma inendstagepatients on hemodialysissurveyrenalJ“Gastroenterol Hepatolnationwidediseasefora Krokidis M Spiliopoulos S Jarzabek Met al Percutaneous radiofrequency ablationof small renal tumours in patients with asingle functioning kidney longterm resultsEur Radiol “ Lim HK Lee Dupuy DE Monchik JM Decrea C et alRadiofrequency ablation of regional recurrence from welldifferentiated thyroid malignancy Surgery “JH Ha EJalRadiofrequency ablation of benign nonfunctioning thyroid nodules 4year followup results for patients Eur Radiol “et Braga M Cavalcanti TC Collaco LM et alEfficacy of ultrasoundguided fineneedleaspiration biopsy in the diagnosis of complex thyroid nodules J Clin EndocrinolMetab “ Kakiuchi Y Idota N Nakamura M et alA fatal case of cervical hemorrhage after fineneedle aspiration and core needle biopsy ofthe thyroid gland Am J Forensic Med Pathol “ Donatini G Masoni T Ricci V et al Acuterespiratory distress following fine needleaspiration of thyroid nodule case reportand review of the literature G Chir “ Roh JL Intrathyroid hemorrhage and acuteupper airway obstruction after 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guidelines J Vasc Interv Radiol S199“S202 Goldberg SN Radiofrequency tumor ablation principles and techniques Eur JUltrasound “ Rhim H Goldberg SN Dodd GR et alEssential techniques for successful radiofrequency thermal ablation of malignanthepatic tumors Radiographics Spec No S17“S35 S36S39 Korkusuz Y Erbelding C Kohlhase Ket al Bipolar Radiofrequency Ablation ofBenign Symptomatic Thyroid Nodules initial Experience Rofo “ Garberoglio R Aliberti C Appetecchia Met al Radiofrequency ablation for thyroidnodules which indications The first Italianopinion statement J Ultrasound “ Baek JH LeeJH Sung JYet alComplications encountered in the treatmentof benign thyroid nodules with USguidedradiofrequencya multicenterstudy Radiology “ablation Chen MH Dai Y Yan KalIntraperitoneal hemorrhage duringandafter percutaneous radiofrequency ablationof hepatic tumors reasons and managementChin Med J Engl “et Rhim H Dodd GR Chintapalli KN et alRadiofrequency thermal ablation of abdominal tumors lessons learned from complications Radiographics “ Terry WI Radium emanations in exophthalmic goiter”blood vessels of adenomas ofthyroid J Am Med Assoc “ Hor T and Lahiri SW Bilateral thyroidhematomas after fineneedle aspiration causing acute airway obstruction Thyroid “ Minami Y Hayaishi S and Kudo MRadiofrequency ablation for hepatic malignancies is needle tract cauterization necessary for preventing iatrogenic bleeding DigDis “ Baek JH Kim YS Lee D et al Benign predominantly solid thyroid nodules prospective study of efficacy of sonographicallyguided radiofrequency ablation versus control condition AJR Am J Roentgenol “ Koda M Murawaki Y Hirooka Y et alComplications of radiofrequency ablationfor hepatocellular carcinoma in a multicenter study an analysis of treated nodules in patients Hepatol Res “ Wiggermann P Wohlgemuth WA Heibl Met al Dynamic evaluation and quantificationof microvascularization during degradablestarch microspheres transarterial chemoembolisation DSMTACE of HCC lesionsusingultrasoundCEUS a feasibility study Clin HemorheolMicrocirc “enhancedcontrast Rosenbaum MA Haridas M and McHenryCR Lifethreatening neck hematoma complicating thyroid and parathyroid surgeryAm J Surg “ 0c'

Thyroid_Cancer

Primary squamous cell carcinoma ofis anextremely rare aggressive malignancy with a poor prognosis However almost noreportthus far has investigated the microvasculature of ThyPSCC imaged usingcontrastenhanced ultrasoundthe thyroid ThyPSCCCase Report A 59yearold male patient presented to our hospital with progressivelyworsening hoarse voice symptoms for days and was diagnosed with left unilateralvocal fold palsy Ultrasonography revealed a solitary marked hypoechoic thyroid nodulewith an unclear boundary in the inferior part of the left lobe Color Doppler flow imagingshowed a poor blood flow signalinside this nodule Contrastenhanced ultrasoundimages showed a persistent low peak enhancement of the nodule from its periphery to itscenter The timeintensity curve displayed a washin time of s a time to peak of s apeak signal intensity of and a washout time of s for the thyroid tumor Finallyleft hemithyroidectomy of the thyroid tumor was performed and histopathologic andimmunohistochemical evaluations confirmed the diagnosis of ThyPSCC Postoperativelythe patient received a combination therapy of chemotherapy radiotherapy and targetedtherapy but the patient died months after surgeryPrimary squamous cell carcinoma ofConclusionthe thyroid is a rare butaggressive malignancy of the thyroid Herein we reported a case of ThyPSCC and itsultrasonography and pathologic findingsKeywords thyroid cancer thyroid nodules TNs thyroid ultrasound US primary squamous cell carcinomacontrast enhanced ultrasound CEUSINTRODUCTIONPrimary squamous cell carcinoma of the thyroid ThyPSCC is a rare thyroid malignancy withhigh aggressiveness and poor prognosis comprising ˆ¼“ of all primary thyroid carcinomas“ Owing to the rapidly progressing and highly invasive nature of the malignancy patients withThyPSCC often present at an advanced stage and are difficult to diagnose in the early stage becauseof its rare incidence and lack of typical imaging findings Thyroid ultrasonography and fineneedle aspiration biopsy FNAB are the diagnostic tools ofchoice for evaluating patients with suspected thyroid nodules Contrastenhanced ultrasoundCEUS as a relatively novel US technique is used to investigate the microvasculature of thyroidnodules and improve the diagnostic accuracy of thyroid nodules accompanied by the use of ThyroidEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byPasqualino MalandrinoUniversity of Catania ItalyDaniela PasqualiUniversity of Campania LuigiVanvitelli ItalyCorrespondenceChengcheng NiuniuchengchengcsueducnSpecialty sectionThis was submitted toCancer Endocrinologya section of the journalFrontiers in EndocrinologyReceived February Accepted June Published August CitationChen S Peng Q Zhang Q and Niu C ContrastEnhanced Ultrasoundof Primary Squamous Cell Carcinomaof the Thyroid A Case ReportFront Endocrinol 103389fendo202000512Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaImaging Reporting and Data Systems for ultrasonographicfeatures “ However very few published studies havereported the use of ultrasonography for ThyPSCC To ourknowledge this is the first case describing the CEUS findingsof ThyPSCCreached its peak [time to peak TTP] at s with a peakintensity of Then the nodule slowly declined until allthe microbubbles washed out at s Figures 1CD Based onits malignant conventional ultrasound features and the poormicrovasculature revealed by CEUS we inferred that the nodulewas a malignant tumorCASE REPORTA 59yearold male patient presented to our hospital withprogressively worsening hoarse voice symptoms for daysand was diagnosed with left unilateral vocal fold palsy Ahighresolution ultrasound instrument Siemens Acuson S3000Mountain View CA USA equipped with a to 9MHz linearprobe was used Thyroid ultrasonography revealed a solitary — — 26cm3 marked hypoechoic thyroid nodule with anunclear boundary in the inferior part of the left lobe AThis nodule exhibited many malignant ultrasound featuressuch as solid components hypoechogenicity and microlobulatedmargins Color Doppler flow imaging CDFI showed poorblood flow signals in the nodule B Contrastenhancedultrasound was performed with a bolus intravenous injectionof mL of SonoVue Bracco Milan Italy followed by mLof saline Contrast pulse sequencing technology was used andthe timeintensity curves TICs of the nodule were calculatedThe nodule began to be slowly enhanced from the peripheryto the center at s washin time and the enhancementAfterneckthepositronultrasonographyemissiontomography“computed tomography was carried for evaluatingthesituation of distant metastases Positron emissiontomography“computed tomography showed a mass withincreased glucose metabolism in the inferior part of the leftthyroid lobe A which indicated it as a malignantmass whereas there was no evidence of lymph nodes metastasisand distant metastases Then ultrasonographyguided FNABwas performed for the left thyroid mass immediately Cytologicexamination by fineneedle aspiration FNA revealed sheets oftumor cells with giant deepstained nuclei Bethesda categoryV B Finally a left hemithyroidectomy of the thyroidtumor was undertaken The lower edge of the tumor reachedthe upper mediastinum and the depth of the tumor invadedthe esophagus and trachea which could not be completelyremoved According to the eighth edition of the AmericanJoint Committee on CancerTumor Lymph Node MetastasisTNM staging system the patient was in TNM stage IIIT4a N0 M0 Histopathological examination of hematoxylinand eosin staining showed that a carcinoma in the inferiorFIGURE Ultrasonography images of primary squamous cell carcinoma of the thyroid A Longitudinal grayscale sonography revealed a solid marked hypoechoicthyroid nodule in the inferior part of the left lobe B Color Doppler flow imaging showed a poor blood flow signal inside this nodule C Contrastenhanced ultrasoundimage showed a persistent low peak enhancement of the nodule at s D Timeintensity curves displayed the washin time of s TTP of s peak signalintensity of and washout time of s for the thyroid tumorFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE A A positron emission tomography“computed tomography scan showed increased 18Ffluorodeoxyglucose metabolism in the left neck mass BPreoperative fineneedle aspiration cytology of the mass demonstrated a few sheets of malignantlooking tumor cells with giant deep stained nuclei hematoxylin andeosin magnification — FIGURE Hematoxylin and eosin staining of primary squamous cell carcinoma of the thyroid A magnification — B magnification — C magnification — D magnification — part of the thyroid lobe A had no obvious palisadearrangementintercellular bridges or keratinization with acancer pearl Figures 3B“D Immunohistochemically tumorcells were positive for cytokeratin CK19 Acytokeratin and CK56 B epithelial membraneantigen EMA C p40 D p63 Aand Ki67 B and negative for thyroglobulinTG C and thyroid transcription factor TTF1D In view of these findings the tumor was diagnosedas poorly diï¬erentiated ThyPSCC Postoperatively the patientreceived two cycles of chemotherapy with docetaxelcisplatinintensitymodulated radiotherapy and nimotuzumabtargetedtherapy However the patient died months after surgeryDISCUSSIONPrimary squamous cell carcinoma of the thyroid is a thyroidmalignancy with extremely rare incidence and the clinicaldiagnosis and treatment guidelines for this disease have noconsensus The biological behavior of ThyPSCC is aggressiveFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A CK19 BCK56 C EMA D p40 all of which were deeply stained positiveFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A p63 B Ki C TG D TTF1 and p63 was deeply stain positive Ki67 proliferation index was TG and TTF1 did not stain negativeFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell Carcinomaand the prognosis is poor with a median overall survival of “ months which depends on the diï¬erent tumor grades Yang et al using the Surveillance Epidemiology and End ResultsProgram database reported that poorly diï¬erentiated tumrade occupied the highest percentages of all graded tumors andthe median survival was months which is similar to the survivaltime in our case Highfrequency ultrasound as the basic imaging modality inthe diagnosis of thyroid nodules has found gradually increasingdiï¬erentiated thyroid cancers over recent years Theultrasonography imaging findings of ThyPSCC have seldombeen published Regarding the ultrasonography findings Chenet al reported that ThyPSCC presented as a thyroid masswith eggshell calcification peripheral soft tissue with a blurredmargin and minimal vascular signals on CDFI sonographyIn the case of Jang et al ThyPSCC presented as a largewelldefined lobulated heterogeneously hypoechoic mass withdiï¬use microcalcifications on ultrasonography Kondo et al reported that a welldiï¬erentiated ThyPSCC showed acystic hypoechoic mass with a smooth margin and rapidlygrew with margin change blurring in year In our case thispoorly diï¬erentiated ThyPSCC presented as a solitary markedhypoechoic thyroid mass with an irregular margin and unclearboundary with a normal thyroid The irregular margin andunclear boundary with normal thyroid corresponded to tumorinvasion with adjacent tissue infiltration which is consistentwith the findings during the operation that tumor invasion withthe esophagus cannot be completely removed Poor blood flowsignals on CDFI sonography and persistent hypoenhancement onCEUS of the mass are consistent with squamous cell carcinomawhich has no obvious vascularity on pathologic examinationMany studies have investigated the application of CEUS toimprove the diagnostic accuracy of thyroid nodules despiteits usage in ThyPSCC being scarce Zhang et al foundthat highcircularequal enhancement indicated benign thyroidnodules and low enhancement indicated malignant thyroidnodules Ma et al investigated whether incomplete noring or heterogeneous enhancement later washin time andlow peak intensity on CEUS were independent risk factorsin predicting malignantthyroid nodules Deng et al detected that papillary thyroid carcinomas PTCs exhibited lowenhancement a lower peak signal intensity and a lower areaunder the curve AUC than peripheral thyroid parenchyma onCEUS In our study the TICs of CEUS for ThyPSCCshowed a washin time of s a TTP of s a peak signalintensity as low as and a washout time of s Thisis similar to the results of PTCs with a slow washin time alower peak signal intensity and a lower AUC as in previousreports To our knowledge no reports on CEUS imagingfindings of ThyPSCC have appeared in the Englishlanguageliterature According to Jang et al ThyPSCC showed a largeheterogeneously enhancing thyroid mass with a large centralnonenhancing portion on enhanced CT which correspondedwell with the squamous cell carcinoma portion with a necroticportion in pathologic staining Because of the rapid growth ofsquamous tumor cells relatively few interstitial blood vessels intumors were related to the low peak signal intensity and low AUCon CEUSisusefulstainingWith increasing malignancy in squamous cell carcinoma thetypical squamous cell carcinoma findings of intercellular bridgesand keratinized cancer pearl can decrease or disappearImmunohistochemicalin diagnosingprimary thyroid cancer In this case positivity for CK56and EMA and negativity for TTF1 and TG expressionpredicted squamous cell carcinoma derivation and excludedthe possibility ofthese common tumors Furtherpositivity for p63 and Ki67 expression as poor prognosticmarkers was associated with its poorly diï¬erentiated tumrade CONCLUSIONPrimary squamous cell carcinoma of the thyroid is an extremelyrare tumor and very few studies describe its ultrasonographicimaging findings It is difficult to establish a clinical guidelinefor diagnosis Our case presents the CEUS features of ThyPSCCindicating that the TICs of ThyPSCC are similar to the enhancingparameters of PTCs with a slow washin time a lower peak signalintensity and a lower AUCDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studies involving human participants were reviewed andapproved by the Ethics Committee of Second Xiangya HospitalCentral South University China The patientsparticipantsprovided their written informed consentto participate inthis study Written informed consent was obtained from theindividuals for the publication of any potentially identifiableimages or data included in this AUTHOR CONTRIBUTIONSAll authors listed have made a substantial direct and intellectualcontribution to the work and approved it for publicationFUNDINGof ChinaThis project was funded by the National Natural ScienceFoundationProvincialNatural Science Foundation of China 2018JJ2575 andHunan Provincial Health Commission Research FoundationProject B2019166 HunanFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alREFERENCES Yang S Li C Shi X Ma B Xu W Jiang H et al Primary squamous cellcarcinoma in the thyroid gland a populationbased analysis using the SEERdatabase World J Surg “ 101007s00268019049062 Limberg J Ullmann TM Stefanova D Finnerty BM Beninato T Fahey TJet al Prognostic characteristics of primary squamous cell carcinoma of thethyroid a national cancer database analysis World J Surg “ 101007s00268019050985Thyroid Primary Squamous Cell Carcinomacontrastenhanced ultrasound Ultrasound Med Biol “ 1016jultrasmedbio201810020 Casella C Ministrini S Galani A Mastriale F Cappelli C Portolani NThe new TNM staging system for thyroid cancer and the risk of diseasedownstaging Front Endocrinol 103389fendo201800541 Zhang Y Zhou P Tian SM Zhao YF Li JL Li L Usefulness of combineduse of contrastenhanced ultrasound and TIRADS classification for thediï¬erentiation of benign from malignant lesions of thyroid nodules EurRadiol “ 101007s003300164508y Koyama S Fujiwara K Nosaka K Fukuhara T Morisaki T MiyakeN et al Immunohistochemical features of primary pure squamous cellcarcinoma in the thyroid an autopsy case Case Rep Oncol “ Zhang YZ Xu T Gong HY Li CY Ye XH Lin HJ et al Application ofhighresolution ultrasound realtime elastography and contrastenhancedultrasound in diï¬erentiating solid thyroid nodules Medicine95e5329 101097MD00000000000053290000579220161108000016 Wang SS Ye DX Wang B Xie C The expressions of keratins andP63 in primary squamous cell carcinoma ofthe thyroid gland anapplication of raman spectroscopy Onco Targets Ther “ 102147OTTS229436 Chen CY Tseng HS Lee CH Chan PW Primary squamous cellcarcinoma of the thyroid gland with eggshell calcification sonographicand computed tomographic findings J Ultrasound Med “ 107863jum201029111667 Yasumatsu R Sato M Uchi R Nakano T Hashimoto K Kogo R et al Thetreatment and outcome analysis of primary squamous cell carcinoma of thethyroid Auris Nasus Larynx “ 101016janl201707009 Kao NH Tan CS H Koh AJ The utility of immunohistochemistry indiï¬erentiating metastatic primary squamous cell carcinoma of the thyroidfrom a primary lung squamous cell carcinoma Case Rep Endocrinol “ Jang JY Kwon KW Kim SW Youn I Primary squamouscarcinoma ofandtomographic“ 1014366usg13022cellrecurrence ultrasonographicthyroid gland with localUltrasonographycomputedfindings Raggio B Barrcarcinomacell 1031486toj180002J Ghandour Z Friedlander P Primary squamousof“thyroid OchsnertheJ Kondo T Matsuyoshi A Matsuyoshi H Goto R Ono K Honda Y et alA case of primary thyroid squamous cell cancer transformation frombenign tumour associated with chronic thyroiditis BMJ Case Rep 2009bcr1020081137 101136bcr1020081137 Ma JJ Ding H Xu BH Xu C Song LJ Huang BJ et al Diagnosticand contrastmalignant101089thy201performances ofenhancedultrasonographythyroid nodules Thyroid“ color dopplergrayscalepredictingfindingsvariousin Deng J Zhou P Tian SM Zhang L Liofefficacydiagnosticofradiationdiï¬erentiating9e90674 101371journalpone0090674PONED1330329imagingthyroidandnodulescontrastenhancedimpulseforcesolidfocalJL Qian Y ComparisonacousticinuseultrasoundcombinedPLoS ONEtheir Haugen BR Alexander EK Bible KC Doherty GM Mandel SJ Nikiforov YEet al American thyroid association management guidelines for adultpatients with thyroid nodules and diï¬erentiated thyroid cancer the Americanthyroid association guidelines task force on thyroid nodules and diï¬erentiatedthyroid cancer Thyroid “ 101089thy20150020 Tessler FN Middleton WD Grant EG Hoang JK Berland LL Teefey SAet al ACR thyroid imaging reporting and data system TIRADS whitepaper of the ACR TIRADS committee J Am Coll Radiol “ 101016jjacr201701046 Kwak JY Han KH Yoon JH Moon HJ Son EJ Park SH et al Thyroidimaging reporting and data system for US features of nodules a step inestablishing better stratification of cancer risk Radiology “ 101148radiol11110206radiol11110206 Peng Q Niu C Zhang Q Zhang M Chen S Mummified thyroid nodulesconventional and contrastenhanced ultrasound features J Ultrasound Med “ 101002jum14712 Peng Q Niu C Zhang M Chen S Sonographic characteristics ofpapillary thyroid carcinoma with coexistent hashimoto™sthyroiditisconventional ultrasound acoustic radiation force impulse imaging and Struller F Senne M Falch C Kirschniak A Konigsrainer A Mullerthe thyroid case report andS Primary squamous cell carcinoma ofsystematic review of the literature Int J Surg Case Rep “ 101016jijscr201706011 Wang W Ouyang Q Meng C Jing L Li X Treatment optimization andprognostic considerations for primary squamous cell carcinoma of thethyroid Gland Surg “ 1021037gs20191107Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Chen Peng Zhang and Niu This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer

LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathwaysXue Wu123 XiaoFeng Li123 Qian Wu4 RuiQi Ma123 Jiang Qian123 Rui Zhang123·Basic Research·1Department of Ophthalmology Eye ENT Hospital of Fudan University Shanghai China2NHC Key Laboratory of Myopia Fudan University Shanghai China 3Laboratory of Myopia Chinese Academy of Medical Sciences Shanghai China4Department of Pathology West China Hospital Sichuan University Chengdu Sichuan Province ChinaCofirst authors Xue Wu and XiaoFeng LiCorrespondence to Rui Zhang Department of Ophthalmology Eye ENT Hospital of Fudan University Fen Yang Road Shanghai China zhangrui936163comReceived Accepted Our research suggests that SNHG15 may play a vital role as a potential marker in UM that predicts poor prognosis Besides GSEA indicates the underlying signaling pathways enriched differentially in SNHG15 high expression phenotype KEYWORDS SNHG15 uveal melanoma the Cancer Genome Atlas pathology prognosis Gene Set Enrichment Analysis1018240ijo20200804Citation Wu X Li XF Wu Q Ma RQ Qian J Zhang R LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathways Int J Ophthalmol Abstract— AIM To evaluate the role of long noncoding RNA lncRNA SNHG15 and its potential pathways in uveal melanoma UM METHODS The SNHG15 mRNA expression level and corresponding clinicopathological characteristics of patients with UM were obtained from the Cancer Genome Atlas TCGA database and further analyzed The SPSS statistical software package was used for statistical analyses To investigate the potential function of SNHG15 in UM we conducted indepth research on Gene Set Enrichment Analysis GSEA— RESULTS The univariate analysis revealed that the age tumor diameter pathological type extrascleral extension cancer status and high expression of SNHG15 were statistical risk factors for death from all causes The multivariate analysis suggested that the mRNA expression level of SNHG15 was an independent risk factor for death from all causes as was age and pathological type KaplanMeier survival analysis confirmed that UM patients with high SNHG15 expression might have a poor prognosis In addition SNHG15 was significantly differentially expressed in the different groups of tumor pathologic stage metastasis and living status Besides the logistic regression analysis indicated that high SNHG15 expression group in UM was significantly associated with cancer status pathologic stage metastasis and living status Moreover the GSEA indicated the potential pathways regulated by SNHG15 in UM INTRODUCTIONU veal melanoma UM the most common intraocular cancer in adult worldwide[] is a malignant tumor that originates in melanocytes of the choroid plexus ciliary body and iris of the eye At present despite definitive radiotherapy or removal of the primary lesion numerous patients eventually develop metastases and subsequently prognosis is significantly poor[] In addition UM tends to metastasize to liver through hematogenous pathway a distant site relative to their origins in the eye[] There is an incubation period between the enucleation of the primary tumor and the emergence of metastasis which can range from a few months to several decades[] Despite the advancement of UM management there are currently no effective therapy once the metastases occurred[] Therefore close followup and further research on the pathogenesis and novel makers exploration of UM are of great significance for accurate diagnosis appropriate therapy and prognosis prediction Long noncoding RNA lncRNA is a class of noncoding transcripts with a length of larger than nucleotides[] which has been involved widely in biological processes of different cancers including cell cycle apoptosis cell differentiation[] In the development of UM lncRNA is also reported to play a vital role in cell cycle cell proliferation apoptosis invasion and autophagy[] For example silencing of lncRNA PVT1 prevents the development of UM by impairing microRNA173pdependent MDM2 upregulation[] ZNNT1 can suppress Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomathe progression of UM by inducing the expression of crucial autophagy gene[] The lncRNA RHPN1AS1 facilitates the tumorigenesis of UM by influencing cell proliferation and migration[] However the study of vital lncRNAs in UM still remains to be exploredSNHG15 a novel lncRNA located on chromosome 7p13[] is identified to play a key role in many types of human tumors such as osteosarcoma[] papillary thyroid carcinoma[] pancreatic ductal adenocarcinoma[] colorectal carcinoma[] hepatocellular carcinoma[] prostate cancer[] and breast cancer[] To our knowledge the potential impact of SNHG15 on the tumorigenesis of UM seems unclear recently Thus the purpose of this study was to evaluate the pivotal role of SNHG15 in the progression of UM In addition the relationship between SNHG15 expression and clinicopathologic characteristics in UM was preliminarily demonstrated To explore the underlying mechanisms of the biological pathways involved in UM we conducted a research on Gene Set Enrichment Analysis GSEA MATERIALS AND METHODSEthical Approval The study protocol was approved by the Ethics Committee of the Eye ENT Hospital of Fudan University and all procedures were complied with the principles of the Declaration of Helsinki All datasets of our present study were downloaded from an database TCGA so there was no written informed consent from participantsRNASequencing Patient Data and Bioinformatics Analysis The RNASeq gene expression level and clinicopathological characteristics including cases were obtained from the official website of the Cancer Genome Atlas TCGA UM project portalgdccancergov Patients with UM were classified as two groups based on the median SNHG15 expression level cutoff value794 FPKM Finally patients with UM were retained and their clinicopathological characteristics were further analyzed including the detailed information of age gender tumor diameter thickness pathological type extrascleral extension cancer status pathological stage metastasis living status SNHG15 expressionGene Set Enrichment Analysis GSEA is a common bioanalysis used to interpret and analyze microarray and other similar data and to speculate related pathways that can significantly enrich regulatory genes[] Through TCGA UM project we obtained the RNASeq gene expression level of UM patients And the analysis was conducted using GSEA v30 software In this study according to the association with SNHG15 expression the ordered gene list was generated firstly by GSEA Subsequently GSEA was conducted to clarify statistically significant differences between the two groups with high and low SNHG15 expression A total of permutations were performed The SNHG15 expression level was identified as a phenotype label The related pathways statistically enriched in each phenotype were selected with the nominal P005 and an false discovery rate FDR Statistical Analysis The SPSS statistical software package SPSS Inc USA was used for statistical analyses Both the univariate and multivariate analyses using Cox analysis were performed to demonstrate independent prognostic biomarkers for UM patients The survival curve was generated by conducting KaplanMeier method To compare the significant differences in overall survival OS the logrank test was conducted The plot chart was performed to visualize the difference of SNHG15 expression level for diverse variables through Graphpad The relationship between the SNHG15 expression and clinicopathological characteristics were analyzed using logistic regression The median value of SNHG15 expression was selected as the cutoff value P005 was considered statistically significantRESULTSPatient Characteristics The records of primary UM with both RNASeq gene expression level and clinicopathological characteristics were obtained from TCGA database The mean age of UM patients was years old including males and females The mean value of tumor diameter and thickness were and mm respectively In our study cohort the pathological type of UM included epithelioid cell dominant type and spindle cell dominant type of tumors were epithelioid cell dominant and were spindle cell dominant There were cases without extrascleral extension and cases with extrascleral extension The cancer status included tumorfree cases and cases with tumor Pathologic stage II was found in cases and stage IIIIV in cases And of cases had metastases of cases had no metastases Of cases cases died of all causes Survival Outcomes and Multivariate Analysis Prognostic factors of UM were analyzed using univariate and multivariate Cox regression The univariate analysis suggested that high SNHG15 expression was a risk factor for death from all causes Other clinicopathologic variables related to poor prognosis included age tumor diameter pathological type extrascleral extension cancer status Table In a multivariate analysis SNHG15 was an independent risk factor for death from all causes as was age and pathological typeSNHG15 Expression Associated with Clinical Pathological Characteristics A total of UM cases with SNHG15 expression data and clinicopathologic characteristics were analyzed from TCGA KaplanMeier survival analysis 0cTable Prognostic parameters in UM were analyzed using univariate and multivariate Cox regressionDeath from all causesParametersnmeanUnivariate analysisPHR95CIAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelioid cell dominantSpindle cell dominantExtrascleral extensionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVSNHG15HighLowUM Uveal melanomaMultivariate analysisPHR95CIdemonstrated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group Figure 1A P005 As shown in Figure 1B1D SNHG15 was statistically differentially expressed in diverse groups of the tumor pathologic stage stage II vs IIIIV P00257 metastasis P00071 living status P00017 To clarify the clinicopathologic impact of SNHG15 we also used logistic regression and concluded that the SNHG15 expression based on median value of FPKM as a categorical variable was statistically related to clinicopathologic features Table High SNHG15 expression was significantly related to cancer status pathologic stage metastasis living status in UM all P005 Table These results demonstrated that UM with high SNHG15 expression were prone to progress to cancer status of survival with tumor a more advanced stage metastasis and poor living status when compared to the low SNHG15 expression group However there was no statistically significant difference in age gender tumor diameter thickness pathological type extrascleral invasionMain Enriched Pathways in UM Tissues with High SNHG15 Expression To explore the SNHG15related potential signaling pathways activated in UM GSEA was performed In the current study based on the association with SNHG15 expression the gene list was generated firstly Figure The SNHG15 expression was associated with clinical pathological characteristics A Patients with high SNHG15 expression had a shorter OS when compared with the low SNHG15 expression group P002 BD The expression of SNHG15 was statistically different in diverse groups of the tumor pathologic stage P00257 metastasis P00071 living status P00017 aP005 bP001by GSEA To clarify the statistically significant differences between high and low SNHG15 expression groups GSEA was Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaTable Association between SNHG15 expression and clinicopathologic variables using logistic regressionParametersAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelialNonepithelialExtrascleral invasionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVMetastasesNoYesLiving statusAliveDeadnmeanSNHG15 expressionLowHighPOR95CINESNominal PvalESTable Enriched pathways for differential SNHG15 expression in UMNameSpliceosomeCell cyclePyrimidine metabolismDNA replicationNucleotide excision repairRNA degradationHomologous recombinationMismatch repairUM Uveal melanoma ES Enrichment score NES Normal enrichment score FDR False discovery rateFDR Qvalconducted subsequently The results indicated that there were significant differences in spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair among patients with high SNHG15 expression phenotype Figure Table DISCUSSIONAccumulating evidences indicate that SNHG15 plays a dual role in the tumorigenesis and development of different tumors[] Previously SNHG15 has been demonstrated as a carcinogenic lncRNA which is usually upregulated in tumor tissues compared with normal tissues[] It exerts 0cFigure Enrichment plots from GSEA Spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair are enriched significantly in SNHG15 high expression phenotypean oncogenic effect via various epigenetic mechanisms[] For example it can suppress the expression of miR3383p and facilitate the proliferation of colorectal cancer cells[] It plays a carcinogenic role by affecting miR3383pFKBP1A axis in prostate cancer[] It can also enhance hepatocellular carcinoma progression by negative regulation of miR1413p[] However there are reports that SNHG15 has a tumor suppressive effect suggesting that low SNHG15 expression is related to poor prognosis in thyroid cancer and upregulating expression of SNHG15 can significantly suppress cell proliferation[] At present the impact of SNHG15 on UM is still unclear Therefore vital roles and potential biological mechanism of SNHG15 in UM needs to be elucidated In this study we revealed that high SNHG15 expression was related to clinicopathologic features in UM Through RNASeq gene expression level and clinicopathological characteristics obtained from the TCGA UM project we analyzed the relationship among SNHG15 expression clinicopathological features and prognosis of UM The univariate analysis demonstrated that SNHG15 expression level age tumor diameter pathological type extrascleral extension and cancer status were risk factors for death from all causes The multivariate analysis suggested that high SNHG15 expression along with age and pathological type was an independent risk factor for death from all causes Therefore the results demonstrated that high SNHG15 expression was an independent predictor of poor prognosis in UM through univariate and multivariate analysis KaplanMeier survival analysis also indicated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group in UM In addition an analysis was conducted to further explore the relationship between SNHG15 and clinicopathological features The SNHG15 expression was statistically different in diverse groups of the tumor pathologic stage metastasis and living status Besides high SNHG15 expression based on median expression value of FPKM in UM was associated with cancer status of survival with tumor advanced pathologic stage metastasis and living status It demonstrated that high SNHG15 expression in UM was strongly related to poor prognosis The mechanisms of SNHG15 dysregulation in malignant tumors are quite complex and are far from being completely understood Previous studies have suggested that SNHG15 is involved in diverse pathological and physiological processes of many tumors through their abnormal expressions including cell proliferation invasion migration and autophagy[] To explore the biological mechanism of SNHG15 in UM GSEA was conducted It indicated that spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair were all enriched differentially in SNHG15 high expression phenotype Alternative splicing is essential for gene regulation and abnormal splicing plays a vital role in inactivating tumor suppressor genes or activating oncogenes[] SNHG15 may have an impact on the invasion Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaand migration of UM cells by affecting spliceosomal related factors The abnormal cell proliferation of tumor is related to the lack of checkpoint control over the cell cycle which is the basis of genetic instability[] Evidence shows that the lack of homologous recombination may facilitate the disturbance of cell cycle the instability and accumulated mutations of genome during the progression and development[] Mismatch repair proteins have an significant role in DNA hypermethylation alteration and tumorigenesis[] SNHG15 is closely related to DNA replication and mismatch repair demonstrating that SNHG15 may promote the occurrence of UM by affecting DNA replication and DNA mismatch repair It indicated that SNHG15 may be identified as a novel marker of diagnosis therapeutic and prognosis prediction in UM However the related mechanism needs to be further elucidated This research also has some limitations The most important one is the limited number of patients and time of followup In addition some patient characteristics such as ciliary body involvement were not completely recorded in the database In fact ciliary body involvement plays a critical role in UM[]In conclusion this study aims to demonstrate the vital role of SNHG15 in UM and the potential relationship between SNHG15 expression and clinical parameters SNHG15 expression may be a valuable biomarker for poor survival in UM Moreover we have preliminarily explored the crucial pathway associated with SNHG15 in UM However further experimental validation is needed to be performed for clarifying the significant impact of SNHG15 And it is of great significance to further identify its independent prognostic value in a largescale standardized researches on UMACKNOWLEDGEMENTSFoundations Supported by the National Natural Science Foundation of China No81970835 No81800867 Conflicts of Interest Wu X None Li XF None Wu Q None Ma RQ None Qian J None Zhang R NoneREFERENCES van Raamsdonk CD Griewank KG Crosby MB Garrido MC Vemula S Wiesner T Obenauf AC Wackernagel W Green G Bouvier N Sozen MM Baimukanova G Roy R Heguy A Dolgalev I Khanin R Busam K Speicher MR O€™Brien J Bastian BC Mutations in GNA11 in uveal melanoma N Engl J Med Carvajal RD Sosman JA Quevedo JF Milhem MM Joshua AM Kudchadkar RR Linette GP Gajewski TF Lutzky J Lawson DH Lao CD Flynn PJ Albertini MR Sato T Lewis K Doyle A Ancell K Panageas KS Bluth M Hedvat C Erinjeri J Ambrosini G Marr B Abramson DH Dickson MA Wolchok JD Chapman PB Schwartz GK Effect of selumetinib vs chemotherapy on 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Long noncoding RNA OCC1 suppresses cell growth through destabilizing HuR protein in colorectal cancer Nucleic Acids Res Cao CH Sun JY Zhang DY Guo XJ Xie LW Li X Wu DH Liu L The long intergenic noncoding RNA UFC1 a target of microRNA 34a interacts with the mRNA stabilizing protein HuR to increase levels of βcatenin in HCC cells Gastroenterology 20151482415426e18 Wang P Xue YQ Han YM Lin L Wu C Xu S Jiang ZP Xu JF Liu QY Cao XT The STAT3binding long noncoding RNA lncDC controls human dendritic cell differentiation Science Zheng XL Tang HW Zhao XF Sun YM Jiang YF Liu YH Long noncoding RNA FTH1P3 facilitates uveal melanoma cell growth and invasion through miR2245p PLoS One 20171211e0184746 Lu QK Zhao N Zha GP Wang HY Tong QH Xin SH LncRNA HOXA11AS exerts oncogenic functions by repressing p21 and miR in uveal melanoma DNA Cell Biol Lu LN Yu XY Zhang LL Ding X Pan H Wen XY Xu SQ Xing Y Fan JY Ge SF Zhang H Jia RB Fan XQ The long noncoding RNA RHPN1AS1 promotes uveal melanoma progression Int J Mol Sci Wu S Chen H Han N Zhang CX Yan HT Long noncoding RNA PVT1 silencing prevents the development of uveal melanoma by impairing MicroRNA173pdependent MDM2 upregulation Invest Ophthalmol Vis Sci Li P He J Yang Z Ge SF Zhang H Zhong Q Fan XQ ZNNT1 long noncoding RNA induces autophagy to inhibit tumorigenesis of uveal melanoma by regulating key autophagy gene expression Autophagy Dong YZ Meng XM Li GS Long noncoding RNA SNHG15 indicates poor prognosis of nonsmall cell lung cancer and promotes 0ccell proliferation and invasion Eur Rev Med Pharmacol Sci SNHG15 serves as an oncogene and predicts poor prognosis in epithelial ovarian cancer Onco Targets Ther Liu K Hou Y Liu YK Zheng J LncRNA SNHG15 contributes to proliferation invasion and autophagy in osteosarcoma cells by sponging miR141 J Biomed Sci Wu DM Wang S Wen X Han XR Wang YJ Shen M Fan SH Zhang ZF Shan Q Li MQ Hu B Lu J Chen GQ Zheng YL LncRNA SNHG15 acts as a ceRNA to regulate YAP1Hippo signaling pathway by sponging miR200a3p in papillary thyroid carcinoma Cell Death Dis Guo XB Yin HS Wang JY Evaluating the diagnostic and prognostic value of long noncoding RNA SNHG15 in pancreatic ductal adenocarcinoma Eur Rev Med Pharmacol Sci Sun XT Bai Y Yang C Hu SY Hou ZL Wang GX Long noncoding RNA SNHG15 enhances the development of colorectal carcinoma via functioning as a ceRNA through miR141SIRT1Wntβcatenin axis Artif Cells Nanomed Biotechnol Ye JF Tan LD Fu Y Xu HJ Wen LJ Deng Y Liu K LncRNA SNHG15 promotes hepatocellular carcinoma progression by sponging miR1413p J Cell Biochem Zhang JH Wei HW Yang HG Long noncoding RNA SNHG15 a potential prognostic biomarker for hepatocellular carcinoma Eur Rev Med Pharmacol Sci Zhang YL Zhang DH Lv J Wang S Zhang Q LncRNA SNHG15 Acts as an oncogene in prostate cancer by regulating miR3383pFKBP1A axis Gene Kong QL Qiu M Long noncoding RNA SNHG15 promotes human breast cancer proliferation migration and invasion by sponging miR2113p Biochem Biophys Res Commun Wang TQ Sun HB Bao Y En R Tian YJ Zhao W Jia LZ POM121 overexpression is related to a poor prognosis in colorectal cancer Expert Rev Mol Diagn Shuai Y Ma ZH Lu JW Feng JF LncRNA SNHG15 a new budding star in human cancers Cell Prolif 2020531e12716 Qu C Dai CM Guo YH Qin R Liu JB Long noncoding RNA Ma YW Xue YX Liu XB Qu CB Cai H Wang P Li ZQ Li Z Liu YH SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR153 Oncol Rep Li M Bian ZH Jin GY Zhang J Yao SR Feng YY Wang X Yin Y Fei BJ You QJ Huang ZH LncRNASNHG15 enhances cell proliferation in colorectal cancer by inhibiting miR3383p Cancer Med Liu YC Li JL Li F Li M Shao Y Wu LP SNHG15 functions as a tumor suppressor in thyroid cancer J Cell Biochem Liu YC Li JL Li M Li F Shao Y Wu LP microRNA5105p promotes thyroid cancer cell proliferation migration and invasion through suppressing SNHG15 J Cell Biochem Li YW Guo HY Jin CJ Qiu CP Gao M Zhang L Liu ZJ Kong BH Spliceosomeassociated factor CTNNBL1 promotes proliferation and invasion in ovarian cancer Exp Cell Res Williams GH Stoeber K The cell cycle and cancer J Pathol Yu B Ding YM Liao XF Wang CH Wang B Chen XY Overexpression of PARPBP correlates with tumor progression and poor prognosis in hepatocellular carcinoma Dig Dis Sci Maiuri AR Peng M Podicheti R Sriramkumar S Kamplain CM Rusch DB DeStefano Shields CE Sears CL O€™Hagan HM Mismatch repair proteins initiate epigenetic alterations during inflammationdriven tumorigenesis Cancer Res Berry D Seider M Stinnett S Mruthyunjaya P Schefler AC Ocular Oncology Study Consortium Relationship of clinical features and baseline tumor size with gene expression profile status in uveal melanoma a Multiinstitutional study Retina Jiang ZM Yu FH Li M Upregulation of BCL2 kD proteininteracting protein BNIP3 is predictive of unfavorable prognosis in uveal melanoma Med Sci Monit Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0c'

Thyroid_Cancer

Comparison of different calculationtechniques for absorbed dose assessment inpatient specific peptide receptor radionuclidetherapyDomenico Finocchiaro12 Salvatore Berenato3 Valentina Bertolini1 Gastone Castellani2Nico Lanconelli2 Annibale Versari4 Emiliano Spezi35 Mauro Iori1 Federica FioroniID1Elisa Grassi1 Azienda Unit  Sanitaria Locale di Reggio Emilia”IRCCS Medical Physics Unit Reggio Emilia Italy Department of Physics and Astronomy University of Bologna Bologna Italy Department of MedicalPhysics Velindre Cancer Centre Cardiff United Kingdom Azienda Unit  Sanitaria Locale di Reggio Emilia”IRCCS Nuclear Medicine Unit Reggio Emilia Italy School of Engineering Cardiff University CardiffUnited Kingdom federicafioroniauslreita1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSAbstractCitation Finocchiaro D Berenato S Bertolini VCastellani G Lanconelli N Versari A Comparison of different calculation techniques forabsorbed dose assessment in patient specificpeptide receptor radionuclide therapy e0236466 101371journalpone0236466Editor Choonsik Lee National Institute of HealthUNITED STATESReceived July Accepted July Published August Copyright Finocchiaro This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscriptFunding This work was supported by theEuropean Metrology Programme For InnovationAnd Research EMPIR joint research project15HLT06 Metrology for clinical implementation ofdosimetry in molecular radiotherapyMRTDosimetry which has received funding fromthe European Union The EMPIR initiative is cofunded by the European Union™s Horizon AimThe present work concerns the comparison of the performances of three systems for dosimetry in RPT that use different techniques for absorbed dose calculation anlevel dosimetry voxellevel dose kernel convolution and Monte Carlo simulations The aim was toassess the importance of the choice of the most adequate calculation modality providingrecommendations about the choice of the computation toolMethodsThe performances were evaluated both on phantoms and patients in a multilevel approachDifferent phantoms filled with a 177Luradioactive solution were used a homogeneous cylindrical phantom a phantom with anshaped inserts and two cylindrical phantoms withinserts different for shape and volume A total of patients with NETs treated by PRRTwith 177LuDOTATOC were retrospectively analysedResultsThe comparisons were performed mainly between the mean values of the absorbed dose inthe regions of interest A general better agreement was obtained between Dose kernel convolution and Monte Carlo simulations results rather than between either of these two andanlevel dosimetry both for phantoms and patients Phantoms measurements alsoshowed the discrepancies mainly depend on the geometry of the inserts eg shape and volume For patients differences were more pronounced than phantoms and higher interintrapatient variability was observedPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTresearch and innovation programme and theEMPIR Participating States SB acknowledgesfunding from Cancer Research Wales throughgrant No Competing interests The authors have declaredthat no competing interests existConclusionThis study suggests that voxellevel techniques for dosimetry calculation are potentiallymore accurate and personalized than anlevel methods In particular a voxelconvolution method provides good results in a short time of calculation while Monte Carlo basedcomputation should be conducted with very fast calculation systems for a possible use inclinics despite its intrinsic higher accuracy Attention to the calculation modality is recommended in case of clinical regions of interest with irregular shape and far from sphericalgeometry in which Monte Carlo seems to be more accurate than voxelconvolutionmethodsIntroductionRadiopharmaceutical therapy RPT as defined in ICRP [] is based on the use of specificpharmaceuticals labelled with radionuclides to deliver a lethal dose of radiation to tumourareas Radiopharmaceuticals are specifically designed to have high affinity with given tumoursites so that ionizing radiations such as ps and photons emitted by the isotopes maydeposit energy inside or close to unhealthy tissues saving surrounding healthy tissues Thisapproach produced very encouraging results in the treatment of neuroendocrine tumoursNET in the last decades in particular in therapies which make use of somatostatin analogueslabelled with 90Y or 177Lu [] such as the recently registered Lutathera [] Different responserates and a large interpatient variability of the outcome were however reported by someauthors eg Campana D and Vinjamuri S [ ]The wellestablished experience with external beam radiation therapy EBRT has providedstrong evidence that tumour response and normal an toxicity is related to absorbed dosesFor this reason it was supposed that the treatment outcome correlates with the absorbed dosedelivered to tumours even in RPT [ ] Yet a dosimetry as more accurate and personalized aspossible is needed to this purpose to provide clinicians with reliable resultsDespite the general demand for a more individualized treatment based on pretherapeuticdosimetry study in NET dosimetry is not conducted always in the clinical routine This ismostly because dosimetry is often considered time consuming a lot of time required for imaging expensive costs for every image scan and every measurement and sometimes inaccuratefor the lack of standardization and harmonization mainly At present a standard procedurefor calculating the absorbed dose is not well defined for every kind of radionuclide therapy Inrelation to NET the evidence of prolonged survival has been demonstrated only recently [] ina subgroup of NETDifferent methods have been developed to perform dosimetry since its beginnings Techniques based on standardized reference models were first developed thanks to their simplicityof implementation and have been used for many years These models assume uniform activityie homogeneous uptake in the source regions However evidence indicates that deterministic biological effects including tumour response and normal tissue toxicity may not be wellpredicted by the mean absorbed dose in the region and may be significantly influenced bynonuniform doses [] To take into account this aspect voxelbased techniques were considered similarly to those which have been also used for decades as standard of care in EBRT [] Contrary to what happens for EBRT however in which plenty of software for therapyplanning are available on the market in RPT only few systems which are adequate toPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTdosimetry for Peptide Receptor Radionuclide Therapy PRRT and which can work with multiple 3D imaging have been officially released in the last few years [“] For this reasonmany dosimetry software and tools are in use worldwide but only some of them are commercially available Several of them are homemade tools which were developed before the commercial software were finalized [“] and have been fully customized by clinical users in themeantimeAt present standardization and harmonization of the calculation systems are importantTherefore it is essential to compare the various results obtained with the most advanced existing homemadecommercial software and other less advanced still used worldwide methodsYet both categories should be tested on a larger sample of cases than ever done before Thesetests should provide an example of the most accurate methodology for 3D dosimetry in RPTthanks to the gained experience in the last decades giving recommendations about the appropriate use and the limitations of each methodA few studies presenting some comparisons have already been published [“] Howeverthese works either did not report dosimetry studies performed completely at the voxel level[] or considered a limited number of clinical cases [] or showed a comparison based onthe dose factors and not based on absorbed doses [] Therefore more studies are needed tofully evaluate calculation performances in clinically relevant conditions considering a highnumber of casesIn this context the main objective of the present work is to compare different modalitiesfor absorbed dose calculation to point out the pros and the cons in each modality and to provide recommendations about the choice of the most adequate computation technique for thesingle clinical or research centres approaching the methodology The modalities here considered include the most used techniques in this field worldwide ranging from the less advancedand less personalised to the most accurate and patient specificThe considered modalities listed in growing complexity are an level dosimetry based onstandardized reference models such as OLINDA version [] which has been used fordecades before the recent release of the new updated commercial version OLINDA version [] voxellevel dosimetry based on dose kernel convolution VoxelMed20 [] and voxellevel dosimetry based on Monte Carlo MC simulations RAYDOSE [] OLINDA11 waschosen because it is still widely used for RPT dosimetry VoxelMed20 was chosen because itwas designed to achieve a good compromise between calculation accuracy and easy applicability in clinical practice RAYDOSE was considered because MC techniques are considered toprovide the most accurate approach to dose estimate []The comparison was performed on 3D images of specifically designed phantoms and onmultiple 3D dataset of images of a high number of clinical cases This multiapproach methodbased both on phantoms and patients allowed to investigate the differences of performancebetween the calculation modalities depending on the shape and the volume of the activity distribution and to provide a valuable comparison based on a conspicuous number of clinicalcasesMaterials and methodsThis study involves human participants All participants were enrolled in a clinical trialEUDRACT at Azienda USLIRCCS of Reggio Emilia Italy The study wasapproved by the ethics committee of Azienda USLIRCCS of Reggio Emilia Italy and eachpatient gave written informed consent for the study conductionThe following sections describe in detail the specific phantoms the image set the softwareand the data elaboration approachPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTPreparation of phantomsThree different phantoms filled with 177Lu radiolabelled peptides leftover from the clinicalapplication were used¢ a ˜Cylindrical phantom™ filled with a homogeneous radioactive solution Jaszczak Data Spectrum Corporation USA shown in Fig 1A Details are included in Table ¢ a cylindrical phantom and a set of fillable plastic inserts arranged in two different configurations to originate a couple of ˜Geometrical phantoms™ The inserts different for shapetoroidal pearshaped tubular and ellipsoidal and volume are depicted in Fig 1C Insertstake the name from the shape and the equivalent diameter ie the diameter for a spherewith the same volume as shown in Table Each insert was filled with the same activityconcentration and placed in a nonradioactive water background Details of volume andactivity concentration are shown in Table ¢ an ˜Anthropomorphic phantom™ with an shaped inserts LiquiPhill The Phantom Laboratory Greenwich NY shown in Fig 1B Details are included in Table Every insert wasfilled with an activity concentration typical of real ans in clinical cases and placed in aradioactive water backgroundTo accurately measure the volumes the weight of the phantoms and of the insertsbefore and after refilling was taken with a calibrated scale The density of the waterbasedsolution was of 1gml HCl M was used as a carrier solution to prevent radioactive177Lu deposition on the phantom walls and to guarantee a homogenous radionuclidesolutionEvery phantom was scanned once and the timeactivity curve was generated using the physical decay of the isotope All specific data regarding the volumes of inserts and phantoms andthe activity used are reported in Table Fig CT scans of phantoms used in this study a Cylindrical phantom b Anthropomorphic phantom c Insertswith different shapes placed in the Geometrical phantom101371journalpone0236466g001PLOS ONE 101371journalpone0236466 August PLOS ONE 0cTable Description of phantoms used to test the dosimetry toolsPhantomPhantom volumeInsert nameInsert volumeInsert activity concentration MBqBackground activity concentration MBqComparison of different absorbed dose calculation methods in MRTCylindricalGeometricalmlAnthropomorphicNATo17aTo26E20E30E38To17bP38P39aP39bTu38aTu38bLesionPancreasLeft kidneyRightkidneySpleenLivermlNA101371journalpone0236466t001Clinical trialmlmlNANAThe clinical cases considered in the present work were all extracted from a preexisting clinicalPRRT trial including patients and conducted by Azienda USLIRCCS of Reggio EmiliaItalyAll considered patients were previously enrolled in the trial EUDRACT between and The clinical trial design established that every patient had to besequentially administered with either 177Lu labelled radiopeptides 177LuDOTATOC or 90Ylabelled radiopeptides 90YDOTATOC up to a maximum of infusions or cycles Dosimetry was mandatory in the clinical trial and was to schedule during the first cycle of therapyafter a therapeutic administration of 177LuDOTATOC Each patient underwent SPECTCT scans at h post injection According to the trial design clinical absorbedTable Legend of the insert acronyms for the Geometrical phantomInsert geometryEquivalent diameter mmInsert nameTorusTorusTorusEllipsoidEllipsoidEllipsoidPearPearPearTubeTube101371journalpone0236466t002To17aTo17bTo26E20E30E38P38P39aP39bTu38aTu39bPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTdoses for 177Lu and 90Y labelled radiopeptides for liver spleen and kidneys were calculatedEach an was manually contoured and absorbed doses were calculated in compliance withthe MIRD scheme [] at anlevel from images The number of cycles the isotope and theactivity chosen for every injection were planned by an expert physician on the basis of thedosimetry results The activity prescription had to be determined based on the BiologicalEffective Dose BED delivered to kidneys Kidneys are regarded as the principal ans at riskin PRRT [ ] As suggested by different works [“] in this clinical trial the cumulativedose limit to kidneys was set to Gy of BED for patients with no risk factors hypertensiondiabetes renal failure are considered risk factors for this therapy and at 28Gy for patients withrisk factorsIn the present work absorbed doses to kidneys spleen and liver were calculated to comparethe three dosimetric methodsImage acquisition and reconstructionAll activity measurements were performed with an accurate activity calibrator for 177Lu Aktivimeter Isomed Nuklear Medizintechnik Germany and all image acquisitions were performed through a SPECTCT scanner Symbia T2 Siemens Medical Germany  NaITldetector previously calibrated [] The standard clinical protocol for body studies was usedboth for phantoms and patients with the following SPECT settings MEHR collimators matrix x zoom views x timeview s step and shoot mode degree of rotation ˚ noncircular orbit detector configuration ˚The first CT acquisition per patient was performed with the following parameters 130kVand max mAs using tube current modulation The subsequent CT images were acquiredwith kV and 40mAs for radiation protection safety of patients The CT reconstructed slicethickness was mm and a smooth reconstruction kernel was used B08s Siemens MedicalSolution Germany The higher image quality of the first CT scan is necessary for contouringvolumes of interest more accuratelyThe SPECT projections were reconstructed by an iterative algorithm including CT attenuation correction scatter correction and full collimatordetector response in Siemens ESoftworkstation Syngo MI Application version 32B Siemens Medical Solution Germany withFlash 3D iterative algorithm iterations subsets Gaussian filter cutoff mm mmcubic voxel []All cases of Sample A were rigidly registered to the first CT image of the sequence in Siemens Esoft workstation Images of patients included in Sample B were registered using adeformable multipass algorithm with the Velocity Advanced Imaging workstation Varian Medical Systems Palo Alto USA [] The registration procedures rescaled the originalvoxel size to 39x39x35 mm3The Volumes Of Interest VOI for each phantom and each patient were manually drawnon the reference CT image as recommended by Uribe [] using the VelocityworkstationSoftware for image processing and dosimetry calculationsOLINDA11 OLINDA version [] is an an level dosimetry software based on theMIRD methodology [] for internal dose estimation This is the method adopted in the clinical trial the clinical cases of this work are extracted from Absorbed doses to ans and tolesions can be calculated by using different models in the software human phantom modelsie mathematical representations of the human body to represent ans and whole body andsphere models ie mathematical representations of spheres to represent lesions [ ]PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTUnlike VoxelMed and RAYDOSE OLINDA needs timeintegrated activity A values ofVOIs as input parameters [] which were calculated with VoxelMed20 which will bedescribed in the next section and then inserted in OLINDAOLINDA sphere model commonly used to calculate doses to lesions was used to generatethe results for the inserts placed in the Geometrical phantom and for the dummy lesion housedin the anthropomorphic phantom while OLINDA an model adult male was used for thedummy ans placed in the anthropomorphic phantom Real insert volumes were used forcalculationsThe human models adult male or adult female were used to calculate dosimetry of thecohort of patients Doses were scaled using the true patient weight and the true an massesVoxelMed20 VoxelMed is a homemade software for dose calculation developed atAzienda USLIRCCS research hospital Reggio Emilia Italy It was developed in the MatlabThe Mathworks Natick MA programming environment and designed on the CERR platform wwwcerrinfo It performs voxellevel dosimetry based on the MIRD guidelines []The first version of the software along with the S value matrices for voxel dosimetry used incalculations were described in detail elsewhere []VoxelMed version includes a graphical user interface the possibility to export the resultsof calculations to Microsoft Excel file the visualization of the fitting curves both mono andbiexponential a module for renal BED calculation following the model suggested by StrigariL [] and the possibility to correct activity for partial volume effect PVE as presentedin [] Moreover VoxelMed20 provides the user with the timeintegrated activity A at VOIlevel which can be used for dosimetry with OLINDA version both for ans and lesionsTo calculate the number of disintegrations VoxelMed integrates the timeactivity curvewith the trapezoidal method in the time interval between the first and the last acquisitionBeyond this timeinterval the integration is performed analytically and the timeactivity curveis extrapolated using the effective halflife or the physical halflife it is chosen by the userThe effective halflife of the an or lesion is derived with a biexponential fit of the activitiesin the VOI the physical halflife is known from the selected isotope Timeintegrated activityis calculated in each voxel or in the whole an depending on the modality of dose calculationselected ie voxel level or an levelRAYDOSE RAYDOSE is a software package developed at Cardiff University School ofEngineering Cardiff University UK and designed to carry out 3D patientspecific imagebased dosimetry for RPT RAYDOSE provides personalized 3D dose map performing MonteCarlo simulations on radiation transport based on the Geant4 MC toolkit CERN Switzerland Geant4 is the stateoftheart package for the simulation of the transport of psthrough matter [] RAYDOSE generates voxellevel dose maps using anatomical and physiological data taken from morphologic and functional images []In order to obtain the area under the timeactivity curve RAYDOSE allows to use differentfitting modalities monoexponential decay linear uptake plus monoexponential decay or thetrapezoidal method In this study for the dose calculation of the clinical cases we used thetrapezoidal method at the voxel level up to the last time acquisition point while the timeactivity curve beyond the last scan time was extrapolated from the monoexponential curve fittingof the whole an activities in the VOI For dose calculation in phantoms we used the physical halflife of the isotope to extrapolate the activity from the scan time upwardsData and statistical analysisTwo groups of patients were considered for the purpose of this workPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTTable Demographic and baselines clinical characteristics of all patients�CharacteristicSample A N Sample B N Gender NoMaleFemaleAge yHeight cmWeight kgPrimary tumour site NoIleumPancreasLungThyroidRectumOthers177Lu activity for dosimetry MBq   ± ± ± ± ± ± ± N\\AN\\AN\\A ± � Plusminus values are means ± standard deviation  Injected activity at the first cycle of therapy Dosimetry was performed after the first injection101371journalpone0236466t003A first subgroup of cases named as œSample A was extracted by random samplingfrom the original clinical trial to adequately represent the whole population The number ofcases was calculated safely adopting a margin of error of and a standard deviation of A second independent subgroup of patients named as Sample B was extracted toofrom the original clinical trial similarly to Sample A A sample of cases was considered adequate in relation to the aim of the experiment conducted on Sample BPatient baseline characteristics for Sample A and Sample B are reported in Table The study type the image registration and the software used for the dose calculations of theclinical cases in sample A and sample B are summarised in Table Absorbed doses were calculated separately with OLINDA11 VoxelMed20 and RAYDOSEusing the same set of imagesKidney liver and spleen absorbed doses were calculated for each patient Two differentcomparison studies were performed The first study involved only comparison between VoxelMed and OLINDA based on absorbed dose calculations of patients in Sample A The secondstudy involved comparison between all the three software VoxelMed OLINDA and RAYDOSE based on absorbed dose calculations of patients in Sample B Furthermore in order toreduce the contribution of the fitting of the activitytime curves in the comparison of the software the VoxelMed dosimetry calculations for Sample B were repeated using the same effective halflife applied in RAYDOSE RAYDOSE estimates the effective halflife by fitting theTable Summary of phantom and patient studies performedStudy typePhantomObject of studyImage registrationSoftwareHomogeneous phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEGeometrical phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEAnthropomorphic phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEClinicalSample A patientsRigid registrationOLINDA11”VoxelMedSample B patientsDeformable registrationOLINDA11”VoxelMed VoxelMedλ RD”RAYDOSE101371journalpone0236466t004PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Clinical comparison study workflow Procedure flow of absorbed dose calculation for each patient of sample A top of the image and sample B lowerpart of the image101371journalpone0236466g002an activities against time as previously described Flow chart in Fig illustrates methodology in clinical studyDosevolume histograms DVH were evaluated to compare spatial dose distribution atvoxellevelMean values of absorbed dose were used to compare an level and voxel level techniquesComparison between the different dosimetry methods was statistically evaluated using theLin™s concordance correlation coefficient CCC and the BlandAltman plot [] The CCCsymbolized by ρc allows to evaluate the degree of concordance between two measures whilethe BlandAltman plot is used to analyse the agreement between two quantities The CCC wascalculated using SAS SAS Institute Cary NC USA A value of ρc equal to denotes perfect concordance a value equal to perfect discordance while a value of no correlationResultsPhysical phantom studyThe values of mean absorbed dose for the physical phantoms calculated with OLINDA11VoxelMed and RAYDOSE are reported in Table Visual representation of the same data isprovided in Fig Similar DVH curves were generated with VoxelMed and RAYDOSE both for the Cylindrical phantom Fig and for the other two phantoms Fig Fig shows DHVs only for theinserts in the Geometrical and the Anthropomorphic phantoms with the smallest and the largest relative differences of mean absorbed dose respectivelyClinical studyAbsorbed dose for kidneys liver and spleen of the sample A of patients calculated withOLINDA11 and VoxelMed are shown in Table The absorbed doses to liver and to spleenwere found to be highly correlated while lower correlation was found for kidneys The CCCPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTTable Mean absorbed dose Gy calculated with OLINDA11 VoxelMed and RAYDOSE for all of the three phantoms The absorbed dose calculated withOLINDA11 was performed using either the an model and the Sphere model Absorbed doses to Pancreas Kidneys Spleen and Liver were calculated using the anmodel otherwise the Sphere model was usedPhantomCylindricalGeometricalAnthropomorphicInsert nameOLINDA11VoxelMedRAYDOSENATo17aTo17bTo26E20E30E38P38P39aP39bTu38aTu39bLesionPancreasKidneysSpleenLiver101371journalpone0236466t005[ confidence interval] values were ρc liver [ ] ρc spleen [ ]ρc kidneys [ ] The BlandAltman plot is shown in Fig OLINDA11 VoxelMed VoxelMedλ RD and RAYDOSE calculated mean absorbed dosefor patients of Sample B are shown in Table while the BlandAltman plot is shown in Fig The absorbed doses calculated with VoxelMed and RAYDOSE were highly correlated withρc kidneys [ ] ρc liver [ ] and ρc spleen [ ] andalmost complete agreement were found between VoxelMedλ RD and RAYDOSE withρc kidneys [ ] ρc liver [ ] and ρc spleen [ ]DiscussionIn this study we compared the performances of three tools for dosimetry calculationsOLINDA11 VoxelMed20 and RAYDOSE with the primary aim to evaluate the influence ofthe calculation modality on absorbed dose assessment anlevel based voxellevel dose kernel convolution based and Monte Carlo simulations based respectively The secondary aimwas to give some recommendations about the choice of the adequate technique for dosimetrycalculation to be implemented in a hospital in a research centre or in an academic instituteclinical or research purpose small or large number of patients clinical trials only or standardprocedures This analysis was performed in standard conditions by acquisition and processing of radioactive phantoms provided with inserts of specific volume and geometry and inclinical conditions over a large cohort of patients a selection of clinical cases taken from a clinical trial in which dosimetry had already been calculated Clinical conditions are indeed quitedistant from and more complicated than the standard conditions achievable in a phantom forseveral reasons biological kinetics in place of only physical decay of activity serial acquisitionsof functional images and associated issues related to image registration [] motion of thepatient that creates artefacts in images irregular shape of volumes of interest inhomogeneousactivity distributionPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Comparison of mean absorbed dose Gy calculated using OLINDA11 VoxelMed and RAYDOSE a Homogeneous phantom b Anthropomorphicphantom and cf Geometrical phantom Note the Lesion insert in the Anthropomorphic phantom is missing because beyond the range of dose visualized in thegraph101371journalpone0236466g003Therefore to consider a large sample of clinical cases was of great interest since many studies about methods for dosimetry calculation are based on smaller groups of patients [“]and in a small group the inter patient variability cannot be properly investigatedThe quantitative intercomparison between all the three software was performed betweenthe mean values of absorbed doses In fact OLINDA provides only mean values while RAYDOSE and VoxelMed20 voxelbased tools provide the dose distribution that can be represented with DVHs from which the mean dose values can be derived To compare thetechniques of calculation the relative differences and the correlation between data pairs wereevaluatedFor standard conditions we evaluated discrepancies of calculated absorbed dose in a cylindrical phantom and in differently shaped inserts filled with a homogeneous radioactive solution Table shows the values of absorbed dose obtained with OLINDA11 VoxelMed andRAYDOSE in each of the phantoms These values are also plotted in Fig Lower values ofabsorbed dose were generally calculated using OLINDA in comparison with the dose calculated with other voxel modalities In the case of the cylindrical phantom a good agreementwas obtained between VoxelMed20 and RAYDOSE discrepancy equal to while largerdifference was observed between VoxelMed20 and OLINDA Absorbed dose map provided by VoxelMed and RAYDOSE showed similar spatial distribution close values of standard deviation across voxels around and analogues slope in DVHs Fig PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Comparison of DVHs calculated using VoxelMed continuous line and RAYDOSE dotted line for the Cylindrical phantom101371journalpone0236466g004To compare the calculation techniques in different conditions of volume and geometry theGeometrical phantoms were acquired Relative differences in absorbed dose depend on theshape and on the volume of the inserts smaller is the volume and further from a regular sphereis the shape more the relative difference is higher In the Geometrical phantom the toroidalinserts provided the greatest discordance relative difference with VoxelMed dose rangingfrom to for OLINDA and from to for RAYDOSE while in the otherinserts differences ranged between [ ] for OLINDA11 and [ ] for RAYDOSEOn one hand the insert dose calculations in OLINDA were performed using the spheremodel since OLINDA only allows to perform dosimetry calculation for specified models ieans or spheres This approximation might explain the huge discrepancies obtained withthe voxelbased methods On the other hand a reason for the difference between RAYDOSEand VoxelMed is that the latter applies a mask before the convolution while RAYDOSE doesnot This contribution affects calculations in so far as the geometry and the volume of theinsert may influence the activity distribution and leave empty spaces around or inside theobjects This effect is especially pronounced for example in the case of the toroid The application of a mask also implies the lack of photon cross irradiation contribution between insertswhich has an impact on dose calculation contribution around [] Discrepancies ofPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calc

Thyroid_Cancer

"Deregulated circular RNAs circRNAs are associated with the development of cancer and therapyresistance However functional research of circRNAs mostly focus on potential miRNA or protein binding and morepotential regulation of circRNA on host gene DNA in cancers are yet to be inspectedMethod We performed total RNA sequencing on clinical breast cancer samples and identified the expressionpatterns of circRNAs and corresponding host genes in patient blood tumor and adjacent normal tissues qPCRnorthern blot and in situ hybridization were used to validate the dysregulation of circRNA circSMARCA5 A series ofprocedures including Rloop dotblotting DNARNA immunoprecipitation and mass spectrum etc were conductedto explore the regulation of circSMARCA5 on the transcription of exon of SMARCA5 Moreoverimmunofluorescence and in vivo experiments were executed to investigate the overexpression of circSMARCA5with drug sensitivitiesResults We found that circRNAs has average higher expression over its host linear genes in peripheral bloodCompared to adjacent normal tissues circSMARCA5 is decreased in breast cancer tissues contrary to host geneSMARCA5 The enforced expression of circSMARCA5 induced drug sensitivity of breast cancer cell lines in vitro andin vivo Furthermore we demonstrated that circSMARCA5 can bind to its parent gene locus forming an Rloopwhich results in transcriptional pausing at exon of SMARCA5 CircSMARCA5 expression resulted in thedownregulation of SMARCA5 and the production of a truncated nonfunctional protein and the overexpression ofcircSMARCA5 was sufficient to improve sensitivity to cytotoxic drugsConclusion Our results revealed a new regulatory mechanism for circRNA on its host gene and provided evidencethat circSMARCA5 may serve as a therapeutic target for drugresistant breast cancer patientsKeywords Breast cancer circRNA DNA damage repair Rloop Host gene Correspondence kechenhusteducn chewhueducnleiweifrhotmailcom Xiaolong Xu Jingwei Zhang Yihao Tian and Yang Gao contributed equallyto this work3Department of Urology Tongji Hospital Tongji Medical College HuazhongUniversity of Science and Technology Wuhan China1School of Basic Medical Sciences Wuhan University Wuhan HubeiChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cXu Molecular Cancer Page of IntroductionCircular RNAs circRNAs are novel RNAs that havebeen ubiquitously discovered in many species by highthroughput sequencing in recent years [ ] CircRNAsare generated by the backsplicing of intronic exonic orintergenic regions circRNAs are resistant to RNase Rand the stability of their structures makes these molecules ideal candidates for disease [] Extensive studieshave revealed that dysregulated circRNAs are involvedin the development of various cancers In gastric cancercircRNAssuch as circPVT1 circLARP4 has_circ_ and circ_100269 have been shown to play arole in promoting tumor growth and their expression iscorrelated with high TNM stage and poor prognosis [“]In colon and hepatic carcinoma ciRS7 promoted tumordevelopment and progression by activating the EGFR andPI3KAkt pathway [ ] CircRNAs such as circKIF4Ahsa_circ_0001944 hsa_circ_0001481 and circRNA_0025202have been implicated in molecular typing brain metastasisand drug resistance in breast cancer [“] Although greatprogress has been made the roles of circRNA and relevantmolecular mechanisms remain largely unknownPrevious studies have shown that circRNAs exert theirfunctions in different ways As noncoding RNAs circRNAs regulate the expression of other genes by servingas sponges for microRNA and RNAbinding proteins[ ] In addition some circRNAs have been shownto be translated into functional proteins [ ] Inaddition circRNAs have also been shown to directlyinteract with the genomic DNA of the host gene inplant which results in altered parent gene expression[] However the interaction of circRNAs and hostgene DNA were less studied in human cancersSMARCA5 is a member of the SWISNF complex withATPdependent chromatin remodeling activity [“]In the process of DNA damage repair SMARCA5 isinvolved in chromatin remodeling in DNA damageregions providing a structural basis for the recruitmentof DNA damage repair factors [ ] In tumorsSMARCA5 is highly expressed in hepatic carcinoma andprostate cancer and its expression levelis inverselyrelated to tumor radiosensitivity [ ]In this study we established circRNAs have averagehigher expression than their host genes in peripheralblood comparing to tissues Then we identified acircRNA derived from SMARCA5 circSMARCA5 issignificantly decreased in breast cancer cell lines andbreast cancer samples Differentto previous worksrevealing circSMARCA5 can also function as a competing endogenous RNAs by binding with miRNAmoleculesour mechanism explorationdisplayed circSMARCA5 is involved in regulating DNArepair capacity by binding exon DNA directly Andfurther functionalinvestigation of this circRNA may[“]contribute to the therapeutic implications for cytotoxicdrugresistant breast cancer patientsResultsIdentification of expression of circRNAs in breast cancerWe performed high throughput sequencing on tumorT and adjacent normal tissue AN and peripheralblood B of six breast cancer patients Total RNA withrRNAdepleted library wereconstructed and thencircRNAs expressed in those samples were identifiedCompared to tumor and adjacent normal tissue we observed average higher CIRCscore expression of circRNA linear host genes in blood than both tumor andadjacent normal tissue In all circRNAs which wereexpressed across all six patients we observed averageCIRCscores from to in blood which is higherthan tumor to and adjacent normal tissue to in six patients Fig 1a This result indicated average higher expression of circRNAs than theirhost genes in peripheral blood comparing to tissueswhich might contribute to the exploration of diagnosticbiomarker for breast cancer We then selected sixcircRNAs with high CIRCscores average to in patients and performed further experimental validation in patients Realtime PCR results establishedtwo circRNAs circHIPK3 and circSMARCA5 weresignificantly differentially expressed between tumor andadjacent normal tissue Fig 1b and Figure S1A Especially circSMARCA5 was lower expressed in tumorsamples and less studied in previous work Furthermorethe ratio of circtolinear expression of circRNA linearhost genes of circSMARCA5 in blood sample of healthvolunteers were significantly higher than those of breast cancer patients P Fig 1c and Figure S1BWe nextcirctolinear ofcircSMARCA5 and clinical relevance in patients withbreast cancer and observed significant difference in thedistribution of the patients according to pathologic T P Table S1 Together these results indicating thepotential function and candidate biomarker attributes ofcircSMARCA5 in breast cancerexamined theratio ofand functionallyinvestigatefound thatTo characterizecircSMARCA5 we firstly detected the expression of circSMARCA5 in cell lines circSMARCA5 is derived fromthe backsplicing of exon and exon of SMARCA5Fig 1d As expected endogenous circSMARCA5 butto RNase R digestionnot premRNA was resistantFig 1dthe ntcircSMARCA5 was further confirmed by Northern blotassaycircSMARCA5 was mainly present in the nucleus whereasits parent mRNA was present exclusively in the cytoplasm as evidenced by qPCR Northern blotting andRNA in situ hybridization Fig 1fh and Figure S2Fig 1e Furthermore wethe existence ofIn addition 0cXu Molecular Cancer Page of Fig See legend on next page 0cXu Molecular Cancer Page of See figure on previous pageFig Identification of circRNAs in breast cancer a Heatmap of CIRCscore FBPcircFBPlinear in tumor T adjacent normal tissue AN and bloodsample B from six breast cancer patients b Expression of six circRNAs with high CIRCscore were validated by RTqPCR assay in breast tumor andadjacent normal tissue represents P CircRNAs IDs are according to circBase through their genomic coordinates c The ratio of circtolinear ofcircSMARCA5 in blood sample of breast cancer patients and health volunteers Total RNA from blood sample of breast cancer patients and healthvolunteers was extracted and detected by RTqPCR The expression level was normalized with βactin as reference P was consideredstatistically significant d Schematic illustration showing the genomic region of circSMARCA5 derived from exons and of the SMARCA5 geneConvergent gray and divergent black primers were designed to amplify the linear or backsplicing products upper Total RNA from MCF7 cellswith or without RNase R treatment was subjected to RTPCR lower and further validated by Sanger sequencing Right e Northern blot using ajunctionspecific probe or an exon probe showing the endogenous existence of circSMARCA5 and SMARCA5 mRNA from MCF7 cells with orwithout RNaseR treatment R or R The bp marker indicates the SMARCA5 fulllength transcript transcribed in vitro The bp markerindicates exon and exon of SMARCA5 transcribed in vitro f The nucleus and cytoplasm mRNA of MCF7 were extracted and SMARCA5 andcircSMARCA5 expression levels were quantitated by RTPCR GAPDH and hU6 serve as internal references of the cytoplasm and nucleus respectivelyœ indicates P g The nucleus and cytoplasm mRNA of MCF7 were extracted SMARCA5 and circSMARCA5 were examined by Northernblotting and the SMARCA5 exon probe was applied in this experiment h Subcellular localization of circSMARCA5 and SMARCA5 in MCF7 cellsThe signals were examined by indirect RNA FISH and confocal microscopy The nucleus was counterstained with DAPI The circSMARCA5 probe waslabled by biotin while the SMARCA5 probe was labled by DIG They were stained with red and green fluorescent secondary antibodies respectively IThe expression of circSMARCA5 detected by northern blot MDAMB231 BT474 MCF7 SKBR3 are breast cancer cell lines MCF10A are normal breastcell line N1N2N3N4N5 are adjacent normal tissues T1T2T3T4 are breast cancer tissues œ indicates P Next we examined the expression of circSMARCA5 invarious breast cancer cell lines MCF7 SKBR3 BT474MDAMB231 and immortalized but nontransformedbreast epithelial cells MCF10A as well as in adjacentnormal tissues and breast cancer tissues Northern blotresultsthe expression levels of circSMARCA5 in MCF10A and normal adjacent tissuesare higher than breast cancer cell lines and cancer tissues Fig 1i These results indicated that circSMARCA5is downregulated in breast cancer tissues and cellsrevealed thatsequencecircSMARCA5 decreases the expression of SMARCA5 incancer cellsTo clarify the mechanisms of circSMARCA5 we investigated its effects on the expression of its parent geneSMARCA5 The expression levels of circSMARCA5 andSMARCA5 were detected by the primers of junctionsequence and “ exonsrespectivelyKnockdown of circSMARCA5 increased both mRNAand protein levels of SMARCA5 while converselycircSMARCA5 overexpression decreased SMARCA5levels Fig 2ac and Figure S3 Consistently the proteinof SMARCA5 was high expressed in breasttumorsamples as compared with the corresponding controlsFigure S4 Moreoverthe ratio of circtolinear ofcircSMARCA5 was significantly lower in breast andrenaltumor tissue than the corresponding adjacenttissue specimens Fig 2de and Figure S1C Besides asignificant negative correlation was also found betweencircSMARCA5 and SMARCA5 expression in various celllines and primary cancer tissues Fig 2df and FigureS5 which corroborates our observation that circSMARCA5 decreased the expression of SMARCA5 incancer cellscirSMARCA5 terminates the transcription of SMARCA5 atexon We further investigated the mechanism of circSMARCA5in regulating the expression of SMARCA5 Interestinglywe found that the overexpression of circSMARCA5 indeed decreased the expression of SMARCA5 exons “but had minimal effects on the expression of exons “Fig 3a Next we designed a primer location in exon for the amplification of ² cDNA ends by rapid amplification of cDNA ends RACE PCR Fig 3b left As shownin Fig 3b SMARCA5 can give rise to multiple isoformsImportantly we found a decrease in a band of bpupon circSMARCA5 overexpression while an bpband displayed the opposite phenomenon Fig 3b rightSanger sequencing showed that the bp band andthe bp band are derived from fulllength and truncated mRNA exons to respectively of the SMARCA5 gene Fig 3c Consistent with the RACE resultsNorthern blot assay further demonstrated that ectopiccircSMARCA5 expression decreased SMARCA5 levelsand promoted truncated mRNA levels Fig 3d The observations gathered thus far have led us to hypothesizethat circSMARCA5 prevents transcription from exon of SMARCA5 Indeed ChIP analysis indicated that thebinding of pol II to exons “ of SMARCA5 was higherthan that to exons “ Fig 3e left and the ectopic expression of circSMARCA5 decreased the binding of Pol IIto exons “ of SMARCA5 Fig 3e right To furtheraddress whether circSMARCA5 could terminate the transcriptional elongation of SMARCA5 we cloned a series ofexons of SMARCA5 in a luciferase plasmid reporterFig 4a upper The transient transfection of these luciferase reporters containing the “ exon sequence revealed that luciferase activity was significantly decreasedwhen circSMARCA5 was overexpressed Fig 4a lower 0cXu Molecular Cancer Page of Fig circSMARCA5 decreases the expression of SMARCA5 in cells a Generation of circSMARCA5knockdown and circSMARCA5overexpressingcells MCF7 cells were infected with lentiviruses expressing shRNA against circSMARCA5 shcircSMARCA5 three different oligonucleotides orcircSMARCA5 pLCDHcircSMARCA5 RTqPCR was performed to evaluate the expression of circSMARCA5 GAPDH was used as an internal controlb RTqPCR showing the levels of circSMARCA5 and SMARCA5 in MCF7 cells stably expressing shNC shcircSMARCA5 pLCDHciR control orpLCDHcircSMARCA5 c Western blot showing the levels of SMARCA5 in MCF7 cells stably expressing shNC shcircSMARCA5 pLCDHciRcontrol pLCDHcircSMARCA5 pLCDHcircSMARCA5Δ without splicinginducing sequence GAPDH was used as an internal control DF Theratio of circtolinear of circSMARCA5 in tumor tissue were significantly lower than normal tissue in breast cancer samples d and RCC samplese P A negative correlation between circSMARCA5 and SMARCA5 expression was observed in breast cancer samples d RCC samplese and various cell lines fTo further confirm the effect of circSMARCA5 on thetranscriptional elongation of SMARCA5 we insertedexons of SMARCA5 between DsRED and EGFP as indicated Fig 4b upper The EGFP level was significantly decreased by circSMARCA5 when exons “were present Fig 4b lower We further investigatedthe role of circSMARCA5 in the regulation of SMARCA5 at the protein level As expected circSMARCA5overexpression downregulated the protein levels ofSMARCA5 and upregulated truncated SMARCA5ΔSMARCA5 protein levels Fig 4c and Figure S6which was confirmed by mass spectrometry Fig 4dMoreover we found that ΔSMARCA5 is more susceptible to proteolysis by the proteasome than SMARCA5 Fig 4e Together these results show the roleof circSMARCA5 in the termination of transcriptionalelongation at exon of SMARCA5circSMARCA5 can form Rloops with its parent geneTo further dissect the mechanism of SMARCA5 transcriptional termination mediated by circSMARCA5 weinvestigated whether circSMARCA5 can bind genomic 0cXu Molecular Cancer Page of Fig cirSMARCA5 terminates the transcription of SMARCA5 at exon a RTqPCR analysis of the expression of SMARCA5 in MCF7 cells using aseries of paired primers œ indicates P b Rapid amplification of cDNA ends RACE PCR analysis of SMARCAC5 transcripts The PCRproducts were readily identified by agarose gel electrophoresis Each set of samples was repeated three times c Sanger sequencing of twotranscripts of SMARCAC5 that are regulated by circSMARCA5 overexpression d Northern blotting using the junctionspecific probes for exons and to show the expression levels of the transcripts of SMARCAC5 mRNA from MCF7 cells stably expressing control vector or pLCDHcircSMARCA5 circOE e CircSMARCA5 prevents transcription from exon of SMARCA5 ChIPseq analysis showing that the binding of pol II toexons of SMARCA5 was higher than that to exons ChIPqPCR showed that the ectopic expression of circSMARCA5 decreased thebinding of Pol II to exons of SMARCA5SMARCA5 DNA to form an Rloop Dotblotting withRloopspecific S96 antibody supported our hypothesisthat circSMARCA5 can bind exons “ of SMARCA5genomic DNA Fig 5a Additionally we performedDNARNA immunoprecipitation DRIP qPCR and confirmed the interaction between circSMARCA5 andexons “ pretreatment with RNase H ablated thisinteraction confirming that the interaction is Rloopspecific Fig b and Figure S7 The interaction of circSMARCA5 with the DNA of SMARCA5 was directlyverified by fluorescence in situ hybridization Fig 5cConsistent with previous findings [] dotblotting ofthe genome without RNA digest revealed that the binding of circRNA to genomic DNA may be widely presentin cancer cells Fig 5d We next determined the specificsequence of exons “ required for Rloop formationA series of fragments from exons “ were hybridizedwith circSMARCA5 for the dotblotting assay As shownin Fig 5e the bp fragment of the ² end of exon plays important role in interacting with circSMARCA5Moreoverthe secondary structure of circSMARCA5was determined by the software MFOLD [] whichrevealed the sequence ²AACAAAAUUGGGAAAGAUGAAAUGCUUCAAAU3² from the ² end of exon located in the loop region of circSMARCA5 Fig 6aWe thus hypothesized that this sequence might play akey role in mediating the circSMARCA5DNA interaction To this end we synthesized the wildtype andmutant phosphorylated DNA fragments ANT andANTmut respectively corresponding to this sequenceFig 6b Dotblotting demonstrated that wildtypeoligonucleotides ANT can bind to circSMARCA5 but 0cXu Molecular Cancer Page of Fig See legend on next page 0cXu Molecular Cancer Page of See figure on previous pageFig cirSMARCA5 blocks the transcription of SMARCA5 and promotes the generation of a truncated SMARCA5 protein ΔSMARCA5 aSchematics of luciferase reporter constructs containing the SMARCA5 exon sequence as indicated upper The SMARCA5 exon sequenceplays an important negative role in mediating the effect of circSMARCA5 overexpression on luciferase activity lower b Schematics offluorescence reporter constructs containing the SMARCA5 exon sequence as indicated upper MCF7 cells were transiently transfected withthese fluorescence reporters along with or without circSMARCA5 cooverexpression After transfection for hours the reporter transcriptionactivities were measured by flow cytometry assay c circSMARCA5 overexpression downregulated the protein levels of SMARCA5 whileupregulating truncated SMARCA5 ΔSMARCA5 protein levels MCF7 cells stably overexpressing circSMARCA5 or control cells were treated withDMSO or MG132 Western blot analysis was performed using an antibody targeting the Nterminus of SMARCA5 to evaluate the expression ofSMARCA5 and ΔSMARCA5 GAPDH was used as an internal control d The ΔSMARCA5 protein was identified by mass spectrometry and detectedSMARCA5 peptides were showed in the map The redlabeled portion is the amino acid sequence of the translated defective transcript e MCF7cells expressing FlagSMARCA5 and FlagΔSMARCA5 were treated with cycloheximide CHX μgml The cell lysates were subsequentlyharvested at sequential time points or h after treatment and then the cell lysates were immunoblotted with antiFlag or antiActin antibodymutant oligonucleotides ANTmut cannot bind to circSMARCA5 Fig 6c As expected DRIPqPCR showedthat ANT inhibited circSMARCA5 binding to the DNAat exons “ whereas ANTmut had no effect on thisinteraction Fig 6d Furthermore the transfection ofANT prevented the decrease in SMARCA5 proteinlevels in MCF7 cells stably expressing circSMARCA5whereas ANTmut had no effect on SMARCA5 proteinlevels Fig 6e Importantly the mutation of the keysequence in circSMARCA5 impaired the interactionwith its parent gene which was confirmed by dotblotting and DRIPqPCR assays Fig 6fh and FigureS8 Unlike circSMARCA5 circSMARCA5mut had littleeffect on SMARCA5 protein levels Fig 6i Theseresults suggested that circSMARCA5 formed Rloopswith its parent gene to inhibit the expression of SMARCA5 in cancer cellscircSMARCA5 inhibits DNA damage repair functionTo explore the roles of circSMARCA5 in cancer progression we overexpressed and depleted circSMARCA5in MCF7 cells by lentiviral vectors and then examinedthe effect of circSMARCA5 on cell proliferation migration and apoptosis However the results showed thatboth overexpressed and depleted circSMARCA5 had noeffect on these three activities Figure S9 Previousstudies have indicated that SMARCA5 plays an important role in regulating the DNA repair process and main[ “]taining theConsistent with previous reports SMARCA5 overexpression improved DNA repair capacity and reducedthe expression of Chk1 and Chk2 after DNA damagerepair Figure S10A Given that circSMARCA5 canpromote the production of the truncated ΔSMARCA5protein we tested whether the truncated protein is alsofunctional The overexpression of FlagΔSMARCA5had a minimal effect on the expression of Chk1 andChk2 after DNA damage repair Figure S10B suggesting that ΔSMARCA5 isa nonfunctional proteinproduct We next assessed whether circSMARCA5 canthe genomestability ofshowedlowerthan thatsignificantlyformation assaysaffect the function of DNA damage repair capacityCCK8 and clonerevealed thatcircSMARCA5 overexpression increased sensitivity tocisplatin or bleomycin in MCF7 cells Fig 7a b NextMCF7 cells were treated with the indicated concentration of cisplatin or bleomycin for h and then theDNA damage was evaluated by single cell gel electrophoresis SCGE at and h MCF7 cells expressingcircSMARCA5repaircapacity than did control cells Fig 7c In parallelDNA damage was examined after h of treatmentwith cisplatin or bleomycin by using an antiγH2AXantibody Consistent with the SCGE results the γH2AXin MCF7 cells expressing circSMARCA5 wassignalsignificantly higherin MCF7 cells asevidenced by immunostaining Fig 7d Consistentlycisplatin significantly enhanced the levels of DNAdamage response proteins Chk1 and Chk2 in MCF7cellsexpressing circSMARCA5 Fig 7e whereasseveral key cellcycle genes were reduced specificallyupon circSMARCA5 overexpression Fig 7f To testwhether circSMARCA5 Rloop formation is necessaryfor its DNA repair function we transfected ANT orANTmutinto circSMARCA5expressing cells TheSCGE assay and γH2AX measurement showed thatANT significantly enhanced the DNA repair capacitywhile ANTmut had no effect on this activity Fig 7gand Figure S11 Furthermore ANT significantlydecreased the degree of colocalization between circSMARCA5 and its cognate DNA locus Figure S12In addition unlike circSMARCA5 the overexpressionof circSMARCA5mut had little effect on the DNArepair rate Fig 7h and Figure S13A B Next we determined whether SMARCA5 could mediate the effects ofcircSMARCA5 in preventing DNA damage repair Asshown in Fig 7i the γH2AX signal was much lower incircSMARCA5expressing cellscomplemented withSMARCA5 than that in cells expressing circSMARCA5alone As expected ΔSMARCA5 could not rescue theinhibition of DNA damage repair function induced by 0cXu Molecular Cancer Page of Fig See legend on next page 0cXu Molecular Cancer Page of See figure on previous pageFig circSMARCA5 interacts with its site of transcription a circSMARCA5 interacts with the exon sequence of the SMARCA5 locus A seriesof exon DNA fragments were hybridized with circSMARCA in vitro The DNARNA hybridization strength was quantified by dotblot with Rloopspecific S96 antibody Hybridization stringency was altered by decreasing ionic strength mM NaCl b DRIPqPCR analysis of the exon sequence of SMARCA5 to detect the association of circSMARCA5 in MCF7 cells RNase Htreated andor DRIPqPCR analysis of the exon sequence as a control c CircSMARCA5 partially localized at its site of transcription Double FISH of circSMARCA5 red and its parent DNAregion green The nucleus was stained by DAPI d Dotblot of Rloops in MCF7 cell genomic DNA preparations treated with DNase I RNase Hor RNase R The DNARNA hybrids in genome DNA were analyzed by S96 antibody e Mapping of the Rloop formation region of circSMARCA5A series of exon deletion mutants were hybridized with circSMARCA5 for the dotblotting assay The DNARNA hybridization intensity wasanalyzed by dotblot with an S96 antibody targeting the DNARNA hybrid strand Hybridization stringency was altered by decreasing ionicstrength mM NaClcircSMARCA5 Figure S13C D Moreover the overexpression of SMARCA5 could significantly rescue thegrowth defects of cells expressing circSMARCA5 asdemonstrated by a colony formation assay Fig jTogetherthese results demonstrated the roles ofcircSMARCA5 in regulating the DNA repair process inMCF7 cellstheevaluatecircSMARCA5 overexpression enhances the cisplatinresponse in breast cancerTo furthertherapeutic potential ofcircSMARCA5 in breast cancer in vivo we establishedcircSMARCA5 overexpression clones in MCF7 cells Asshown in Fig 8a the overexpression of circSMARCA5efficiently enhanced the sensitivity of MCF7 xenograftsto concurrent cisplatin treatment Fig 8a b The overexpression of circSMARCA5 was confirmed by in situhybridization and qPCR analysis Fig 8c along with decreased SMARCA5 protein levels and increased γH2AXlevels Fig 8d In addition qPCR analysis demonstratedthat circSMARCA5 can be detected in the bloodsuggesting that circSMARCA5 is a secretory moleculeCollectively these data demonstrate that circSMARCA5could serve as a potential therapeutic target to restoresensitivity to cisplatin therapy in breast cancerDiscussionPrevious studies have indicated that circRNAs have multiple functions in cancer development and progression[“] In this study we identified multiple expressedcircRNAs in breast cancer samples and observed averagehigher abundance of circRNAs over their host genes inperipheral blood than tissues which might contribute tothe exploration of diagnostic biomarkerfor breastcancer We then identified that circSMARCA5 is significantly decreased in breast cancer tissues using RNAseqMore importantly we define a critical role for circSMARCA5 in the regulation of DNA damage repaircapacity and the drug sensitivity of breast cancer cellsin vitro and in vivo through the negative regulation ofits parent gene SMARCA5 These findings are of highclinical relevance because chemotherapy with cisplatinand bleomycin remains the standard of care in breastcancer [“] Hence the restoration of circSMARCA5levels provides an approach to overcome treatmentresistance in breast cancer patientsSMARCA5 also known as SNF2H is a member of theSWISNF chromatinremodeling complex During DNAdamage repair processes SMARCA5 is recruited toDNA damage sites where it induces the ubiquitinationand phosphorylation of histone H2A which facilitateschromatin remodeling and DNA damage repair [ ]In this study we show that circSMARCA5 expressionresulted in the downregulation of SMARCA5 and theeffect of circSMARCA5 overexpression on DNA repaircapacity was reversed by concomitant SMARCA5 overexpression suggesting that the effect of circSMARCA5on DNA repair capacity is mediated through SMARCA5circRNAs exert functions in various ways such as forming an Rloop with DNA to regulate splicing and transcriptional pausing [] For example circSEPALLATA3regulates the splicing of its parent mRNA through Rloop formation [] In addition circRNAs are a novelclass of ceRNAs that sponge miRNAs thus positivelyregulating gene expression [ ] Additionally circRNAs such as exonintron circRNAs regulate geneexpression through specific RNARNA interactions withU1 snRNA [] Furthermore circRNAs also exertfunctions by binding to proteins and regulating theiractivities [] We identified one mechanism by whichcircSMARCA5 regulates the drug sensitivity of breastcancer cells to cisplatin and bleomycin through thedownregulation of SMARC5 circSMARCA5 is recruitedto its parent gene locus leading to Rloop formationtranscriptiontruncatedΔSMARCA5 protein upregulation and decreased SMARCA5 expression This regulatory mechanism has alsobeen verified in cervical cancer Hela cells Figure S14However our evidence demonstrates that circSMARCA5has no significant effect on the proliferation migrationand apoptosis of breast cancer cells suggesting that thismolecule functions in a celltype and contextdependentmanner Notablythatnonfunctionalterminationweprovideevidence 0cXu Molecular Cancer Page of Fig circSMARCA5 can form an Rloop with its parent gene a Secondary structure prediction for circSMARCA5 using the Mfold program Thesequence KEY shared by the minimum free energy structure and the thermodynamic ensemble structure is marked by red b Thethiophosphorus nucleic acid analog ANT complementary to KEY and its mutant ANTmut were synthesized in vitro c Dotblot verifying theinteraction between circSMARCA and ANT or ANTmut d DRIPqPCR analysis on exon or exon sequences of SMARCA5 to detect theassociation of circSMARCA5 in MCF7 cells overexpressing ANT or ANTmut RNase Htreated andor DRIPqPCR analysis of the exonsequence as a control œ indicates P e Western blot analysis shows that transfection of ANT into circSMARCA5overexpressing cells canrestore SMARCA5 protein levels but ANTmut cannot f g Dotblot analysis quantifying Rloop strength between the SMARCA5 locus andcircSMARCA5 or circSMARCA5mut guanine converted to cytosine of the KEY sequence h DRIPqPCR in MCF7 cells transfected withcircSMARCA5 or circSMARCA5mut RNase Htreated genomic DNA and qPCR of exon1314 were treated as controls œ indicates P iWestern blot analysis shows that overexpression of circSMARCA5 to MCF7 cells can decrease SMARCA5 protein levels but circSMARCA5mutcannot 0cXu Molecular Cancer Page of Fig circSMARCA5 decreases DNA repair capacity a circSMARCA5 increases sensitivity to cisplatin or bleomycin in MCF7 cells MCF7 cellsstably expressing control vector or pLCDHcircSMARCA5 were treated with cisplatin or bleomycin for h and CCK8 was used to measure cellviability b Relative colony formation units of MCF7 cells stably expressing control vector or pLCDHcircSMARCA5 treated with μM cisplatin or μgml bleomycin After hours the drugs were replaced by fresh medium The number of colonies was quantified c d e Single cell gelelectrophoresis SCGE assay indicating that circSMARCA5 overexpression inhibits cell recovery from DNA damage MCF7 cells stably expressingcontrol vector or pLCDHcircSMARCA5 treated with μM cisplatin or μgml bleomycin After incubation for h the cells were recoveredwith fresh medium for or hours and then collected for SCGE analysis c immunofluorescence assay using an antiγH2AX antibody d andwestern blot assay with the indicated antibodies e f RTqPCR assay showing the relative levels of several key cell cycle genes in MCF7 cellsstably expressing control vector or pLCDHcircSMARCA5 treated with DMSO or μM cisplatin for h and replaced with fresh medium for hœ indicates P g SCGE assay showing that the cotransfection of ANT in circSMARCA5overexpressing cells can restore the DNA repaircapacity

Thyroid_Cancer

Signal Transduction and Targeted Therapywwwnaturecomsigtrans REVIEW The potent roles of saltinducible kinases SIKs in metabolichomeostasis and tumorigenesisZicheng Sun12 Qiwei Jiang1 Jie Li2 and Jianping Guo1Saltinducible kinases SIKs belong to AMPactivated protein kinase AMPK family and functions mainly involve in regulatingenergy responserelated physiological processes such as gluconeogenesis and lipid metabolism However compared with anotherwellestablished energyresponse kinase AMPK SIK roles in human diseases especially in diabetes and tumorigenesis are rarelyinvestigated Recently the pilot roles of SIKs in tumorigenesis have begun to attract more attention due to the finding that thetumor suppressor role of LKB1 in nonsmallcell lung cancers NSCLCs is unexpectedly mediated by the SIK but not AMPK kinasesThus here we tend to comprehensively summarize the emerging upstream regulators downstream substrates mouse modelsclinical relevance and candidate inhibitors for SIKs and shed light on SIKs as the potential therapeutic targets for cancer therapiesSignal Transduction and Targeted Therapy 101038s4139202000265wINTRODUCTIONSaltinducible kinase SIK was first identified in the adrenalglands of high salt dietfed rats in Further the SIK familyincluding SIK1“SIK3 are characterized as serinemembersthreonine kinases that belong to AMPactivated protein kinaseAMPK family23 Later SIKs have shown selfphosphorylationand play an important role in regulating adrenocortical functionunder the stimulation of high salt or adrenocorticotropichormone ACTH1 Of note the SIK1 is abundantly expressedin the adrenal cortex as well as in the adipose and neuraltissues3“ while both SIK2 and SIK3 are ubiquitous in humansand mainly expressed in adipose and neural tissues respectively3 In addition these SIK family members are dysregulatedin various cancers including ovarian breast prostate and lungcancers indicating that SIKs may execute crucial roles in tumoroccurrence or progression36In recent years although the roles of SIKs in tumorigenesis havedrawn much attention due to their association with TGFSmadAKT Hippo NFκb and other signaling pathways involved incancer progression6“ similar to the AMPK kinases the potentialroles of SIKs in tumorigenesis are still controversial as oncogene ortumor suppressor in a tissue context dependent manner Therefore the purpose of this review is to comprehensively summarizethe upstream regulators downstream effectors clinical relevanceas well as candidate inhibitors of SIKs to highlight the potentialstrategy to target SIKs for cancer therapiesTHE UPSTREAM REGULATORS AND DOWNSTREAMSUBSTRATES OF SIKSSIK1 gene is located in human chromosome while SIK2 andSIK3 genes are both located on chromosome SIKs sharesimilar structures to AMPKrelated kinases including AMPKα1α2in SIKsspecificallySADAB MARK1“ NUAK12 and SNRK all of which can bephosphorylated and activated by liver kinase B1 LKB1 GenerallyAMPKrelated kinases consist of two common domains possessing an Nterminal serinethreonine kinase domain KD followedby a ubiquitinassociated UBA domain18“ Beyond that SIKs arealso composed of a central sucrose non fermenting SNF1homology SNH domain and a long Cterminal domainFig 1a2022 The Nterminal KD contains a LKB1 phosphorylationsite and is relatively conserved across SIK family However theSNH domain is distinctthe similaritypercentage of SIK2 and SIK3 compared that of SIK1 is and respectively The Cterminal domain contains multipleprotein kinase A PKA phosphorylation sites and is highlyconserved between SIK1 and SIK222 Like other AMPK familymembers an activation loop Tloop exists in the KD of SIKswhich near the substratebinding pocket and is phosphorylatedand activated by LKB1 Fig 1a b1922 In addition there is also anautophosphorylation residue in the Tloop which is essential forthe kinase activity of SIK1 and SIK223 On the other hand a UBAdomain has also been defined within the SNH domain24 andmutations derived from the UBA domain notably decreased LKB1mediated SIK phosphorylation and kinase activation24 partially viapreventing SIK interacting with adapter protein to promoteSIK nuclear transport2425 Similar to AMPK kinases the Thr322residue in SIK1 SNH domain could also be activated by calciumdependent protein kinase CaMKmediated phosphorylation2026similar results were observed in SIK2 kinase and resulted in SIK2degradation27 SIKs are considered rapid turnover proteins due tothe phosphorylation by PKA PKC and tyrosine kinase in theirCterminal region Fig 1a1720 Thus SIK family members share asimilar structure and play redundant and distinctroles inregulating biological processes especially in metabolic homeostasis which will be further summarized in the following sections1Institute of Precision Medicine the First Affiliated Hospital Sun YatSen University Guangzhou Guangdong China and 2Department of Breast and Thyroid Surgery theFirst Affiliated Hospital Sun YatSen University Guangzhou Guangdong ChinaCorrespondence Jie Li Lijie78mailsysueducn or Jianping Guo guojp6mailsysueducnReceived May Accepted July The Authors 0cThe potent roles of saltinducible kinases SIKs in metabolic Sun et alFig The diagram structure of SIKs and related kinases a The structure and phosphorylation residues are illustrated SIKs are composed ofKD kinase domain containing an LKB1 phosphorylation site SNH domain containing UBA ubiquitinassociated domain and Cterminaldomain containing multiple PKA phosphorylation sites b The structure of AMPKrelated family kinases are illustrated These kinases share asimilar structure with SIKs AMPKα subunits are composed of KD AID autoinhibitory domain αlinker containing two αRIM regulatorysubunitinteracting motif and αCTD Cterminal domain SADs are composed of KD UBA domain and KA1 kinaseassociated domain it isNterminal next to the AIS sequence autoinhibitory sequence MARKs are composed of KD UBA domain spacer and tail domain includingthe KA1 domain NUAKs are composed of KD and UBA domain SNRK is composed of KD and UBA domain The phosphorylation sites on theTloop of AMPKrelated family kinases are illustrated The AMPKrelated family kinases can be directly phosphorylated and activated by LKB1on their Tloop right panel c SIK and AMPK downstream substrate phosphorylations are illustrated SIKs phosphorylated LXRKHSTXSXXXL motif underlined phosphorylated residue X any residue and the identified AMPK substrates phosphorylation sites reside in theknown AMPK phosphorylation consensus sequence LMIXRKHXXSXXXLVIF are illustratedsuch as energy deprivationSIK upstream regulatorsActing as AMPKrelated kinases SIKs exhibit a similar activationproperty with AMPK1922 in an LKB1mediated phosphorylationdependent manner Fig 1a b18“ Importantly physiologicalchangesinsulin or glucagonperturbation all manipulate SIK kinase activity29 For exampleinsulin stimulation or chronic hyperglycemia could increase SIKprotein level and kinase activity30“ By contrast Patel et al33reported that insulin did not regulate SIK2 phosphorylation andactivity Different from other AMPKrelated family members SIKscould be specifically activated by the sodium homeostasis2 As aresult sodium intakeinduced calcium ‚ux affected by NaCa2exchange system NCE1 could cause CaMKmediated SIK1phosphorylation and activation263435 which was argued byanother study36Liver kinase B1LKB1 protein kinase was initially identified inPeutz Jeghers syndrome PJS37 and later it has been considered amaster serinethreonine kinase involved in diverse physiologicalsuppressorfunctionsin LBK1 tumorprocesses38 Accumulating evidence has demonstrated that LKB1can phosphorylate and activate many AMPKrelated kinases ontheir Tloop Fig 1b18“ Genetically deletions of LBK1 arefrequently occurred in NSCLCs especially in KRASG12DbearingNSCLCs3940 indicating that LBK1 is a potent tumor suppressene Although previous efforts mainly devoted to the studies ofAMPK rolesrecentlydepletion of AMPKα1 or AMPKα2 could not markedly impairLKB1 tumor suppressive roles in KRASG12Ddriven NSCLC models41indicating that other substrates will play more important roles inmediating LBK1 tumor suppressor functions As such SIK1 andSIK3 have been revealed as the predominant downstream targetsof LKB1 in mediating antitumorigenesis effect in NSCLC4243While some studies provided that SIK2 underwent autophosphorylation and activation in vitro independent on the presenceof LKB17 Therefore whether other members of AMPK subfamilymediating LBK1 functions in metabolic homeostasis and tumorigenesis need more investigations especially in combination withtheir conditional KO mouse modelsSignal Transduction and Targeted Therapy 0ctriphosphate IP3Ca2“CaMK Ca2“CaMK is another important upstream regulator of SIKs in an LKB1 independent manner744 In the absence ofLKB1 there is still a residual activation of SIK1 which may be dueto the activation by CaMK4243 Phospholipase C PLC can boostCa2 ‚ux from endoplasmic reticulum ER to the cytoplasmvia inositolthus activating theCaMK which leads to the phosphorylation and activation ofSIK2 at Ser358745 PKA can also phosphorylate SIK2 at Ser358720But PKA is not involved in PLCmediated SIK2 phosphorylation atSer358 and activation7 As discussed earlier sodium mediatedSIK1 activation is also through CaMK263435 Interestingly a studyfound that CaMK IIV phosphorylated SIK2 at Thr484 leading toSIK2 degradation and promoting CREBmediated transcriptionFig 1a27receptorProtein kinase A PKA one of the members of AGC kinase is atetrameric holoenzyme composed of homodimer including twokinds of regulatory subunits RIα and RI RIIα and RII and threecatalytic subunits Cα C or Cγ4647 PKA activity depends onthe binding of cAMP with the regulatory subunits leading to therelease of active catalytic subunits and then phosphorylatingdiverse substrates46 Pathologically mutations in PKA catalyticsubunit promoted adrenal cortical tumorigenesis and Cushing™ssyndrome4849 Although PKA is not considered as an oncogenePKA has an active role in several cancers50“ and induce thetransformation of human mammary stromal cells into epithelialcells MET53 Until now all three SIK family members have beendiscovered to undergo PKAmediated phosphorylation andinhibition Bioinformatic analyses imply that SIKs contain multiplemotifs harboring PKA phosphorylation and binding sitesRSXSXP underlined phosphorylated residue X any residue202954 When these potential phosphorylation residues areabolished the binding of SIKs with is largely eliminatedwhich markedly antagonizes PKA inhibitory roles on SIKs2954Notably changes in these residues do not affect LKB1mediatedSIKs activation Biologically PKA can phosphorylate SIK1 topromote its nucleus translocation55“ which could be efficientlyblocked by mutating these two arginine residues within thephosphorylation motif56 Similarly PKA directly phosphorylatesSIK2 to regulate its stabilization and relocation by modulating itsinteraction with Meanwhile the deletion of PKA notonly promotes SIK1 protein stability but also transcriptionallyaccelerates SIK1 expression59 Hence PKA would be a criticalnegative upstream regulator of SIKs to compete with LKB1 ingoverning SIK physiological or pathological functionsOther upstream regulators Aside from phosphorylation otherposttranslational modifications PTMs such as acetylation alsoplay important roles in governing SIK activity Of note p300mediated acetylation inhibited ATP binding with and activating ofSIK2 by disturbing its phosphorylation at Thr175 conversely SIK2can also directly phosphorylate and regulate p300 acetyltransferase60 In addition HDAC6 has been identified to activate SIK2 byremoving its acetylation modification9 In addition RNF2 an E3ligase has been revealed to ubiquitinate and in turn degrade SIK1in hepatoma cells61 Consequentlythe specific regulatorymechanisms of other PTMs to SIKs need to be further exploredfor fully understanding the upstream regulation for SIKsSIK downstream substratesSimilar to AMPK in recognizing the substrate motif LMIXRKHXXSXXXLVIF6263 SIKs phosphorylates substrates containing LXFig 1c5455 A variety of metabolicRKHSTXSXXXL motifregulatorsincluding CRTC and class IIa HDACs are commonsubstrates of both AMPK and SIKs29 Importantly SIKs but AMPKscan directly phosphorylate some specific substratesincludingCRTCCREB and PPase methylesterase1 PME1 to involve inmetabolic homeostasis34Signal Transduction and Targeted Therapy The potent roles of saltinducible kinases SIKs in metabolic Sun et alHDAC Histone deacetylases functionally remove the acetylationmodification from both histone and nonhistone proteins64Among the histone deacetylases class IIa HDACs HDAC4 and are inhibitors of different transcription factors especially forMEF2 family64 All three SIK family members have emerged as newkinases for class IIa HDACs5765 SIKmediated HDAC phosphorylation promotes its binding with and facilitates its transportfrom nucleus to cytoplasm and then represses MEF2dependenttranscription5765 Therefore SIKs can regulate the development ofskeletal muscle skeleton regulatory T cells as well as leukemiaand other pathological processes via manipulating class IIa HDACsas discussed above2957596466CREBregulated transcription coactivatorcAMP response elementbinding proteincAMP response elementbinding protein CREBand CREBregulated transcription coactivator CRTC affect cellproliferation differentiation metabolism and other biologicalprocesses67 Increased CREB activity confers to tumor progressionchemotherapy resistance and reduced survival68 CREB is anotherwellestablished SIK downstream effector Although SIKs could notdirectly phosphorylate CREB they can inhibit CREB in a kinasedependent manner69 CRTC is a coactivator of CREB includingCRTC1“ and favors to stabilize CREB or directly contacts withCREB promoters70 CRTC is also helpful for the recruitment ofhistone acetyltransferase p300 for CREB transcriptional activitySIKs can directly phosphorylate CRTC block its association with and inhibit its nuclear transport where CRTC binds andenhances CREB driven gene transcription820313271 SIKs alsopromote COP1mediated CRTC1 ubiquitination and degradationby phosphorylating its multiple residues67 In keeping with thesefindings SIK2 could abrogate CRTC2 acetylation by phosphorylating p300 and integrate with the phosphorylation of CRTC2 tofacilitate COP1mediated CRTC2 ubiquitination and subsequentdegradation3272 Of note CREB could transcriptionally boost theexpression of Sik1 by binding its enhancer in mouse skeletalmuscle cells57 Thereforeit is possible that there is a negativefeedback loop between SIKs and CRTC“CREB signaling pathway to‚uence cellular malignanciesPME1Na KATPaseThe NaKATPase is widely distributedon the cell membrane and functions to transport sodium andpotassium ions and maintain the balance of osmotic pressure73The activated SIK1 phosphorylates PME1 causing its dissociationfrom the complex of PP2APME1 NaKATPase34 as a resultPP2A dephosphorylates NaKATPase and attains its catalyticactivity34 On the other hand SIKs also transcriptionally regulatethe NaKATPase either by directly inhibiting the entry of CRTCinto the nucleus to transcript ATP1B1 gene which encodes a NaKATPase subunit2 or by indirectly repressing the hormonesinduced NaKATPase expression74 via an increased CYP11Aand StAR mRNA levels to promote the adrenocorticotropichormone production5575“Other downstream signaling pathwaysTGFSmad pathwayIn normal epithelial cells TGFSmadsignaling pathway is considered to play an anticancer role byinducing cell cycle arrest and apoptosis78 However during thelate stage of tumorigenesis TGFSmad promotes cancer cell EMTand plays a pilot role in promoting cancer79 SIK1 is considered asa transcriptional substrate of TGFSmad pathway80 meanwhileactivated SIK1 may regulate the contraction phenotype of vascularsmooth muscle cells by inhibiting TGF1 signaling to prevent highsalt intakecaused hypertension81 Recent studies also indicatethat SIKs function as a negative feedback in the TGF signal byformatting the SIK1“Smad7“SMURF2 SMADspecific E3 ubiquitinprotein ligase complex to ubiquitinate ALK5 to repress TGFsignaling pathway8082 Notably high glucosemediated downregulation of SIKs results in the stabilization of ALK5 in mesangial 0cThe potent roles of saltinducible kinases SIKs in metabolic Sun et alFig The roles of SIKs in the regulation of glucose lipid metabolism and ‚ammation are illustrated SIKs™ regulatory effect is mainlythrough phosphorylating CRTC and class IIa HDACs to retain them in the cytoplasm Therefore SIKs repress various gene expression and theninhibit gluconeogenesis lipogenesis steroidogenesis and the production of IL10 Besides SIK2 upregulates GLUT4 expression by inhibitingtranscriptional repressor ATF3 leading to glucose uptake SIKs promote NFκb signaling pathway and production of ‚ammatory factors suchas IL1 IL6 IL12 TNFα and iNOS through downstream substrates CRTC However SIK1 and SIK3 inhibit the interaction of TRAF6 and TAB2and then repress NFκb signaling pathway Energy deprivation and hormone insulin glucagon and ACTH presence all control the activity ofSIKs kinase and regulate their effect on metabolismcells83 Furthermore one study suggested that SIK1 phosphorylated polarity protein partitioningdefective Par3 to promote itsdegradation via both proteasome and lysosome manners84 Arecent research also revealed that SIK inhibitors could repress theTGFmediated transcriptional capability of plasminogen activator inhibitor PAI1 and cellular apoptosis without affecting thephosphorylation or nuclear translocation of R“Smads complex85Of note this might be via SIK1 but not SIK2 or SIK3 to controlSmadassociated transcriptional cofactors via phosphorylatingCRTC85Hippo pathwayThe Hippo signaling pathway was conservativeand initially identified in drosophila which plays a major role incontrolling an size86 SIK2 and SIK3 have been proved to beupstream regulators of the Hippo signaling pathway in drosophilaMechanisticallythey can directly phosphorylate the scaffoldprotein Salvador Sav a core component of Hippo complex toprevent the oncogene driven inhibition of Yki an ortholog of Yesassociated protein YAP87 As an important hub of Hipposignaling pathway YAP activation leads to the inhibition of cellcontact and facilitates tumor cell metastasis8688 Specifically SIK2can directly trigger YkiYAP transcription activity to increase theYkiYAP target gene expression and promote tissue overgrowth87indicating the potential oncogenic role of SIK2 in tumorigenesisNFκb signaling pathway NFκb signaling pathway is one of thewellestablished ‚ammatory pathways by which SIKs couldmanipulate the production of‚ammatory factors Fig Meanwhile CRTC and class IIa HDACs two important downstreamregulate NFκb signalingsubstratespathway89“ However it is noteworthy that the effects of SIK1and SIK3 on NFκb signaling pathway seem to be distinct theyprefer to inhibit the binding of TAB2TRAF6 to repress the NFκbsignal92negativelyofSIKsPI3KAKT signaling pathwayTo date the correlation betweenSIKs and AKT signaling pathway is focused on SIK2 Fig SIK2 leads to a decrease of AKT phosphorylationwhich may be due to the SIK2mediated IRS1 phosphorylationthus inhibiting the insulin signaling pathway30 However in tumorcells the effect of SIK2 on the AKT signaling pathway seems to bechanged to elevating PI3KAKT activity7101193 MechanisticallySIK2 can directly phosphorylate p85 a regulatory subunit of PI3Kcomplex to activate the AKT kinase activity7 As a result SIKinhibitors such as ARN3236 can efficiently reduce AKT phosphorylation and activation in ovarian cancer cells10 Howeverthere is no compelling evidence proven that SIK2 could directlybind and regulate AKT kinase activity7101193 therefore the directconnection between SIKs especially the SIK1 and SIK3 and AKTneed to be further exploredBIOLOGICAL FUNCTIONS OF SIKS IN METABOLICHOMEOSTASISSIK functions in gluconeogenesisGluconeogenesis is a biological process in which noncarbohydrateprecursors including lactic acid glycerin amino acids etc aretransformed into carbohydrates including glucose or glycogenwhich can be manipulated by insulin and glucagon controlled theexpression of glucose6phosphatase G6Pase and phosphoenolpyruvate carboxykinase PEPCK293194 Specifically PEPCK andG6Pase control the initial and final steps of gluconeogenesisrespectively95 Proliferatoractivated receptor gamma coactivatorPGC1α a directtarget of CREB can largely elevate theexpression of PEPCK and G6Pase96 Meanwhile PGC1α is alsoassociated with histone acetyltransferase HAT p30097 and servesas a key regulator in liver gluconeogenesis and the focal target ofcAMPPKACREB axis98 On the other hand insulin can block theeffect of PGC1α and interfere with the activation of gluconeogenesis through AKTmediated forkhead box FoxO1 phosphorylation99 Furthermore insulin can also regulate the activity of PGC1α by governing its acetylation and phosphorylation100101SIKs and their substrates such as CRTC and class IIa HDAC arelargely involved in gluconeogenesis Fig SIK1 was firstfound to inhibit gluconeogenesis in the hepatocytes and itsmRNA and protein levels under fasting conditions increasedSignal Transduction and Targeted Therapy 0cfourfold relative to feeding conditions31 Moreover overexpression of SIK1 in primary hepatocytes suppressed forskolin or cAMPinduced an increase in Pck1 gene expression via phosphorylationof CRTC231 Subsequent studies have proven that SIK2 and SIK3have a similar effect3233102 Dentin et al reported that SIK2 is adownstream substrate of PI3KAKT signaling pathway response toinsulin subsequently followed by CRTC2 phosphorylation32 Itohet al102 showed that SIK3 knockout in hepatocytes was associatedwith elevated mRNA of Pgc1a Pepck and G6pc gene Collectivelyall three SIK isoforms can inhibit gluconeogenesis possibly via SIKmediated CRTC phosphorylation and restriction in the cytoplasm2054 CRTC played a key role in gluconeogenic by bindingCREB to transcriptionally promote gluconeogenic genes expression such as G6PC PEPCK1 and PGC1α gene29103 By contrast inthe case of starvation glucagon can also inhibit the catalyticactivity of SIKs via PKAmediated phosphorylation and facilitategluconeogenesis29 On the other hand SIKs directly phosphorylated class IIa HDACs to block their nuclear translocation2029 andinteraction with FoxO1 on PEPCK and G6Pase promoter regionsthereby stimulating gluconeogenesis104 Conceivably SIK inhibitors could compromise the phosphorylation of CRTC23 andHDAC45leading to gluconeogenic gene expression andglucose production203354 As such lossoffunction mutations ofSIKs or deficiency ofantagonizegluconeogenesis202933could efficientlyLKB1While SIKs have markedly involved in diverse signaling pathways to regulate gluconeogenesis several studies demonstratedthat SIK1 and SIK2 did not impact gluconeogenesis alone inmouse model33105 Of note in liver specific Sik1 and Sik2 doubleKO mice CRTC phosphorylation and gluconeogenesis were not‚uenced33105 instead SIK3 plays a key role in regulatinggluconeogenesis rather than SIK1 and SIK2102 Under the conditions of lactateinduced gluconeogenesis the blood glucose levelof Sik3 but not Sik1 and Sik2 KO mice were rapidly increased thanthat of WT mice indicating that Sik3 plays an important role inmouse gluconeogenesis66102 Though SIKs display a controversialrole in gluconeogenesisit is generally accepted that SIKs canreduce insulin sensitivity and promote energy storage byinhibiting gluconeogenesisareThese proteinsSIK functions in glucose uptakeThe process of glucose uptake mainly depends on the expressionof sodiumdependent glucose transporter and glucose transporterGLUT106 The majority of peripheral glucose uptake in adiposetissue and skeletal muscle are achieved by insulinresponsiveglucose transporter GLUT4106 Importantly GLUT4 expressionhas been negatively regulated by various upstream regulatorsincluding but notlimited to HDAC4 CRTC23 and proteinphosphatase 2A PP2A107all wellestablished SIK2 downstream substrates indicating that SIK2 is apositive regulator of glucose intake by upregulating GLUT4expression Fig “ Meanwhile CREB upregulated theexpression of transcriptional repressor activating transcriptionfactor ATF3 and thereby downregulated the GLUT4 resultingin promoting insulin resistance107110 Consistently inactivating SIKpharmacologically or genetically could reduce GLUT4 expressionand glucose uptake107“ However SIK1 promotes insulinresistance and inhibits glucose uptake in skeletal muscle possiblyvia directly phosphorylating insulin receptor substrate IRS Sik1 KO did not lead to hyperglycemia and gluconeogenesissignificantly improved glucose toleranceperipheral insulin sensitivity and skeletal muscle glucose uptakeupon highfat diet due to elevated expression of GLUT4 GLUT1and GLUT12105in vivo butSIK functions in lipid metabolismIn addition to its role in glucose metabolism SIKs also seems tofunction as a negative regulator of lipid metabolism Fig LipidSignal Transduction and Targeted Therapy The potent roles of saltinducible kinases SIKs in metabolic Sun et alis an important source of energy and substance for cell homeostasis and its metabolic process is tightly regulated by a complexnetwork112 The fatty acid a vital and raw materialfortriglycerides112 is synthesized mainly by two key enzymesacetylCoA carboxylase ACC and fatty acid synthase FAS112SIK1 represses lipogenic gene expression such as Acaca acetylCoA carboxylase Fasn FAS Srebf1 sterol regulatory elementbinding transcription factor and Scd1 stearoylCoA desaturase possibly via an SREBPs sterol regulatory elementbindingproteinmediated transcriptional regulation113 Overexpression ofSIK1 in hepatocytes induced high mRNA levels of the lipogenicgene Srebf1 Fasn and Scd1 and high protein levels of ACC andFAS113 SREBP1c is directly phosphorylated by SIK1 at Ser329which is proposed to be required for SIK1 in repressing lipogenicgene expression113 Steroids are another kind of lipid includingestrogen progesterone and adrenocorticotropic hormone77114Steroidogenic acute regulatory protein StAR and cytochromeP450 cholesterol side chain cleavage P450scc are two keyenzymes in steroidogenesis77114 StAR regulates the transport ofcholesterol from the outer membrane to the inner membrane inmitochondria which is the key ratelimiting step of steroidsynthesis114 In addition CYP11A gene encodes P450scc acholesterol side chain cleavage enzyme that catalyzes theconversion of cholesterolto pregnenolone a precursor ofsteroid77 SIK1 plays a key role in steroidogenesis and adipogenesis mediated by governing ACTH signaling pathway2255115 ThemRNA levels of SIK1 in mouse adrenal cortex cells Y1 cellsstimulated by ACTH peaked rapidly within h then decreasedgradually and returned to the basic level after h However themRNA levels of P450scc and StAR began to rise after a few hoursreaching the highest levels after h115 The transcription of SIK1occurred before the ACTH stimulated StAR and P450scc transcription so it can be speculated that SIK1 is associated withsteroidogenic gene expression115 On the other hand SIK1overexpression significantlyrepressed the ACTHdependentexpression of P450scc and StAR in Y1 cells115 Subsequent studiesdemonstrated that SIK1 repressed the efficient operation of theCREB transcription activation complex thereby inhibiting the CREdriven transcription of the CYP11A gene and the StAR gene inY1 cells5575“Du et al116 found that similar to SIK1 SIK2 can also repress theexpression of lipogenic genes FAS ACC2 and SCD1 and thiseffect can be reversed by depleting SREBP1In addition SIK2promotes fatty acid synthesis by upregulating SREBP1c expression thus promoting the transcription of Fasn in ovarian cancercells93 Meantime SIK2 also promotes cholesterol synthesis byupregulating SREBP2 expressionto transcriptionally elevatecholesterol synthetase 3hydroxy3methylglutarylcoenzyme Areductase HMGCR93Importantly SIK2 phosphorylated andinhibited p300 activity leading to the decreased acetylation ofcarbohydrate response elementbinding protein ChREBP whichplays a positive role in lipogenic and gluconeogenesis60 SIK2 alsophosphorylated IRS1 to attenuate insulin driven lipogenesis inhuman adipocytes55 Another study also showed depletion of SIK2promoted increased adipogenic potential and insulin resistance inpreadipocytes in a CRTC2dependent manner110 SIK2 controlledFAO in liver and skeletal muscle as such Sik2 KO mice displayedthe decreased key enzymes in the process of FAO such ascarnitine carnitine palmitoyltransferase CPT1 mitochondrialmedium chain acyl COA dehydrogenase MCAD and peroxisomalacylCoA oxidase ACOX1110 Inconsistently SIK2 promotes FAOby phosphorylating ACC1 and inhibiting CPT1A in ovarian cancercells resulting in promoting abdominal metastasis7 In additionsome studies showed that Sik2 KO mice do not impact lipidmetabolism in vivo33SIK3 has been reported as a new energy regulator by promotinglipid storage in Drosophila through compromising the activity ofHDAC4 and CRTC20117 SIK3 also regulated cholesterol and bile 0cThe potent roles of saltinducible kinases SIKs in metabolic Sun et alTable The summary of SIK mouse modelsSIK members Mouse model typesFunctional characteristicsSIK1SIK1SIK1SIK1SIK1SIK2SIK2SIK2SIK2SIK3SIK3SIK3SIK3SIK3SIK3Sik1flflGDF9Cre miceSik1flflTBGCre miceSik1flflMyf5Cre miceSik1ˆ’ˆ’ mice generated from Sik1 KO ES cellsKSik1 KrasLSLG12DR26LSLluclucSik1flfl miceKTH11LSLCas9 KrasLSLG12DR26LSLTomatoH11LSLCas9 miceSik1ˆ’ˆ’ miceSik2ˆ’ˆ’ mice Sik2lacZSik2flflCreˆ’ miceSik2ˆ’ˆ’ mice generated from Sik2 KO ES cellsSik2ˆ’ˆ’ mice generated from Sik2 KO ES cellsSik3ˆ’ˆ’ mice generated from Sik3 KO ES cellsSik3ˆ’ˆ’ mice generated from Sik3 KO ES cellsSik3flflCol11α211EnhCre and Col11α2ERCre miceSik3ˆ’ˆ’ mice generated from Sik3 KO ES cellsSik3ˆ’ˆ’ mice generated from Sik3 KO ES cellsSIK1T182A SIK2T175A and SIK3T163A single anddouble KI mice created by ES cells gene targetingtechnologiesAbnormal glucose metabolismElevated insulin secretion and more osteogenic potentialIncreased tumor size and burdenIncreased tumor sizeHigh blood pressureHyperglycemia and hypertriglyceridemianormal glycemiaEnhanced neuronal survivalPreventing left ventricular hypertrophyDystrophic including lipodystrophy hypolipidemia hypoglycemiaand hyperinsulinemia with cholestasis and cholelithiasisphenotypeDwarfism and skeletal deformitiesAchondroplasia and resistance to the osteoarthritisAbnormal circadian rhythmsPro‚ammatory phenotypeAnti‚ammatory phenotypeReferencesacid metabolism by combining with retinoic acid metabolism andmight alter energy storage in mice118 Inhibition of fatty acidsynthesis was observed in Sik3 KO mice118 however the roles ofSIK3 in regulating lipid metabolism are not good evaluated inmammal animals20 Based on these observations although SIKshave been considered to play important roles in lipid metabolismthe mechanism of SIKs regulating lipid metabolism has not beenwell elucidated yet Thus more efforts are desired in the future toexplore the diverse and distinct roles of SIK family members inlipid metabolic homeostasisSIK functions in ‚ammationInflammation is an important pathological change tightly relatedto tumorigenesis Inflammation predominantly changes the tumormicroenvironment and accelerates tumor occurrence growth andmetastasis119 An important aspect of controlling ‚ammation isreprogramming macrophages to promote transformation fromclassic activated macrophage M1 macrophage to regulatorymacrophage M2 macrophage120 Of note SIKs act as molecularin regulating M1“M2 macrophage transformationswitchesFig The observation that SIK inhibitors compromisedCRTC3 phosphorylation in TLRstimulated macrophagesled toincreased CREBdependent gene expression including IL10 andreduced pro‚ammatory cytokine expression such as TNFα andIL6871 Importantly IL10 drives an anti‚ammatory function bypromoting the expression ofregulatory M2b macrophagemarkers such as SPHK1 LIGHT and Arg18120 Similar results werealso observed in dendritic cells DCs71 On the other hand SIKinhibitors decreased the production of pro‚ammatory cytokines but not IL10 in IL1mediated macrophages possibly dueto the insufficient CRTC3 phosphorylation71 Moreover otherupstream regulators for example prostaglandin E2 PGE2 alsoinduced IL10 production via the PKA“SIK“CRTC signaling pathway in the quiescent

Thyroid_Cancer

Genetic alterations in the 3q263132 locus conferan aggressive prostate cancer phenotypeBenjamin S SimpsonSusan Heavey Jason Pitt5 Caroline M Moore6 Hayley C Whitakerœ‰ Niedzica Camacho234 Hayley J Luxton Hayley Pye Ron Finn1Largescale genetic aberrations that underpin prostate cancer development and progressionsuch as copynumber alterations CNAs have been described but the consequences ofspecific changes in many identified loci is limited Germline SNPs in the 3q2631 locus areassociated with aggressive prostate cancer and is the location of NAALADL2 a gene overexpressed in aggressive disease The closest gene to NAALADL2 is TBL1XR1 which is implicated in tumour development and progression Using publiclyavailable cancer genomic datawe report that NAALADL2 and TBL1XR1 gainsamplifications are more prevalent in aggressivesubtypes of prostate cancer when compared to primary cohorts In primary disease gainsamplifications occurred in CI“ and CI“ for NAALADL2 and TBL1XR1 respectively increasing in frequency in higherGleason grade and stage tumours Gainsamplifications result in transcriptional changes andthe development of a proproliferative and aggressive phenotype These results support apivotal role for copynumber gains in this genetic region Molecular Diagnostics and Therapeutics Group Research Department of Targeted Intervention Division of Surgery Interventional Science UniversityCollege London London UK Human Oncology and Pathogenesis Program Memorial Sloan Kettering Cancer Center New York NY USA MarieJoseand Henry R Kravis Center for Molecular Oncology Memorial Sloan Kettering Cancer Center New York NY USA Department of Pathology MemorialSloan Kettering Cancer Center New York New York for Genomics Research Discovery Sciences Biopharmaceutical RD AstraZeneca Cambridge UK Cancer Institute of Singapore National University of Singapore Singapore Singapore Department of Urology UCLH NHS Foundation Trust London UKœ‰email HayleyWhitakeruclacukCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xProstate cancer PCa is the most common noncutaneouscancer in developed countries12 and is defined by dynamicgenome alterations and both its pathological and geneticheterogeneity3 An important pathological predictor of prostatecancer aggressiveness is Gleason grade used to assess risk ofprogression and stratify patients for treatment however theunderlying genomic changes which accompany more aggressivetumours remains incompletely definedOverall copynumber alteration CNA burden has been linkedto poorer prognosis in prostate cancer associating with Gleasongrade biochemical recurrence and prostate cancer specific deathhowever the exact mechanism driving these prognostic changes isunknown and thought to be primarily driven by general chromosomal instability4“ Changes in specific loci have also beenlinked to aggressiveness in particular gains in proliferative geneseg MYC 8q24 and loss of tumour suppressors PTEN 10q23and NKX31 8p2178 Many genetic alterations have been linkedwith prostate cancer such as point mutations in SPOP FOXA1and IDH19 Largescale oncogenic structural rearrangementstranslocations and copynumber changes are also common oftenleading to the coordinated dysregulation of multiple elements forexample the loss of 21q which is associated with the TMPRSSERG fusion rearrangement and the subsequent rearrangement ofSMAD410 Improved understanding of the mechanisms governing disease pathogenesis and progression may allow for bettertherapeutic exploitation for example genetic alterations in theDNA repair machinery have been linked to susceptibility toPARP inhibitors in a range of tumour types and alterations in ARconfer sensitivity or resistance to androgen deprivation therapy inmetastatic castrate resistant prostate cancer mCRPC11NAALADL2 is located on 3q2631 and is a member of theglutamate carboxypeptidase II family along with the widely studied PCa marker PSMA NAALAD112 and its expression haspreviously been associated with prostate tumour stage andgrade13 with expression predicting poor survival following radicalprostatectomy13 A large genomewide association study GWASof prostate cancer cases found rs78943174 a SNP withinthe 3q2631 NAALADL2 locus was associated with high Gleasonsum score14 A further rs10936845 SNP was identified within aGATA2 motif that increases NAALADL2 expression in prostatecancer patients where increased expression also predicted biochemical reccurence15 The same study showed even higherbinding preference to HOXB13 and FOXA1 to this site suggestingcooccupancy by these important transcription factors both ofwhich have been shown to be involved in AR cistromereprogramming1516functionsAdjacent to NAALADL2 in the genome is TBL1XR1 a corecomponent of nuclear receptor corepressor NCoR complex thatacts as a coregulator of nuclear receptors ‚uencing severalcellularand‚ammation17 TBL1XR1 is also an androgen receptor AR coactivator18 Expression of TBL1XR1 has been associated withpoor prognosis in several cancers predicting poor overall survivaland lymph node metastasis in gastric19 and ovarian cancers20 andrecurrence in colorectal21 breast22 and liver cancers23antiapoptosisincludinggrowthHere we utilise largescale publicly available genomic data tobetter characterise the broad somatic copynumber changesoccurring within the 3q263132 locus particularly centredaround gainsamplifications in NAALADL2 and TBL1XR1 andlinking them to the clinical characteristics of aggressive prostatecancerResults3q263132 gain frequency is increased in aggressive PCaCopynumber alterations often alter the expression of the gene inwhich they occur with gene dosage known to correlate withmRNA expression Genetic structural variants are also known toalter transcriptional regulation by altering cisregulatory elementssuch as promotors and enhancers resulting in differentialexpression2425 Increased NAALADL2 and TBL1XR1 expressionhave previously been linked to poor prognosis in cancers leadingus to examine the frequency of somatic copynumber gains inthese genes across various prostate cancer subtypes19“Alteration frequency was assessed using data from cBioportalFig 1a and all study data was processed using a standardisedpipeline to ensure comparable results Alteration frequency wasassessed in a total of patients samples in nonoverlapping studies Appendix eleven studies focused onprimary prostate cancer four on metastatic prostate cancer andone on neuroendocrine and castrateresistant cancers Significantcopynumber increases above a derived background thresholdwere categorised as ˜gains™ and copynumber decreases as ˜deletions™ Overall the distribution of NAALADL2 and TBL1XR1alterations were significantly different between disease subtypesto that which is expected Chisquared goodnessoffittestFig Somatic alteration frequency of NAALADL2 and TBL1XR1 across prostate cancer subtypes in publically available genomic studies n a NAALADL2 genetic alteration frequency across different subtypes of prostate cancer b TBL1XR1 genetic alteration frequency across differentsubtypes of prostate cancer P primary prostate cancer M metastatic prostate cancer NE neuroendocrine prostate cancer and castrate resistantprostate cancer CRPC All annotations were assigned using Genome Nexus and CNAs are called using GISTIC or RAE algorithms Pvalues show theresults of a Chisquared goodnessoffit test to determine if the number of observed patients with each alteration type is different from that which isexpected across each cancer subtype Results detailed in Supplementary data COMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xp — ˆ’ and p — ˆ’ Fig 1a b with gains beingmost frequent in castrateresistant prostate cancer and respectively followed by neuroendocrine and metastatic and then primary prostatecancer and Fig 1a b3q263132 gains extend across an oncogenerich region ofChr3 As CNA™s are known to associate with more aggressivesubtypes of prostate cancer we investigated their association withclinical characteristics to establish if changes can be detected earlyin the life history of cancer predicting more aggressive diseaseWe utilised copynumber data from primary an confineddisease from both the UK and Canadian International CancerGenome Consortium ICGC cohorts and The Cancer GenomeAtlas TCGA These studies use intermediatehigh risk prostatecancer patients with no treatment prior to radical prostatectomyTo allow comparisons between the studies data were reanalysedusing the Genomic Identification of Significant Targets in Cancer GISTIC2 methodfavoured by the broad institute andTCGA27 as it distinguishes between lowlevel copy numberincreases gains and highlevel copynumber increases amplifications Within the three cohorts we found that copynumbergains across both genes were frequent with gains in NAALADL2ranging from Canada to UK and between“ for amplifications Table TBL1XR1 had an almostidentical CNA frequency of between UK to Canada Table We fitted a randomeffects model to more accurately estimatethe frequency of NAALADL2 and TBL1XR1 gainamplificationscombining the data from all three cohorts which estimated thetrue frequencies to be CI “ and CI “ for NAALADL2 and TBL1XR1 respectively Supplementary Fig Leaveone out analysis and adiagnostic plots revealed that the ICGC Canada study was asignificant source of heterogeneity thereforethe study wasremoved and the model refit The final estimated frequency ofNAALADL2 and TBL1XR1 gainsamplifications was CI “ and CI “ respectivelyin primary prostate cancerDue to their close proximity in the genome we investigated ifgainsamplifications in NAALADL2 and TBL1XR1 cooccurred inthe same patients using a genomewide Fisher™s exact test with afalse discovery rate correction NAALADL2 and TBL1XR1significantly coamplified in all three cohorts ICGC UK p — ˆ’ ICGC Canada p 158e — ˆ’ and TCGA p — ˆ’testingconfirmed that widespanning gainsamplifications occurred inneighbouring regions in the majority of patients In the ICGC UKSupplementary Fig Additionallycohort n there was a significant cooccurrence of somaticcopynumber gainsamplifications in NAALADL2 with TBL1XR1FDRcorrected Fisher™s exact test — ˆ’ Fig 2a Gainsin this region also significantly correlated with two regionsspanning chromosomes and both gains previously describedas being abundant in prostate cancer Supplementary Data The Canadian cohort n showed a similar pattern of cooccurrence with gainsamplifications spanning the region surrounding NAALADL2 and TBL1XR1 3p253 to 3q29 Fig 2bThere was also a significant cooccurrence with gains in thebeginning of chromosome as well as some sporadic cooccurrence across the genome Fig 2b Supplementary Data These results were supported by the outcome of the same analysisin the TCGA cohort n although several large spikes ofcooccurrence were also observed in regions nottoNAALADL2 and TBL1XR1 as these spikes were not present inthe other two cohorts they most likely represent artefacts Fig 2cSupplementary Data Overall across the three cohorts therewere was a consistent coamplification in region spanning genes between 3p141 and 3q29 While a number of patients hadmultiple CNAs we found no consistent cooccurrence withcommon CNAs such as MYC gain FGFR1 gain PTEN loss RB1loss or NKX31 loss FDRcorrected Fisher™s exact test p The 3q26 region where NAALADL2 and TBL1XR1 are locatedis rich in oncogenes such as PIK3CA SOX2 ECT2 and PRKCIwhich may act to drive tumorigenesis29 We determined thenumber of known oncogenes within this defined region bycomparing the overlapping genes that coamplified withNAALADL2 and TBL1XR1 in alltheNetwork of Cancer Genes database30 This revealed that of genes are known oncogenes including BCL6 ATRand PI3K family members Supplementary Data These resultsconfirm that a high proportion of prostate cancer patientsdevelop large copynumber gains across multiple oncogenes inthis genetic regionthree cohorts againstlocalGains in 3q263132 associate with adverse clinical featuresCommon prostate cancer CNAs such as those in MYC andPTEN are known to associate with higher Gleason grade31Consistent with these findings we also found NAALADL2 andTBL1XR1 amplifications were highly correlated with GradeGroup GG showing that the frequency of NAALADL2 andTBL1XR1 gains tripling between GG1 and GG2 lesions and morethan doubling between GG2 and Table A Chisquaredgoodnessoffit test showed that the distribution of gainsamplifications between Grade groups was significantly different to thedistribution of diploid patientsfor both NAALADL2 andTBL1XR1 p — ˆ’ and p — ˆ’ WhenTable Alteration frequency of NAALADL2 and TBL1XR1 called via the GISTIC2 method in three nonoverlapping primary anconfined radical prostatectomy cohorts from the International Cancer Genome Consortium ICGC and The Cancer GenomeAtlas TCGAICGC UKICGC CANADATCGANAALADL2TBL1XR1NAALADL2TBL1XR1NAALADL2TBL1XR1AlterationDeep DelShallow DelDiploidGainAmplificationTotalnnnnnnThe degree of copy number alteration is discretised into five categories amplification gain representing low and high level copy number increase diploid no significant CNA and shallow and deepdeletion representing low and high level copy number lossCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xand compared to those without and Moreover ofthe patients who had their lymph nodesexamined the percentage of patients with lymph node positivitydefined through positivity on haematoxylin and eosin stainingHE was more than double in patients with NAALADL2 orTBL1XR1 gains and compared to those withouta gain and Chisquared goodnessoffit test p and p Finally while only one man in the cohorthad evidence of positive findings in his bone scan we did observea significant between the number of equivocal bone scans inpatients with gains and compared to and in those patients without gainsChisquaredgoodnessoffit test p and p for NAALADL2and TBL1XR1 respectively however the number of expectedcases in each of these categories was less than adding someuncertainty to this result We found no significant difference inthe mean age between patients with different copynumbers ofNAALADL2 or TBL1XR1 KruskallWallis rank sum test p and As gainsamplifications in NAALADL2 and TBL1XR1 coincidewith a cluster of known oncogenes and coincide with clinicalvariables linked to more aggressive disease we also compareddiseasefree survival Comparing patients with gainsamplifications in NAALADL2 and TBLXR1 to those with diploid copies weobserved no significant association in the ICGC UK cohort n although there was a trend towards reduced diseasefreesurvival Supplementary Fig 4A In the larger TCGA cohortn there was a significant reduction in diseasefree survivalin patients with a gain in either NAALADL2 Logrank MantelCox p or TBL1XR1 Logrank MantelCox p Supplementary Fig 4BUnivariable Cox regression confirmed that carrying a gainamplification in NAALADL2 and TBL1XR1 in the TCGA cohortresulted in reduction in diseasefree survival hazard ratio HR CI “ p Forreference weperformed a similar analysis of patients with PTEN deletion orMYC gains two common copy number alterations with provenin prostate cancer3233association with diseasefree survivalWhen patients were stratified solely by CNA status and survivalcompared using the KaplanMeier method those patients withMYC gain or PTEN deletion homo or hemizygous showed nosignificant difference in diseasefree survival Logrank MantelCox p and p respectively while those stratified byNAALADL2 gain TBL1XR1 gain or both NAALADL2 andTBL1XR1 gain showed significant differences in survival Logrank MantelCox p Supplementary Fig 5A“E Univariable Cox regression estimated the hazard ratios for thesecopynumber alterations as CI “ CI “ and CI “ for MYCPTEN and NAALADL2TBL1XR1 respectively We also comparedthe diseasefree survival of patients with only a copynumberalteration in each of the four genes where each group wasmutually exclusive Supplementary Fig 5F G This showed thaton the whole patients with CNAs in NAALADL2TBL1XR1 hadreduced or equal diseasefree survival as those with either onlyMYC gain or only PTEN loss Patients with copy number gains inboth had a worse prognosis All clinical data is available inSupplementary Data Fig Genomewide cooccurrence with NAALADL2 and TBL1XR1 gainsamplifications The Y axis shows “log10 qvalues from a Fishers exact testbetween gainamplifications in NAALADL2 and cooccuring genes Thedotted line represents the threshold for statistical significance aftercorrection for multiple testing a Significantly cooccurring gains across thegenome in the ICGC UK cohort b Significantly cooccurring gains acrossthe genome in the ICGC Canada cohort c Significantly cooccurring gainsacross the genome in the TCGA cohort NAALADL2 and TBL1XR1 cytobandpositions are labelled All Fisher tests use NAALADL2 gain or amplificationas the altered group Full results are detailed in Supplementary Data compared to common CNAs such as PTEN loss and MYC gainthe alteration frequency of NAALADL2 and TBL1XR1 was morecorrelated with higher Gleason grade groups Spearmans rho was p p for NAALADL2 and TBL1XR1and p for PTEN and MYC respectivelySupplementary Fig 3AMoreover we also noted the same pattern ofincreasingfrequency of gains with T stage Chisquared goodnessoffit testp and p respectively Table Patients with gains exhibited differences in the location of thetumour within the prostate with and of thosewith NAALADL2 and TBL1XR1 gains having tumoursinoverlapping and multiple zones compared to just and for those without gains Chisquared goodnessoffit testp and p There was also an increased relativenumber of positive surgical margins Chisquared goodnessoffittest p and p in patients with gains As CNAs in NAALADL2 and TBL1XR1 were associated withclinical characteristics such as Gleason grade group and T stagewe used multivariable Cox regression models to confirm that anychanges in survival were driven by these associations and foundthat copy number gains in NAALADL2 and TBL1XR1 were nolonger significant once corrected for Gleason grade and T stagep Supplementary Data These results suggest thatthe differences in diseasefree survival seen when stratified byCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xTable The frequency of NAALADL2 and TBL1XR1 gainamplifications by Gleason Grade Group in the TCGA cohortGrade groupGG1ObservedExpected within GGGG2ObservedExpected within GGGG3ObservedExpected within GGGG4ObservedExpected within GGGG5ObservedExpected within GGTotal nNAALADL2DiploidGainTBL1XR1DiploidGainTotalGrade groups defined as Grade Group Gleason score ‰¤ Grade Group Gleason score Grade Group Gleason score Grade Group Gleason score Grade Group Gleason scores and Displayed are the numbers of patients observed with gain or without diploid a gainamplification in this region in each Grade Group Additionally the expected number ofpatients estimated to be within each category is also shown along with the percentage of each Grade Group which is made up by patients with or without a gain Bold values indicate the overallpercentage of the group with a given copynumber state All clinical data detailed in Supplementary Data Table The frequency of NAALADL2 and TBL1XR1 gainamplifications by T stage in the TCGA cohortT stageT2aObservedExpected within T stageT2bObservedExpected within T stageT2cObservedExpected within T stageT3aObservedExpected within T stageT3bObservedExpected within T stageT4ObservedExpected within T stageTotal nNAALADL2DiploidGainTBL1XR1DiploidGainTotalDisplayed are the numbers of patients observed with gain or without diploid a gainamplification in this region in each T stage Additionally the expected number of patients estimated to be withineach category is also shown along with the percentage of each T stage which is made up by patients with or without a gain Bold values indicate the overall percentage of the group with a given copynumber state All clinical data detailed in Supplementary Data COMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xgainamplification status are driven by strong association withthese clinical variablesIn the ICGC cohortsindividuals with somatic singlebasealterations in NAALADL2 also associated with reduced diseasefree survival in a combined ICGC cohort as well as associatingwith reduced diseasefree and overall survival in an early onsetprostate cancer cohort ICGC EOPC Denmark Singlebasesubstitutions in TBL1XR1 were only associated with diseasefreesurvival in the ICGC EOPC cohort Supplementary Fig Singlebase alterations did not occur with a frequency greater than onein any single base in NAALADL2 or TBL1XR13q263132 gains cooccur with proproliferative transcriptionTo determine the potentialfunctional consequences of gainswithin the NAALADL2 and TBL1XR1 amplicon mRNA expression profiles were explored using the TCGA RNAseq dataDESeq2 was used to determine differentially expressed genesbetween patients with copynumber gains for both NAALADL2and TBL1XR1 compared to those without For NAALADL2 therewere differentially expressed genes DEGs and DEGsfor TBL1XR1 when the two groups were compared FDR Supplementary Data Our previous study on NAALADL2identified nine genes which were reciprocally regulated by overexpression or knockdown of NAALADL226 Of these nine wefound that three cancer antigen XAGE1B adhesionmotiliy regulator SPON2 and AR regulator HN1 were significantly differentially expressed p in patients with a NAALADL2 gainand in the same direction as the overexpression model2634“When comparing the DEGs between patients with and withoutin either NAALADL2 or TBL1XR1 wegainsamplificationsobserved that of the DEGs overlapped between NAALADL2 and TBL1XR1 Fig 3a of the geneswere located within the locus we identified as coamplified withNAALADL2 and TBL1XR1 and were differentially expressedconsistent with a mechanism of selfregulating expression2425TBL1XR1 was one ofsignificant overlapping DEGsNAALADL2 was just on the boundary of statistical significanceFDR corrected Wald test p Supplementary Fig theNAALADL2 has been shown to be coexpressed with numberof androgen regulated proteins and contains a number of ARbinding sites and TBL1XR1 is an AR coactivator and may beinvolved in AR cistrome reprogramming18263738 We thereforelooked at overlap between androgen regulated genes with ARbinding sites full or partial and genes demonstrated to beandrogen regulated following R1881 stimulation in at least twoindependent studies3739 shared genes were differentiallyexpressed in patients with NAALADL2 and TBL1XR1 gainsamplifications that contained AR binding sites and demonstratedandrogen regulation by R1881 genes had either aAR binding motif were androgen regulated in two or morestudies or both Fig 3bOf the overlapping DEGs a total of were knownoncogenesSupplementary Data which may drive anaggressive clinical phenotype Of note was PI3K family membersPIK3C2G PIK3CA PIK3CB PIK3R4 Mucin family membersMUC1 MUC4 and MUC6 and other prostate cancer associatedgenes such as SMAD4 SOX9 and SPOP794041 Additionallyseveral genes which form commercial prognostic assays were alsodifferentially expressed such as the Decipher assay NFIB LASP1ZWILCH THBS2 COL1A2 and COL5A142 Oncotype DX assaySFRP4 COL1A1 KLK2 TPX24344 and the Prolaris assayASPM BUB1B CENPF and FOXM145We inspected of the top most significant shared DEGs usingunsupervised hierarchal clustering Fig 3b SupplementaryData DEGs mostly displayed upregulation consistent with agenedosage effect Fig 3b24 Enrichment for biological processes was assessed by Geneset enrichment analysis GSEA forNAALADL2 and TBL1XR1 gainsseparately and by overrepresentation analysis ORA on the shared DEG list usingWebGestalt46GSEA on the individual lists of DEGs showed that despite a largeoverlap the enriched biological processes did differ between the twogenes patients with a gain in NAALADL2 showed enrichment inprocesses related to NADH dehydrogenase complex assemblyFDR mitochondrial respiratory chain complex assemblyFDR translational initiation FDR cytochromecomplex assembly FDR protein localisation toendoplasmic reticulum FDR and cytoplasmic translationFDR Supplementary Data Patients with a gain inTBL1XR1 showed enrichment in mitotic cell cycle phase transitionchromosome segregation actin filamentbased movement microtubule cytoskeleton anisation involved in mitosis regulation ofcell cycle phase transition cell cycle G1S phase transition FDR as well as a number of other processes SupplementaryData To understand the combined effect of gainsamplification inthese genes we investigated overrepresentation of processes in theDEGs which were common to both NAALADL2 and TBL1XR1In the shared DEG list the significantly enriched Gene OntologyGO biological processes were all involved in the cell cycle cyclepathway including mitotic regulation and chromosome segregation Fig 3c Supplementary Data These findings support ahypothesis whereby gains in NAALADL2 and TBL1XR1 concomitantly bring about mRNA expression changes which supportan aggressive proproliferative phenotype in primary prostatecancerDiscussionIn this study we present evidence that somatic copynumber gainsin NAALADL2 and TBL1XR1 are more frequent in high gradeand aggressive forms of prostate cancer These results are bolstered by studies which have identified CNAs in this region inmCRPC however to our knowledge this is the first time thesegains have been reported in neuroendocrine disease47 We alsodemonstrate that NAALADL2 and TBL1XR1 gains occur in anearlier setting cooccurring with gains in neighbouring genes Amajor barrier to the adoption of CNA based tests in the clinic isthe reliance on expensive NGS approaches as well as sufficientsequencing depth and coverage to assess overall copynumberburden The discovery of smaller clinically significant loci couldallow for cheaper quicker targeted approaches particularly if asingle loci can elude to gainsamplifications in a larger regionsurvivalto diseasefreeIn primary prostate cancer Gainsamplifications in this regionassociated with Gleason grade tumour stage number of positivelymph nodes bone scan results and as these variables contributetostratified byNAALADL2TBL1XR1 status also have altered diseasefree survival times Our work is supported by previous studies that haveeluded to the clinical significance of this locus particularly asgermline SNPs within this locus have been associated with higherGleason grade tumours and more aggressive disease14 This alsosupports smaller studies such as those by HeselmeyerHaddadet al who identified two out of seven patients with gains inTBL1XR1 in recurrent prostate cancer48 However these studiesinvestigated these genes in isolation na¯ve to the larger context inwhich these alterations occur Here we have found that gainsamplifications atthis locus not only coamplify with otherdescribed oncogenes but associate with much larger transcriptional changes which are consistent with the observed aggressiveclinical phenotypetimepatientsCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xFig Transcriptomic changes in patients with NAALADL2TBL1XR1 gains a Venn diagram showing the number and percentage of overlapping DEGsbetween patients with NAALADL2 gainamplification and TBL1XR1 gainamplification overlap b Venn diagram showing the number ofNAALADL2 and TBL1XR1 DEGs and genes with identified AR binding sites determined through ChIPSeq and AR knockdown and genes shown to beandrogen regulated following R1881 stimulation c Unsupervised hierarchal clustering of the top most significant DEGs bar beneath upper dendrogramshows copynumber status of patients where red is patients with a gain in both NAALADL2 and TBL1XR1 and grey represents those without gainamplification in these genes Heatmap represents meancentred z scores derived from RKPM values d Chord diagram showing significantly overrepresented GO biological processes and key genes within these processes All clinical data detailed in Supplementary DataCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xOverall changes in copynumber burden have been shown to beindicative of genetically unstable tumours and predict prostatecancer relapse5 Many single CNAs have already been describedthat predict PSA recurrence after radical prostatectomy includingPTEN loss cooccurrence of PTEN FAS 10q2331 and PAPSS210q232“10q2331 loss a loss of 16q with or without a loss ofPTEN a loss within 6q 13q gains in MYC 11q1317 7q and aconcurrent loss of 8p22 with a gain of 8q2487“ Compared towellknown CNAs such as PTEN loss and MYC amplification wehave observed that Gainsamplifications in NAALADL2TBL1XR1equally or better segregate patients who will have reduced diseasefree survivalThe gainsamplifications in NAALADL2TBL1XR1 also corresponded to a significantincrease in both NAALADL2 andTBL1XR1 mRNA supporting previous studies that have described upregulation of these genes and linked them to poor prognosis in various cancers19“ This suggests that gains in thesegenes may cause increased expression of NAALADL2 andTBL1XR1 in cancers We also noted a number of the differentiallyexpressed genes between patients with and without a gainamplification in NAALADL2TBL1XR1 have been shown to beandrogen regulated however further work is required to determine if gainsamplifications in this region cause changes in ARtranscriptional regulation through cis regulatory elements or as adirect consequence of the genes altered in this region1837In those patients with these gains we noted transcriptionalchanges in several genes associated with aggressive prostatecancer including differential expression of genes appertaining toprognostic assays such as Decipher Oncotype DX and Prolaris aswell as families such as mucins50“ This may explain theaggressive clinical phenotype observed in these patients We alsoobserved that when weighted individually there were differencesin enrichment of biological processes between those with NAALADL2 gains and TBL1XR1 gains suggesting that each generesults in some unique cellular changesOur finding that gains in the 3q26 locus result in concurrentexpression of oncogenes located within this region and theirdownstream targets identifies multiple potential therapeutic avenues warranting further investigations This study centred aroundtwo genes NAALADL2 and TBL1XR1 both of which areattractive therapeutic targets with TBL1XR1 previously suggestedas a potential cancer target operating via the TGFβ signallingpathway and potentially regulating AR signalling5354 Additionally the tumour specificity of NAALADL2 and basal membranouslocalisation makes it potentially accessible using antibodydrugconjugates13 This approach may be feasible if like other familymembers such as PSMA antibody binding results in subcellularinternalisation12 Moreover several of the oncogenes in whichgains cooccur as well as the downstream oncogenes activatedfrom gains in the 3q26 region such as ATR PI3K family members PIK3C2G PIK3CA PIK3CB PIK3R4 MUC4 BCL6 SOX9can be therapeutically targeted or have been suggested as therapeutic targets in cancer5155“ In the PI3K pathway PIK3CBspecific inhibitors may have utility in patients with mutationsamplifications andor fusion of this gene59 These findings mayhave clinical relevence as it has been reported by de Bono et althat many individuals who had durable year responses toPIK3CBspecific inhibition harboured activating mutation oramplification in PIK3CB60 and phase II trials of ipatasertib anAkt inhibitor targeting the PI3KAkt axis has shown promise inlate stage mCRPC61 Together our results suggest that largescalegenomic gainsamplifications occur in the 3q26 region in a hi

Thyroid_Cancer

peripheral serum metabolomic profiles inform central cognitive impairmentJingye Wang1 Runmin Wei12 Guoxiang Xie1 Matthias Arnold Alexandra Kueider‘Paisley Gregory Louie Siamak Mahmoudian Dehkordi3 colette Blach5 Rebecca Baillie Xianlin Han7 Philip L De Jager David A Bennett9 Rima Kaddurah‘Daouk Wei Jia The incidence of Alzheimer™s disease AD increases with age and is becoming a significant cause of worldwide morbidity and mortality However the metabolic perturbation behind the onset of AD remains unclear In this study we performed metabolite profiling in both brain n and matching serum samples n to identify differentially expressed metabolites and metabolic pathways associated with neuropathology and cognitive performance and to identify individuals at high risk of developing cognitive impairment The abundances of metabolites glycolithocholate GLCA petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid and the deoxycholatecholate DCACA ratio along with the dysregulation scores of metabolic pathways primary bile acid biosynthesis fatty acid biosynthesis and biosynthesis of unsaturated fatty acids showed significant differences across both brain and serum diagnostic groups P‘value Significant associations were observed between the levels of differential metabolitespathways and cognitive performance neurofibrillary tangles and neuritic plaque burden Metabolites abundances and personalized metabolic pathways scores were used to derive machine learning models respectively that could be used to differentiate cognitively impaired persons from those without cognitive impairment median area under the receiver operating characteristic curve AUC for the metabolite level model median AUC for the pathway level model Utilizing these two models on the entire baseline control group we identified those who experienced cognitive decline in the later years AUC sensitivity specificity for the metabolite level model AUC sensitivity specificity for the pathway level model and demonstrated their pre‘AD onset prediction potentials Our study provides a proof‘of‘concept that it is possible to discriminate antecedent cognitive impairment in older adults before the onset of overt clinical symptoms using metabolomics Our findings if validated in future studies could enable the earlier detection and intervention of cognitive impairment that may halt its progressionAlzheimer™s disease AD one of the top leading causes of death in the United States is an increasing challenge for health care systems and will result in increased economic burden as increasing numbers of new cases are diagnosed annually12 Currently there is no therapy to prevent or slow AD progression which may be due to the inability to detect AD before its progression into evident cognitive decline Identification of early 1University of Hawaii Cancer Center Ilalo Street Honolulu HI USA 2Department of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI USA 3Department of Psychiatry and Behavioral Sciences Duke University Durham NC USA 4Institute of Computational Biology Helmholtz Zentrum M¼nchen German Research Center for Environmental Health Neuherberg Germany 5Duke Molecular Physiology Institute Duke University Durham NC USA 6Rosa Co LLC San Carlos CA USA 7University of Texas Health Science Center at San Antonio San Antonio TX USA 8Center for Translational Computational Neuroimmunology Columbia University College of Physicians and Surgeons Department of Neurology New York NY USA 9Rush Alzheimer™s Disease Center Rush University Medical Center Chicago IL USA 10Institute of Brain Sciences Duke University Durham NC USA 11Department of Medicine Duke University Durham NC USA email kaddu001mcdukeedu wjiacchawaiieduScientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cbiomarkers associated with preclinical symptoms would allow early intervention or preventive strategies to be developed3 Research has identified multiple neurochemical perturbations in AD including amyloid precursor protein metabolism phosphorylation of tau protein and a wide range of metabolic perturbations4 Unfortunately current biomarkers for early disease including cerebrospinal fluid CSF betaamyloid and tau levels5 structural and functional magnetic resonance imaging6 the recent use of brain amyloid imaging7 or inflammaging8 and CSF markers to track brain atrophy and deposition of cortical betaamyloid and neurofibrillary tangles are limited because they are either invasive timeconsuming or expensiveRecent studies have focused on obtaining biomarkers to identify features that differentiate persons a0with cognitive impairment from persons without cognitive impairment Molecular markers sensitive to the underlying pathogenic factors would be highly relevant to early disease detection and facilitation of disease monitoring and treatment responses Metabolomics is an unbiased approach to study smallmolecule metabolites that offers hope for the discovery of more biomarkers for AD This profiling technology has already been used to identify differential metabolites that can distinguish mild cognitive impairment MCI subjects who will develop AD from stable MCI9 Mounting evidence suggests that AD is closely accompanied with the abnormal bile acid BA metabolism10“ free fatty acid FFA metabolism14“ lipid metabolism1718 and neurotransmitter metabolism19 BAs have become increasingly recognized as important metabolic signaling molecules that modulate lipid glucose and energy metabolism20 More importantly BAs in brain act as neuroactive steroids21 Different classes of BAs can either inhibit or potentiate GABAα a0and inhibit NMDA receptors while also exerting neuroprotective effects Recent crosssectional studies have shown differences in blood BAs in AD compared with noncognitively impaired individuals2425 Additionally researchers found an accumulation of FFAs in the hippocampus and cortex of AD mice compared to control mice2627 Another animal study examined the role of elevated FFA in the pathogenesis of AD and established a potential mechanism of FFA causing hyperphosphorylation of tau through astrogliamediated oxidative stress28 Alterations of FFAs have also been detected in postmortem AD brains tissues14 and serum samples16 which may indicate an alternative fuel source before the onset of clinical symptoms29 These observations have given rise to the possibility that metabolic perturbations could presage the onset of cognitive impairment and therefore aid in the identification of individuals with higher risks by providing additional information to use with standard clinical markersIn this study we performed metabolomic profiling in participants from a large longitudinal cohort with the goal of identifying metabolic changes as well as key metabolic pathways that might serve as new predictors of future cognitive impairment in older adultsMaterials and methodsParticipants The Religious Orders Study ROS which began in is a longitudinal clinicalpathologic cohort study of risk factors of cognitive decline and incident dementia run from the Rush Alzheimer™s Disease Center that is comprised of individuals from religious communities eg Catholic brothers nuns and priests across the USA3031 The Rush Memory and Aging Project MAP which began in includes participants from northeastern Illinois USA with a broader range of socioeconomic status and life experiences31 Participants in both studies enroll without known dementia agree to annual clinical evaluation and an donation Both studies were approved by an Intuitional Review Board of Rush University Medical Center All subjects signed an informed consent an Anatomic Gift Act and a repository consent to allow their biospecimens and data to be used for ancillary studies All research was performed in accordance with relevant guidelinesregulations set forth by the Rush University Medical Center Both studies are conducted by the same team of examiners and share a large common core of data collection at the item level to allow for efficient merging of dataCognitive performance tests Cognitive performance was measured using a battery of cognitive performance tests of which could be summarized in five cognitive domains ie episodic memory working memory semantic memory perceptual orientationvisuospatial ability and perceptual speed Table a0S1 Domains are created by averaging the zscores based on mean and standard deviation from all baseline data for tests in each domain The global cognitive function score is calculated by averaging zscores for all tests to yield a global measure of cognitive function Additionally the MiniMental State Examination was also administered to characterize the cohortClinical diagnoses Medical conditions were documented via selfreport and clinical evaluation Clinical diagnoses each year were determined blinded to previously collected data A threestep process starts with an actuarial decision tree based on the history of cognitive decline and impairment ratings in five cognitive domains based on cutoffs for cognitive tests32 a0followed by clinical judgment by a neuropsychologist for cognitive impairment and determination of dementia and its causes by a clinician ie neurologist geriatrician second neuropsychologist geriatric nurse practitioner33 The diagnosis of AD follows the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer™s Disease and Related Disorders Association NINCDSADRDA34 Participants were categorized as a AD b MCI if diagnosed cognitive impairment by the neuropsychologist but not diagnosed dementia by the clinician32 and c no cognitive impairment NCI if diagnosed without AD or MCI35 At the time of death brain autopsies and histopathological exams were performed by clinicians to confirm the diagnosis After an autopsy was completed a spectrum of neuropathologic diagnoses was obtained such as a pathologic diagnosis of AD as defined using the modified NIA Reagan criteria However many other pathologies were present in the brains of older individuals the mean age of death is a0years old in ROSMAP and they were catalogued for each participantScientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0cNCI converters were those participants who were cognitively normal at the time of blood draw and then experienced the cognitive decline MCI or AD at the time of death while NCI nonconverters were participants who remained cognitively normal during followupNeuropathology Upon death a postmortem neuropathological evaluation was implemented and the procedures follow those outlined by the pathologic dataset recommended by the National Alzheimer™s Disease Coordinating Center Brains of deceased subjects were removed weighed cut into one cmthick coronal slabs and stored Each brain was examined for the neuropathological indices of common pathologies that contribute to cognitive impairment The location age and volume of all macroscopic infarcts were recorded and tissue was obtained for histological confirmation in addition to the identification of microscopic infarctions as previously described3637 AD pathology was identified using the modified Bielschowsky silver stain technique and by use of the Consortium to Establish a Registry for Alzheimer™s Disease CERAD criteria38 and NIAReagan criteria39 while the assessment of neurofibrillary tangles was based on Braak criteria40 as described previously41 The CERAD score a semiquantitative measure of neuritic plaque burden is made of levels no AD possible AD probable AD and definite AD As recommended CERAD scores were reclassified to a binary level score score Seven categories of Braak stages were based on the region and severity of neurofibrillary tangles pathologyMetabolites quantification Using targeted metabolomics protocols42 and profiling protocols43 established in previous studies BAs were quantified by ultraperformance liquid chromatography triple quadrupole mass spectrometry UPLCTQMS Waters XEVO TQS Milford USA and other metabolites were quantified by gas chromatography timeofflight mass spectrometry GCTOFMS Leco Corporation St Joseph USA Details are described in the Supporting InformationStatistical analysis Stratifying by clinical diagnosis continuous demographic variables were expressed as mean [standard deviation SD] and tested by Wilcoxon ranksum test while categorical demographic variables were expressed as n percentage and tested by Chisquare test Missing values in quantitative metabolites due to limits of quantification were regarded as leftcensored missing and imputed by GSimp4445 Individual BA concentrations were normalized to the total BAs concentration ie the proportion of total BAs Metabolites were reported as median quantile quantile and tested by univariate analysis Wilcoxon ranksum test Due to the limited sample size of the AD group participants in serum samples we combined MCI and AD participants into an aggregate group MCIAD for the following data analysis Logtransformed abundances were used in the following data analysis We additionally generated BA ratios based on the BA metabolic pathway topologyTo identify metabolites differentially expressed in participants with cognitive decline we used ordinal logistic regression to compare metabolites across three groups NCI MCI AD for brain samples and logistic regression across two groups NCI MCIAD for serum samples To control the positive false discovery rate Qvalues were calculated based on Pvalues Additionally for serum samples we adjusted for potential confounders eg fasting status supplements diabetic and lipid lowering medications using logistic regressions The relationships between logtransformed brain metabolites levels with neurofibrillary tangle burden and neuritic plaque burden were expressed as boxplots across Braak scores Kruskal“Wallis test and CERAD scores Wilcoxon ranksum test respectively Using Spearman™s rank correlation test we further evaluated the associations between the abundances of each identified metabolite and the global cognitive function score in both brain and serum samples Linear regression models with each individual metabolite used as the predictor and each cognitive test as the response variable adjusted for age gender years of education and presence of APOE ε4 were used to test the associations between metabolite and cognitive function Similar analyses with an additional adjustment of BMI were conducted for serum samples The Wilcoxon ranksum test was carried out to explore whether identified variables were differentially expressed between NCI converters vs NCI nonconverters and between NCI converters vs MCIAD in sera Then we built a random forest RF predictive model to differentiate NCI nonconverters vs MCIAD using glycolithocholate GLCA deoxycholatecholate DCACA ratio petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid and age as the predictorsTo differentiate MCIAD vs NCI nonconverters we randomly split the data into training set and testing set times Each time we trained an RF model on the training set to differentiate the MCIAD from NCI nonconverters and evaluated it on the testing set using the area under the receiver operating characteristic curve AUROC sensitivity SE and specificity SP A final model was built on the whole NCI nonconverters and MCIAD dataTo investigate the preclinical predictive potentials as well as to validate the classification performance of our model we utilized this model on the entire baseline NCI group to identify those NCI converters from NCI nonconverters The differences of RF scores between NCI nonconverters vs NCI converters and NCI nonconverters vs MCIAD groups were tested by the Wilcoxon ranksum test To determine whether RF scores could independently differentiate NCI converters from NCI nonconverters in the presence of potential confounders we used the logistic regression method with RF scores as the predictor adjusting for gender years of education APOE ε4 and BMI Additionally we fit linear mixed effects models to evaluate correlations between RF scores with global cognitive function and each of the five cognitive domains separately with a random effects term for education and BMI and fixed effects terms for RF score gender and APOE ε4For the personalized pathway level analyses we extracted metabolite information from the Human Metabolome Database HMDB46 and metabolic pathway information from the Kyoto Encyclopedia of Genes and Genomes KEGG database47 to map affiliated metabolites to metabolic pathways We used the pathifier Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cOverallNCIMCIADBrain samplesNAge mean SDMale n Education mean SDAPOE ε4carrier n Serum samplesNAge years mean SDMale n Education years mean SDAPOE ε4carrier n Serum NCI samplesNAge years mean SDMale n Education years mean SDAPOE ε4carrier n Overall Overall NCI MCIAD NCI nonconverters NCI converters Table Detailed demographic characteristics of study samples Chisquare test Pvalue comparing AD vs NCI Wilcoxon rank sum test Pvalue comparing MCIAD vs NCI Wilcoxon rank sum test Pvalue comparing NCI converters vs NCI nonconverters Chisquare test Pvalue comparing NCI converter vs NCI nonconverter NCI cognitively normal MCI mild cognitive impairment AD Alzheimer™s disease APOE ε4 apolipoprotein E epsilon allele SD standard deviation algorithm48 to transfer metabolic level information of each sample to pathway level by generating a pathway dysregulation score PDS For each pathway each sample was projected onto a directed principal curve49 which was yielded depending on leading principal components of the pathway to optimally pass through the cloud of samples PDS was the distance along the curve between the projection of each sample and that of NCI Thus PDS could capture the pathwaylevel extent of abnormality increments or decrements for each participant relative to those with NCI We performed similar data analysis on pathway level data to what we did on metabolomics level data We tried to identify differential pathways using ordinal logistic regression across NCI MCI and a0AD groups in brain samples and logistic regression for NCI and MCIAD in serum samples Next we explored the associations between identified pathways with neuropathology Kruskal“Wallis test for Braak scores Wilcoxon ranksum test for CERAD scores and cognitive performance Spearman™s rank correlation test for the global cognitive function linear regression with adjustments for each cognitive test Then we examined the predictive potential of identified pathways in serum samples using univariate analysis Wilcoxon ranksum test for NCI converters vs NCI nonconverters NCI converters vs MCIAD Finally we built RF models on training sets and tested them on testing sets according to times random splitting on the model construction data and applied the final model on the validation data using ROC SE SP as evaluation methods The overall workflow chart of the data and the analysis are shown in Fig a0S9Data were analyzed using R version with packages including pROC pathifier randomForest ggplot2 ggsignif and MASS The statistically significance was determined by a threshold of unadjusted Pvalues and Qvalues ResultsParticipants and characteristics For the joint analyses of the ROSMAP study we measured metabolomics of serum samples NCI MCI and AD at the blood draw and postmortem brain tissues from dorsolateral prefrontal cortex NCI MCI and AD at the time of death Among brain samples and serum samples a total of participants had both brain and blood metabolomics data NCI participants n were further categorized into œNCI converters and œNCI nonconverters NCI converters were those participants who were cognitively normal NCI at the time of blood draw and then experienced the cognitive decline MCI or AD at the time of death while NCI nonconverters were participants who remained cognitively normal during followup Among NCI participants the time between the blood draw and conversion ranged from to a0years with a median of a0years Fig a0S10 Detailed demographic characteristics of the serum samples and postmortem brain samples are included in Table a0 Among participants with postmortem brain samples AD patients tended to have at least one APOE ε4 allele compared to the NCI group as expected The mean age of NCI and MCIAD group at the time of blood draw among serum samples was a0years SD and SD respectively Similarly the age and the percentage of APOE ε4 Scientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0cFigure a0 Brain metabolome and serum metabolome composition and alterations a Left panel the brain metabolome composition Right panel “ a0log10 Pvalue across clinical groups of brain tissues NCI MCI AD b Left panel the serum metabolome composition Right panel “ a0log10 Pvalue across clinical groups of serum tissues NCI MCIADcarriers were higher in the NCI converters group than the NCI nonconverters group We did not observe other significant demographic characteristics differences across clinical groups Table a0Identifying metabolites differentially expressed in participants with cognitive impairment In this study metabolites and metabolites overlapping metabolites were detected in brain tissues and serum samples respectively Tables a0S7 S8 Amino acids BAs carbohydrates anic acids and fatty acids were the predominant types of annotated metabolites accounting for of all the metabolites in brain tissues and in serum samples Fig a01ab left panel A total of seven a0metabolites BA BA ratio anic acid known as a longchain fatty acid fatty acids showed significant differences across clinical groups in both brain and serum samples Pvalue and Qvalue ordinal logistic regression for brain samples logistic regression for serum samples Fig a01ab right panel Tables a0S14 S15 After adjusting for confounders ie fasting status supplement use diabetic and lipid lowering medications most of serum metabolites remained statistically significant Table a0S18 In brain tissues increments of the levels of GLCA DCACA ratio petroselinic acid linoleic acid myristic acid palmitic acid and palmitoleic acid followed the pattern NCI MCI AD We observed increments of GLCA DCACA ratio and decrements of petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid in sera of MCIAD compared to controls Table a0 The trend of increments of identified metabolites in brain samples increments of BAs and decrements of FFAs in serum samples were further validated within individuals with both brain and serum samples From NCI to MCI and AD groups increments of identified metabolites were observed in brain samples Table a0S11 The increasing trend of GLCA DCACA ratio and decreasing trend of FFAs among MCIAD group relative to NCI group were detected in sera Table a0S11The seven brain metabolites were all negatively correlated with global cognitive function where higher scores indicate better cognitive performance ρ ˆ’ a0 for GLCA ρ ˆ’ a0 for DCACA ratio ρ ˆ’ a0 for petroselinic acid ρ ˆ’ a0 for linoleic acid ρ ˆ’ a0 for myristic acid ρ ˆ’ a0 for palmitic acid and ρ ˆ’ a0 for palmitoleic acid using Spearman™s rank correlation analysis Fig a02a Similarly after adjusting for age gender years of education and APOE ε4 all identified metabolites remained negatively correlated with tests in five Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cBrain samplesGLCA median [IQR]DCACA median [IQR]Petroselinic acid median [IQR]Linoleic acid median [IQR]Myristic acid median [IQR]Palmitic acid median [IQR]Palmitoleic acid median [IQR]Serum samplesGLCA median [IQR]DCACA median [IQR]Petroselinic acid median [IQR]Linoleic acid median [IQR]Myristic acid median [IQR]Palmitic acid median [IQR]Palmitoleic acid median [IQR]NCIMCIAD [ ] [ ] [ ] [ ] [ ] [ ] [ ]NCI [ ] [ ] [ ] [ ] [ ] [ ] [ ]MCIAD [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]Table Levels of metabolites differentially expressed in participants a0by diagnostic group Pvalue Pvalue Pvalue by Wilcoxon rank sum test comparing AD vs NCI Pvalue Pvalue Pvalue by Wilcoxon rank sum test comparing MCIAD vs NCI NCI cognitively normal MCI mild cognitive impairment AD Alzheimer™s disease IQR interquartile rangeFigure a0 Associations between metabolites level and global cognitive function a Boxplots showing group differences and P values for identified metabolites across Braak groups for brain tissue abundances b Boxplots showing group differences and significances for identified metabolites across CERAD groups for brain tissue abundances ρ correlation coefficient of Spearman™s rank correlation testScientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0ccognitive domains and the MiniMental State Exam see Table a0S2 for significant correlation pairs The serum concentration of two BAs showed negative correlations with global cognitive function ρ ˆ’ a0 for GLCA ρ ˆ’ a0 for DCACA ratio conversely fatty acids demonstrated positive correlations ρ for petroselinic acid ρ for linoleic acid ρ for myristic acid ρ for palmitic acid and ρ for palmitoleic acid Fig a02b Linear regression revealed similar consistent results in serum samples with adjustment for age gender years of education APOE ε4 and BMI see Table a0S2 Results of associations between identified metabolitesratio and each cognitive performance domains are shown in Table a0S13 Correlations with global cognitive function were further validated in individuals with both brain and serum samples and the directions were consistent with our previous findings among the a0entire cohort Seven identified metabolites were all negatively correlated with global cognitive function in brain samples while two BAs showed negative correlations and five FFAs showed positive correlations in serum samples Fig a0S6 Additionally the serumbrain ratio of identified FFAs were positively correlated with global cognitive function ie lower levels of identified FFAs in serum and higher levels of identified FFAs in brain were associated with worse cognition Fig a0S7Identified metabolites predicted antecedent cognitive impairment before the manifestation of clinical symptoms The concentrations of GLCA and DCACA were significantly lower in the NCI nonconverters group than in the NCI converters group By contrast the abundances of petroselinic acid linoleic acid myristic acid palmitic acid and palmitoleic acid were higher in the NCI nonconverters group than in the NCI converters group Fig a03a There were no significant differences in these metabolites between participants in NCI converters group vs MCIAD group Fig a03a Using the seven metabolites and age we built RF models on the training set according to 100times randomly splitting approach to differentiate MCIAD patients from NCI nonconverters group The median of times AUC on testing set was CIs “ with SE CIs “ and SP CIs “ using Youden™s index to maximize the sum of SE and SP Fig a03b RF models showed decent classification performances in differentiating MCIAD group from NCI nonconvertersNext we were interested in studying the model™s early diagnostic capability for predicting NCI converters before clinical diagnosis The model was thus applied on the entire NCI group at baseline to differentiate NCI converters from NCI nonconverters We achieved an AUC of CIs “ SE SP at the cutoff value of Fig a03c with significant differences in RF scores between NCI converters vs NCI nonconverters between NCI nonconverters vs MCIAD group using the Wilcoxon ranksum test Pvalue Fig a03d After additional adjustment for gender years of education APOE ε4 and BMI fasting status and medications supplements diabetes lipid lowing the RF scores remained significant as an independent predictor with a coefficient of Pvalue Table a0S3 Additionally the RF scores showed significant negative correlations with global cognitive function and the five cognitive domains with the same adjustment in mixed effects models Table a0S12Personalized metabolic pathway‘based study for the association and prediction of cognitive impairment Considering altered metabolite levels were significantly associated with cognitive impairment and showed early predictive value of clinical symptoms onset we then employed the pathifier algorithm to summarize metabolite information to pathways level for further examinations All PDS scores ranged from to where larger scores represent the higher extent of the abnormality in the corresponding metabolic pathway out of metabolites detected in brain tissues and out of metabolites detected in sera were successfully mapped to the KEGG metabolic pathways This method identified metabolic pathways in brain tissues and metabolic pathways in serum samples overlapping pathways Figs a0S1ab left panel Table a0S9 Table a0S10 three of which ie primary BAs biosynthesis FFAs biosynthesis and biosynthesis of unsaturated FFAs were significantly shifted in both brain and serum samples Pvalue and Qvalue ordinal logistic regression for brain samples logistic regression for serum samples Fig a0S1ab right panel Table a0S16 Table a0S17 We noted increased PDS for all three identified pathways from NCI to MCIAD that suggested dysregulation of these metabolic pathways in MCIAD patients compare to NCI Detailed PDS of these pathways stratified by diagnostic groups are described in Table a0S4 Results also indicated that higher PDS were significantly associated with lower global cognitive function ie worse cognitive performance in both brain ρ ˆ’ a0 for primary BAs biosynthesis pathway ρ ˆ’ a0 for FFAs biosynthesis pathway ρ ˆ’ a0 for biosynthesis of unsaturated FFAs pathway Fig a0S4 and serum samples ρ ˆ’ a0 for primary BAs biosynthesis pathway ρ ˆ’ a0 for FFAs biosynthesis pathway ρ ˆ’ a0 for biosynthesis of unsaturated FFAs pathway Fig a0S5 respectively In Table a0S5 we show the significant negative associations between each cognitive test and PDS of three pathways after adjusting for age gender years of education and APOE ε4 additional adjustment for BMI in serum samples Two fatty acid pathways showed significantly different PDS between the NCI nonconverters group and the NCI converters group Pvalue and Pvalue respectively A gradually increasing trend was noted for the BAs pathway across groups ie NCI nonconverters NCI converters and MCIAD Fig a04aWe then constructed a discriminant RF model in training data and tested on testing data based on three identified metabolic pathways along with age to differentiate MCIAD from NCI nonconverters in model construction data using 100times randomly splitting approach The median AUC on the a0testing set was CIs “ with SE CIs “ and SP CIs “ Fig a04b Applying the RF model to the whole NCI data at baseline could successfully discriminate NCI converters from NCI nonconverters with an AUC of CIs “ SE SP cutoff value Fig a04c Similarly predictive RF scores were significantly different between NCI converters vs NCI nonconverters and NCI nonconverters vs MCIAD group Pvalue Fig a04d After adjusting for gender Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cFigure a0 The identified panel of metabolites and its predictive performance a Boxplots showing group differences and P values for identified metabolites across NCI nonconverters NCI converters and MCIAD for serum abundances b ROC curves of metabolite models trained on the training data and tested on the testing data according to 100times randomly training“testing splitting c The ROC curve of the final metabolite model on the validation data d RF scores of the final metabolite model across NCI nonconverters NCI converters and MCIAD Pvalue Pvalue Pvalue Wilcoxon rank sum test The optimal cutoff was determined by the Youden index AD Alzheimer™s disease AUC area under

Thyroid_Cancer

Large cell neuroendocrine carcinoma Descending colon Colonic neuroendocrine neoplasm Colonic adenocarcinoma Neuroendocrine neoplasms are most often found in the small intestine rectum appendix and stomach The colon excluding the appendix and the cecum is a rare location for these neoplasms and often gives rise to highly proliferative poorly differentiated tumors with aggressive features and dismal prognosis A 32yearold male presents with a large cell neuroendocrine carcinoma arising from an unusual location the descending colon The pa tient™s clinical and imaging characteristics resembles those seen in the much more common neoplasm colonic adenocarcinoma Computed tomography and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Pathologic correlation of a sur gical specimen is required to make the correct diagnosis The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 Introduction Neuroendocrine neoplasms NENs are uncommon slow growing neoplasms of the neuroendocrine system that most frequently occur in the ileum the rectum “ the appendix “ and the stomach “ [ ] The colon excluding the appendix and the cecum is a rare ori gin for NENs with a reported incidence of per per sons [] The overall median age at diagnosis is years [] Colonic NENs are not normally associated with hereditary tu mor syndromes such as multiple endocrine neoplasia type [] Colonic NENs are typically poorly differentiated on histol ogy and often appear as a large mass with aggressive growth rapid dissemination and distant metastases at the time of diagnosis [“] Once metastasized the prognosis is dismal with a median survival of months [] Patients with NENs typically present with nonspecific com plaints such as bleeding diarrhea abdominal pain gastroin testinal blood loss or weight loss [ ] Carcinoid syndrome is more often seen in patients with gastric or small intesti nal NENs with liver metastasis In contrast carcinoid syn drome is rare in patients with colonic NENs because these tumors rarely contain serotonin or secrete serotonin precur sors [ ] Urine levels of the serotonin metabolite 5HIAA are not significantly elevated in patients with colonic NENs [] Serum chromogranin A may be elevated in of all gas trointestinal NENs and correlate with tumor burden [] How ever its diagnostic accuracy can be lower for poorly differen tiated NENs [ ] Also it may be falsely elevated in proton œ Competing Interests The authors have declared that no competing interests exist ˆ—Corresponding author Email address alanmlarkinhospitalcom A Mo 101016jradcr202007045 The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 0c R a d i o l o g y C a s e R e p o r t s “ Fig “ Axial a and coronal b images of CT with IV contrast with multiphasic acquisition Irregular circumferential wall thickening of the descending colon — cm heterogeneously with a contiguous enhancing mass — pump inhibitor use atrophic gastritis impaired renal func tion rheumatoid arthritis inflammatory bowel disease and nonneuroendocrine neoplasms such as prostate cancer ovar ian cancer breast cancer and colorectal cancer [] The crosssectional imaging features of colonic NENs include irregular circumferential wall thickening or large polypoidal mass with lymphadenopathy closely resembling those of colonic adenocarcinoma [ ] Metastasis to the liver is common and appear as hypervascular lesions that demonstrate moderatetointense homogenous or pe ripheral rim enhancement during hepatic arterial phase on multiphasic computed tomography or magnetic resonance imaging [ ] In111 octreotide scintigraphy utilizes a synthetic somatostatin analog to characterize NENs [] Neuroendocrine tumors containing somatostatin receptors demonstrate increased radiotracer uptake We present a rare case of large cell neuroendocrine carcinoma in the descend ing colon with metastasis in the liver which demonstrates clinical and imaging features closely resembling those of metastatic colonic adenocarcinoma Case presentation A 32yearold male with a past medical history of depression and schizophrenia presented with constant left abdominal pain radiating down to the hip and groin No pertinent surgi cal or family history was noted The patient admitted to daily use of alcohol and tobacco and denied recreational drug use The review of systems was positive for fatigue intermittent bloodstreak stools and unintentional weight loss of lbs The patient denied fever night sweats decreased appetite nausea vomiting diarrhea cutaneous flushing sweating or bronchospasm The physical exam was unremarkable except for tenderness over the left flank and mid abdomen Computed tomography of the abdomen and pelvis with IV contrast revealed irregular circumferential wall thickening of — cm het the descending colon with a contiguous erogeneously enhancing mass Fig Innumerable hypoat tenuating lesions with hypovascular peripheral enhancement — Fig “ Axial images of CT of the abdomen and pelvis at the level of the right portal vein Noncontrasted axial CT image a demonstrates innumerable illdefined hypoattenuating masses throughout the liver suspicious for metastatic lesions Contrasted axial CT image on arterial phase b demonstrates the same masses with peripheral rim enhancement and a hypoattenuating center The masses remain hypodense to the background hepatic parenchyma Each mass contains a faint hypoattenuating and nonenhancing halo Contrasted axial CT image on portal venous phase c and delayed phase d demonstrate persistent peripheral enhancement with no rapid washout were observed throughout the hepatic parenchyma Fig No skeletal metastasis was appreciated In111 octreotide scan demonstrated multiple phot ic lesions within the liver Fig After an unsuccessful attempt with colonoscopy left hemi colectomy and surgical pathology were pursued Surgical pathology of the colonic mass revealed poorly differentiated large cell neuroendocrine carcinoma with tumoral invasion into the visceral peritoneum and positive of lymph nodes Fig Additionally interventional radiology was consulted for CTguided biopsy of the liver lesions Tissue biopsy of the hepatic lesions confirmed metastasis from the colonic mass Evaluation of 5hydroxyindoleacetic acid 5HIAA in a hour urine specimen was within normal limits at 4mg24h cisplatin The patient was treated with intravenous mgm etoposide for first for weeks in combination with mgm days Discussion Colonic NENs demonstrate similar crosssectional imag ing features as colorectal adenocarcinomas [ ] Both 0cR a d i o l o g y C a s e R e p o r t s “ demonstrating moderatetointense homogenous or periph eral rim enhancement during arterial phase [ ] Hyper vascular hepatic metastasis are nonspecific and can be com monly seen in melanoma renal cell carcinoma choriocarci noma and thyroid carcinoma [] However in the setting of a patient with a colonic mass hypervascular hepatic metastatic lesions may be the differentiating imaging feature to sug gest colonic NENs rather than adenocarcinoma In our case the hepatic metastatic lesions demonstrated hypovascular peripheral enhancement that resemble those typically seen with colonic adenocarcinoma as opposed to those seen with colonic NENs We postulate that the appearance of these le sions may be attributed to the fibrogenic nature of NENs [ ] and central necrosis due to the poor cell differentiation of the mass In111 octreotide scintigraphy is commonly used in diag nosis of NENs with a sensitivity of for all NENs [] It utilizes a synthetic somatostatin analog that binds to somato statin transmembrane receptors which are expressed in of all NENs [] However poorly differentiated NENs may sometimes express fewer somatostatin receptors or may even completely lack them all together producing less reli able results [ ] In our case In111 octreotide scintigraphy demonstrates multiple phot ic lesions in the liver This may be secondary to the absence or fewer numbers of somato statin receptors in poorly differentiated NENs Our patient presents with abdominal pain fatigue weight loss and hematochezia without the characteristic symptoms of carcinoid syndrome despite having substantial hepatic Fig “ hours delayed anterior projection of the chest In111 octreotide scintigraphy demonstrates multiple phot ic lesions within the liver feature irregular circumferential wall thickening or polypoid intramural mass with areas of central necrosis and degener ation [ ] Also both malignancies often metastasize to the liver [] Colonic adenocarcinomas often produce hypo vascular hepatic metastatic lesions In contrast of gas trointestinal NENs metastases feature hypervascular lesions Fig “ HE stain scan power view shows sheetlike growth pattern of tumor cells involving whole layer of colon A On higher magnification B tumor cells show solid growth pattern Note the vesicular nuclei with saltandpepper chromatin Immunohistochemical staining for chromogranin A C and synaptophysin D reveals diffuse positive in tumor cells 0c R a d i o l o g y C a s e R e p o r t s “ metastasis Also laboratory analysis of urine 5HIAA is un remarkable CT and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Ultimately the correct diagnosis is made through immunohistochemical evaluation of the surgical pathologic specimen R E F E R E N C E S [] Turaga KK Kvols LK Recent progress in the understanding diagnosis and treatment of gastroenteropancreatic neuroendocrine tumors CA Cancer J Clinic “ [] Koenig A Krug S Mueller D Barth PJ Koenig U Scharf M et al Clinicopathological hallmarks and biomarkers of colorectal neuroendocrine neoplasms PloS One 20171212e0188876 [] Yao JC Hassan M Phan A Dagohoy C Leary C Mares JE et al One hundred years after ˜carcinoid™ epidemiology of and prognostic factors for neuroendocrine tumors in cases in the United States J Clin Orthodont “ [] Caplin M Sundin A Nillson O Richard PB Klaus JK Fahrettin K et al ENETS consensus guidelines for the management of patients with digestive neuroendocrine neoplasms colorectal neuroendocrine neoplasms Neuroendocrinology “ [] Grabowski P Sch¶nfelder J AhnertHilger G Foss HD Heine B Schindler I et al Expression of Neuroendocrine Markers A Signature of Human Undifferentiated Carcinoma of the Colon and Rectum Virchows Archiv Int J Pathol “ [] Chang S Choi D Lee SJ Lee WJ Park MH Kim SW et al Neuroendocrine neoplasms of the gastrointestinal tract classification pathologic basis and imaging features Radiographics “ [] Ganeshan Dhakshina Bhosale Priya Yang Thomas Kundra Vikas Imaging features of carcinoid tumors of the gastrointestinal tract AJR Am J Roentgenol “ [] Kaltsas GA Besser GM Grossman AB The diagnosis and medical management of advanced neuroendocrine tumors Endocr Rev “ [] Cimitan M Buonadonna A Cannizzaro R Canzonieri V Borsatti E Ruffo R De Apollonia L Somatostatin receptor scintigraphy versus chromogranin a assay in the management of patients with neuroendocrine tumors of different types clinical role Ann Oncol “ [] Gut P Czarnywojtek A Fischbach J B ˛aczyk M Ziemnicka K Wrotkowska E et al Chromogranin a unspecific neuroendocrine marker Clinical utility and potential diagnostic pitfalls Arch Med Sci AMS “ [] Levy AD Sobin LH From the archives of the AFIP gastrointestinal carcinoids imaging features with clinicopathologic comparison Radiographics “ [] Sahani DV Bonaffini PA Castillo CFD Blake MA Gastroenteropancreatic neuroendocrine tumors role of imaging in diagnosis and management Radiology “ [] Bader TR Semelka RC Chiu VC Armao DM Woosley JT MRI of carcinoid tumors spectrum of appearances in the gastrointestinal tract and liver J Magn Reson Imaging JMRI “ [] Intenzo CM Jabbour S Lin HC Miller JL Kim SM Capuzzi DM et al œScintigraphic imaging of body neuroendocrine tumors Radiographics “ [] Namasivayam S Martin DR Saini S Imaging of liver metastases MRI Cancer Imaging “ [] Laskaratos FM Rombouts K Caplin M Toumpanakis C Thirlwell C Mandair D Neuroendocrine tumors and fibrosis an unsolved mystery Cancer “ 0c'

Thyroid_Cancer

LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathwaysXue Wu123 XiaoFeng Li123 Qian Wu4 RuiQi Ma123 Jiang Qian123 Rui Zhang123·Basic Research·1Department of Ophthalmology Eye ENT Hospital of Fudan University Shanghai China2NHC Key Laboratory of Myopia Fudan University Shanghai China 3Laboratory of Myopia Chinese Academy of Medical Sciences Shanghai China4Department of Pathology West China Hospital Sichuan University Chengdu Sichuan Province ChinaCofirst authors Xue Wu and XiaoFeng LiCorrespondence to Rui Zhang Department of Ophthalmology Eye ENT Hospital of Fudan University Fen Yang Road Shanghai China zhangrui936163comReceived Accepted Our research suggests that SNHG15 may play a vital role as a potential marker in UM that predicts poor prognosis Besides GSEA indicates the underlying signaling pathways enriched differentially in SNHG15 high expression phenotype KEYWORDS SNHG15 uveal melanoma the Cancer Genome Atlas pathology prognosis Gene Set Enrichment Analysis1018240ijo20200804Citation Wu X Li XF Wu Q Ma RQ Qian J Zhang R LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathways Int J Ophthalmol Abstract— AIM To evaluate the role of long noncoding RNA lncRNA SNHG15 and its potential pathways in uveal melanoma UM METHODS The SNHG15 mRNA expression level and corresponding clinicopathological characteristics of patients with UM were obtained from the Cancer Genome Atlas TCGA database and further analyzed The SPSS statistical software package was used for statistical analyses To investigate the potential function of SNHG15 in UM we conducted indepth research on Gene Set Enrichment Analysis GSEA— RESULTS The univariate analysis revealed that the age tumor diameter pathological type extrascleral extension cancer status and high expression of SNHG15 were statistical risk factors for death from all causes The multivariate analysis suggested that the mRNA expression level of SNHG15 was an independent risk factor for death from all causes as was age and pathological type KaplanMeier survival analysis confirmed that UM patients with high SNHG15 expression might have a poor prognosis In addition SNHG15 was significantly differentially expressed in the different groups of tumor pathologic stage metastasis and living status Besides the logistic regression analysis indicated that high SNHG15 expression group in UM was significantly associated with cancer status pathologic stage metastasis and living status Moreover the GSEA indicated the potential pathways regulated by SNHG15 in UM INTRODUCTIONU veal melanoma UM the most common intraocular cancer in adult worldwide[] is a malignant tumor that originates in melanocytes of the choroid plexus ciliary body and iris of the eye At present despite definitive radiotherapy or removal of the primary lesion numerous patients eventually develop metastases and subsequently prognosis is significantly poor[] In addition UM tends to metastasize to liver through hematogenous pathway a distant site relative to their origins in the eye[] There is an incubation period between the enucleation of the primary tumor and the emergence of metastasis which can range from a few months to several decades[] Despite the advancement of UM management there are currently no effective therapy once the metastases occurred[] Therefore close followup and further research on the pathogenesis and novel makers exploration of UM are of great significance for accurate diagnosis appropriate therapy and prognosis prediction Long noncoding RNA lncRNA is a class of noncoding transcripts with a length of larger than nucleotides[] which has been involved widely in biological processes of different cancers including cell cycle apoptosis cell differentiation[] In the development of UM lncRNA is also reported to play a vital role in cell cycle cell proliferation apoptosis invasion and autophagy[] For example silencing of lncRNA PVT1 prevents the development of UM by impairing microRNA173pdependent MDM2 upregulation[] ZNNT1 can suppress Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomathe progression of UM by inducing the expression of crucial autophagy gene[] The lncRNA RHPN1AS1 facilitates the tumorigenesis of UM by influencing cell proliferation and migration[] However the study of vital lncRNAs in UM still remains to be exploredSNHG15 a novel lncRNA located on chromosome 7p13[] is identified to play a key role in many types of human tumors such as osteosarcoma[] papillary thyroid carcinoma[] pancreatic ductal adenocarcinoma[] colorectal carcinoma[] hepatocellular carcinoma[] prostate cancer[] and breast cancer[] To our knowledge the potential impact of SNHG15 on the tumorigenesis of UM seems unclear recently Thus the purpose of this study was to evaluate the pivotal role of SNHG15 in the progression of UM In addition the relationship between SNHG15 expression and clinicopathologic characteristics in UM was preliminarily demonstrated To explore the underlying mechanisms of the biological pathways involved in UM we conducted a research on Gene Set Enrichment Analysis GSEA MATERIALS AND METHODSEthical Approval The study protocol was approved by the Ethics Committee of the Eye ENT Hospital of Fudan University and all procedures were complied with the principles of the Declaration of Helsinki All datasets of our present study were downloaded from an database TCGA so there was no written informed consent from participantsRNASequencing Patient Data and Bioinformatics Analysis The RNASeq gene expression level and clinicopathological characteristics including cases were obtained from the official website of the Cancer Genome Atlas TCGA UM project portalgdccancergov Patients with UM were classified as two groups based on the median SNHG15 expression level cutoff value794 FPKM Finally patients with UM were retained and their clinicopathological characteristics were further analyzed including the detailed information of age gender tumor diameter thickness pathological type extrascleral extension cancer status pathological stage metastasis living status SNHG15 expressionGene Set Enrichment Analysis GSEA is a common bioanalysis used to interpret and analyze microarray and other similar data and to speculate related pathways that can significantly enrich regulatory genes[] Through TCGA UM project we obtained the RNASeq gene expression level of UM patients And the analysis was conducted using GSEA v30 software In this study according to the association with SNHG15 expression the ordered gene list was generated firstly by GSEA Subsequently GSEA was conducted to clarify statistically significant differences between the two groups with high and low SNHG15 expression A total of permutations were performed The SNHG15 expression level was identified as a phenotype label The related pathways statistically enriched in each phenotype were selected with the nominal P005 and an false discovery rate FDR Statistical Analysis The SPSS statistical software package SPSS Inc USA was used for statistical analyses Both the univariate and multivariate analyses using Cox analysis were performed to demonstrate independent prognostic biomarkers for UM patients The survival curve was generated by conducting KaplanMeier method To compare the significant differences in overall survival OS the logrank test was conducted The plot chart was performed to visualize the difference of SNHG15 expression level for diverse variables through Graphpad The relationship between the SNHG15 expression and clinicopathological characteristics were analyzed using logistic regression The median value of SNHG15 expression was selected as the cutoff value P005 was considered statistically significantRESULTSPatient Characteristics The records of primary UM with both RNASeq gene expression level and clinicopathological characteristics were obtained from TCGA database The mean age of UM patients was years old including males and females The mean value of tumor diameter and thickness were and mm respectively In our study cohort the pathological type of UM included epithelioid cell dominant type and spindle cell dominant type of tumors were epithelioid cell dominant and were spindle cell dominant There were cases without extrascleral extension and cases with extrascleral extension The cancer status included tumorfree cases and cases with tumor Pathologic stage II was found in cases and stage IIIIV in cases And of cases had metastases of cases had no metastases Of cases cases died of all causes Survival Outcomes and Multivariate Analysis Prognostic factors of UM were analyzed using univariate and multivariate Cox regression The univariate analysis suggested that high SNHG15 expression was a risk factor for death from all causes Other clinicopathologic variables related to poor prognosis included age tumor diameter pathological type extrascleral extension cancer status Table In a multivariate analysis SNHG15 was an independent risk factor for death from all causes as was age and pathological typeSNHG15 Expression Associated with Clinical Pathological Characteristics A total of UM cases with SNHG15 expression data and clinicopathologic characteristics were analyzed from TCGA KaplanMeier survival analysis 0cTable Prognostic parameters in UM were analyzed using univariate and multivariate Cox regressionDeath from all causesParametersnmeanUnivariate analysisPHR95CIAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelioid cell dominantSpindle cell dominantExtrascleral extensionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVSNHG15HighLowUM Uveal melanomaMultivariate analysisPHR95CIdemonstrated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group Figure 1A P005 As shown in Figure 1B1D SNHG15 was statistically differentially expressed in diverse groups of the tumor pathologic stage stage II vs IIIIV P00257 metastasis P00071 living status P00017 To clarify the clinicopathologic impact of SNHG15 we also used logistic regression and concluded that the SNHG15 expression based on median value of FPKM as a categorical variable was statistically related to clinicopathologic features Table High SNHG15 expression was significantly related to cancer status pathologic stage metastasis living status in UM all P005 Table These results demonstrated that UM with high SNHG15 expression were prone to progress to cancer status of survival with tumor a more advanced stage metastasis and poor living status when compared to the low SNHG15 expression group However there was no statistically significant difference in age gender tumor diameter thickness pathological type extrascleral invasionMain Enriched Pathways in UM Tissues with High SNHG15 Expression To explore the SNHG15related potential signaling pathways activated in UM GSEA was performed In the current study based on the association with SNHG15 expression the gene list was generated firstly Figure The SNHG15 expression was associated with clinical pathological characteristics A Patients with high SNHG15 expression had a shorter OS when compared with the low SNHG15 expression group P002 BD The expression of SNHG15 was statistically different in diverse groups of the tumor pathologic stage P00257 metastasis P00071 living status P00017 aP005 bP001by GSEA To clarify the statistically significant differences between high and low SNHG15 expression groups GSEA was Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaTable Association between SNHG15 expression and clinicopathologic variables using logistic regressionParametersAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelialNonepithelialExtrascleral invasionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVMetastasesNoYesLiving statusAliveDeadnmeanSNHG15 expressionLowHighPOR95CINESNominal PvalESTable Enriched pathways for differential SNHG15 expression in UMNameSpliceosomeCell cyclePyrimidine metabolismDNA replicationNucleotide excision repairRNA degradationHomologous recombinationMismatch repairUM Uveal melanoma ES Enrichment score NES Normal enrichment score FDR False discovery rateFDR Qvalconducted subsequently The results indicated that there were significant differences in spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair among patients with high SNHG15 expression phenotype Figure Table DISCUSSIONAccumulating evidences indicate that SNHG15 plays a dual role in the tumorigenesis and development of different tumors[] Previously SNHG15 has been demonstrated as a carcinogenic lncRNA which is usually upregulated in tumor tissues compared with normal tissues[] It exerts 0cFigure Enrichment plots from GSEA Spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair are enriched significantly in SNHG15 high expression phenotypean oncogenic effect via various epigenetic mechanisms[] For example it can suppress the expression of miR3383p and facilitate the proliferation of colorectal cancer cells[] It plays a carcinogenic role by affecting miR3383pFKBP1A axis in prostate cancer[] It can also enhance hepatocellular carcinoma progression by negative regulation of miR1413p[] However there are reports that SNHG15 has a tumor suppressive effect suggesting that low SNHG15 expression is related to poor prognosis in thyroid cancer and upregulating expression of SNHG15 can significantly suppress cell proliferation[] At present the impact of SNHG15 on UM is still unclear Therefore vital roles and potential biological mechanism of SNHG15 in UM needs to be elucidated In this study we revealed that high SNHG15 expression was related to clinicopathologic features in UM Through RNASeq gene expression level and clinicopathological characteristics obtained from the TCGA UM project we analyzed the relationship among SNHG15 expression clinicopathological features and prognosis of UM The univariate analysis demonstrated that SNHG15 expression level age tumor diameter pathological type extrascleral extension and cancer status were risk factors for death from all causes The multivariate analysis suggested that high SNHG15 expression along with age and pathological type was an independent risk factor for death from all causes Therefore the results demonstrated that high SNHG15 expression was an independent predictor of poor prognosis in UM through univariate and multivariate analysis KaplanMeier survival analysis also indicated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group in UM In addition an analysis was conducted to further explore the relationship between SNHG15 and clinicopathological features The SNHG15 expression was statistically different in diverse groups of the tumor pathologic stage metastasis and living status Besides high SNHG15 expression based on median expression value of FPKM in UM was associated with cancer status of survival with tumor advanced pathologic stage metastasis and living status It demonstrated that high SNHG15 expression in UM was strongly related to poor prognosis The mechanisms of SNHG15 dysregulation in malignant tumors are quite complex and are far from being completely understood Previous studies have suggested that SNHG15 is involved in diverse pathological and physiological processes of many tumors through their abnormal expressions including cell proliferation invasion migration and autophagy[] To explore the biological mechanism of SNHG15 in UM GSEA was conducted It indicated that spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair were all enriched differentially in SNHG15 high expression phenotype Alternative splicing is essential for gene regulation and abnormal splicing plays a vital role in inactivating tumor suppressor genes or activating oncogenes[] SNHG15 may have an impact on the invasion Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaand migration of UM cells by affecting spliceosomal related factors The abnormal cell proliferation of tumor is related to the lack of checkpoint control over the cell cycle which is the basis of genetic instability[] Evidence shows that the lack of homologous recombination may facilitate the disturbance of cell cycle the instability and accumulated mutations of genome during the progression and development[] Mismatch repair proteins have an significant role in DNA hypermethylation alteration and tumorigenesis[] SNHG15 is closely related to DNA replication and mismatch repair demonstrating that SNHG15 may promote the occurrence of UM by affecting DNA replication and DNA mismatch repair It indicated that SNHG15 may be identified as a novel marker of diagnosis therapeutic and prognosis prediction in UM However the related mechanism needs to be further elucidated This research also has some limitations The most important one is the limited number of patients and time of followup In addition some patient characteristics such as ciliary body involvement were not completely recorded in the database In fact ciliary body involvement plays a critical role in UM[]In conclusion this study aims to demonstrate the vital role of SNHG15 in UM and the potential relationship between SNHG15 expression and clinical parameters SNHG15 expression may be a valuable biomarker for poor survival in UM Moreover we have preliminarily explored the crucial pathway associated with SNHG15 in UM However further experimental validation is needed to be performed for clarifying the significant impact of SNHG15 And it is of great significance to further identify its independent prognostic value in a largescale standardized researches on UMACKNOWLEDGEMENTSFoundations Supported by the National Natural Science Foundation of China No81970835 No81800867 Conflicts of Interest Wu X None Li XF None Wu Q None Ma RQ None Qian J None Zhang R NoneREFERENCES van Raamsdonk CD Griewank KG Crosby MB Garrido MC Vemula S Wiesner T Obenauf AC Wackernagel W Green G Bouvier N Sozen MM Baimukanova G Roy R Heguy A Dolgalev I Khanin R Busam K Speicher MR O€™Brien J Bastian BC Mutations in GNA11 in uveal melanoma N Engl J Med Carvajal RD Sosman JA Quevedo JF Milhem MM Joshua AM Kudchadkar RR Linette GP Gajewski TF Lutzky J Lawson DH Lao CD Flynn PJ Albertini MR Sato T Lewis K Doyle A Ancell K Panageas KS Bluth M Hedvat C Erinjeri J Ambrosini G Marr B Abramson DH Dickson MA Wolchok JD Chapman PB Schwartz GK Effect of selumetinib vs chemotherapy on progressionfree survival in uveal melanoma a randomized clinical trial JAMA Shain AH Bagger MM Yu R Chang D Liu SS Vemula S Weier JF Wadt K Heegaard S Bastian BC Kiilgaard JF The genetic evolution of metastatic uveal melanoma Nat Genet Bagger M SmidtNielsen I Andersen MK Jensen PK Heegaard S Andersen KK Friis S Kiilgaard JF Longterm metastatic risk after biopsy of posterior uveal melanoma Ophthalmology Kujala E Mäkitie T Kivelä T Very longterm prognosis of patients with malignant uveal melanoma Invest Ophthalmol Vis Sci Chandran SS Somerville RPT Yang JC Sherry RM Klebanoff CA Goff SL Wunderlich JR Danforth DN Zlott D Paria BC Sabesan AC Srivastava AK Xi LQ Pham TH Raffeld M White DE Toomey MA Rosenberg SA Kammula US Treatment of metastatic uveal melanoma with adoptive transfer of tumourinfiltrating lymphocytes a singlecentre twostage singlearm phase study Lancet Oncol Mendell JT Targeting a long noncoding RNA in breast cancer N Engl J Med Lan Y Xiao XW He ZC Luo Y Wu CF Li L Song X Long noncoding RNA OCC1 suppresses cell growth through destabilizing HuR protein in colorectal cancer Nucleic Acids Res Cao CH Sun JY Zhang DY Guo XJ Xie LW Li X Wu DH Liu L The long intergenic noncoding RNA UFC1 a target of microRNA 34a interacts with the mRNA stabilizing protein HuR to increase levels of βcatenin in HCC cells Gastroenterology 20151482415426e18 Wang P Xue YQ Han YM Lin L Wu C Xu S Jiang ZP Xu JF Liu QY Cao XT The STAT3binding long noncoding RNA lncDC controls human dendritic cell differentiation Science Zheng XL Tang HW Zhao XF Sun YM Jiang YF Liu YH Long noncoding RNA FTH1P3 facilitates uveal melanoma cell growth and invasion through miR2245p PLoS One 20171211e0184746 Lu QK Zhao N Zha GP Wang HY Tong QH Xin SH LncRNA HOXA11AS exerts oncogenic functions by repressing p21 and miR in uveal melanoma DNA Cell Biol Lu LN Yu XY Zhang LL Ding X Pan H Wen XY Xu SQ Xing Y Fan JY Ge SF Zhang H Jia RB Fan XQ The long noncoding RNA RHPN1AS1 promotes uveal melanoma progression Int J Mol Sci Wu S Chen H Han N Zhang CX Yan HT Long noncoding RNA PVT1 silencing prevents the development of uveal melanoma by impairing MicroRNA173pdependent MDM2 upregulation Invest Ophthalmol Vis Sci Li P He J Yang Z Ge SF Zhang H Zhong Q Fan XQ ZNNT1 long noncoding RNA induces autophagy to inhibit tumorigenesis of uveal melanoma by regulating key autophagy gene expression Autophagy Dong YZ Meng XM Li GS Long noncoding RNA SNHG15 indicates poor prognosis of nonsmall cell lung cancer and promotes 0ccell proliferation and invasion Eur Rev Med Pharmacol Sci SNHG15 serves as an oncogene and predicts poor prognosis in epithelial ovarian cancer Onco Targets Ther Liu K Hou Y Liu YK Zheng J LncRNA SNHG15 contributes to proliferation invasion and autophagy in osteosarcoma cells by sponging miR141 J Biomed Sci Wu DM Wang S Wen X Han XR Wang YJ Shen M Fan SH Zhang ZF Shan Q Li MQ Hu B Lu J Chen GQ Zheng YL LncRNA SNHG15 acts as a ceRNA to regulate YAP1Hippo signaling pathway by sponging miR200a3p in papillary thyroid carcinoma Cell Death Dis Guo XB Yin HS Wang JY Evaluating the diagnostic and prognostic value of long noncoding RNA SNHG15 in pancreatic ductal adenocarcinoma Eur Rev Med Pharmacol Sci Sun XT Bai Y Yang C Hu SY Hou ZL Wang GX Long noncoding RNA SNHG15 enhances the development of colorectal carcinoma via functioning as a ceRNA through miR141SIRT1Wntβcatenin axis Artif Cells Nanomed Biotechnol Ye JF Tan LD Fu Y Xu HJ Wen LJ Deng Y Liu K LncRNA SNHG15 promotes hepatocellular carcinoma progression by sponging miR1413p J Cell Biochem Zhang JH Wei HW Yang HG Long noncoding RNA SNHG15 a potential prognostic biomarker for hepatocellular carcinoma Eur Rev Med Pharmacol Sci Zhang YL Zhang DH Lv J Wang S Zhang Q LncRNA SNHG15 Acts as an oncogene in prostate cancer by regulating miR3383pFKBP1A axis Gene Kong QL Qiu M Long noncoding RNA SNHG15 promotes human breast cancer proliferation migration and invasion by sponging miR2113p Biochem Biophys Res Commun Wang TQ Sun HB Bao Y En R Tian YJ Zhao W Jia LZ POM121 overexpression is related to a poor prognosis in colorectal cancer Expert Rev Mol Diagn Shuai Y Ma ZH Lu JW Feng JF LncRNA SNHG15 a new budding star in human cancers Cell Prolif 2020531e12716 Qu C Dai CM Guo YH Qin R Liu JB Long noncoding RNA Ma YW Xue YX Liu XB Qu CB Cai H Wang P Li ZQ Li Z Liu YH SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR153 Oncol Rep Li M Bian ZH Jin GY Zhang J Yao SR Feng YY Wang X Yin Y Fei BJ You QJ Huang ZH LncRNASNHG15 enhances cell proliferation in colorectal cancer by inhibiting miR3383p Cancer Med Liu YC Li JL Li F Li M Shao Y Wu LP SNHG15 functions as a tumor suppressor in thyroid cancer J Cell Biochem Liu YC Li JL Li M Li F Shao Y Wu LP microRNA5105p promotes thyroid cancer cell proliferation migration and invasion through suppressing SNHG15 J Cell Biochem Li YW Guo HY Jin CJ Qiu CP Gao M Zhang L Liu ZJ Kong BH Spliceosomeassociated factor CTNNBL1 promotes proliferation and invasion in ovarian cancer Exp Cell Res Williams GH Stoeber K The cell cycle and cancer J Pathol Yu B Ding YM Liao XF Wang CH Wang B Chen XY Overexpression of PARPBP correlates with tumor progression and poor prognosis in hepatocellular carcinoma Dig Dis Sci Maiuri AR Peng M Podicheti R Sriramkumar S Kamplain CM Rusch DB DeStefano Shields CE Sears CL O€™Hagan HM Mismatch repair proteins initiate epigenetic alterations during inflammationdriven tumorigenesis Cancer Res Berry D Seider M Stinnett S Mruthyunjaya P Schefler AC Ocular Oncology Study Consortium Relationship of clinical features and baseline tumor size with gene expression profile status in uveal melanoma a Multiinstitutional study Retina Jiang ZM Yu FH Li M Upregulation of BCL2 kD proteininteracting protein BNIP3 is predictive of unfavorable prognosis in uveal melanoma Med Sci Monit Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0c'

Thyroid_Cancer

SurveillanceWest Nile virus in humans Greece the largest seasonal number of cases years after its emergence in the countryDanai Pervanidou¹ Annita Vakali¹ Theano Geakopoulou¹ Takis Panagiotopoulos2 Eleni Patsoula² Gee Koliopoulos Constantina Politis¹ Kostas Stamoulis´ Elpida Gavanaµ Styliani Pappaµ Maria Mavrouli¶ Maria Emmanouil· Gee Sourvinos¸ Andreas Mentis· Athanassios Tsakris¶ Christos Hadjichristodoulou¹ Sotirios Tsiodras110 Anna Papaµ Hellenic National Public Health anizationformer Hellenic Center for Disease Control Prevention Athens Greece School of Public Health Faculty of Public Health Policy University of West Attica Athens Greece Benaki Phytopathological Institute Athens Greece Hellenic National Blood Transfusion Center Athens Greece National Reference Center for Arboviruses and Haemorrhagic Fever Viruses Department of Microbiology Medical School Aristotle University of Thessaloniki Thessaloniki Greece Department of Microbiology Medical School National and Kapodistrian University of Athens Athens Greece Diagnostic Services Laboratory Public Health Laboratories Hellenic Pasteur Institute Athens Greece Laboratory of Clinical Virology Medical School University of Crete Heraklion Crete Greece Laboratory of Hygiene and Epidemiology Medical School University of Thessaly Larisa Greece National and Kapodistrian University of Athens Athens GreeceCorrespondence Danai Pervanidou dpervanidoueodygovgrCitation style for this Pervanidou Danai Vakali Annita Geakopoulou Theano Panagiotopoulos Takis Patsoula Eleni Koliopoulos Gee Politis Constantina Stamoulis Kostas Gavana Elpida Pappa Styliani Mavrouli Maria Emmanouil Maria Sourvinos Gee Mentis Andreas Tsakris Athanassios Hadjichristodoulou Christos Tsiodras Sotirios Papa Anna West Nile virus in humans Greece the largest seasonal number of cases years after its emergence in the country Euro Surveill 20202532pii1900543 10280715607917ES202025321900543 submitted on Aug accepted on Feb published on Aug Background Human cases of West Nile virus WNV infection are recorded since in Greece with seasonal outbreaks occurring almost annually Enhanced surveillance has been implemented since to promptly characterise cases™ temporal and geographical distribution and inform authorities for implementation of appropriate measures mosquito control health education blood safety Aim We describe the epidemiology of WNV human infections in Greece focusing on the season Methods The National Public Health anization advised physicians to test all suspect WNV infection cases and refer samples to reference laboratories Laboratories notified diagnosed cases on a daily basis Treating physicians patients and infected blood donors were interviewed within hours after diagnosis and the probable infection location was identified Hospitalised cases were followed up until discharge Results A total of autochthonous WNV infection cases were diagnosed in Among them cases had neuroinvasive disease WNND representing a increase of WNND cases compared with the previous most intense season There were deaths Cases started occurring from week earlier than usual Both rural and urban areas were affected with of the total municipalities belonging to seven of the total regions recording cases Two major epicentres were identified in Attica and Central Macedonia regions Conclusions The largest number of human cases of WNV infection ever recorded in Greece occurred in with a wide geographical distribution suggesting intense virus circulation Enhanced surveillance is vital for the early detection of human cases and the prompt implementation of response measuresIntroductionWest Nile virus WNV is a flavivirus primarily transmitted to humans equids and other mammals through the bites of infected mosquitoes mainly of the a0Culex a0genus [] WNV lineages and have been associated with significant outbreaks in humans [] Birds serve as reservoir hosts [] whereas humans and equids are considered deadend hosts [] Transmission through blood transfusion and an transplantation can occasionally occur and other rare modes of transmission have been also recorded such as transmission from mother to child during pregnancy delivery or breastfeeding and in laboratory settings []Most humans infected with WNV remain asymptomatic ca develop a disease with fever andor other influenzalike symptoms known as West Nile fever WNF and less than develop neuroinvasive disease WNND such as encephalitis meningitis or “ more rarely “ acute flaccid paralysis AFP [] Elderly and immunocompromised persons are at higher risk of developing severe disease and having a fatal outcome [] No specific treatment or vaccine exists to cure or prevent the disease in humanswwweurosurveillance 0cWNV infection is considered endemoepidemic in parts of Europe affecting countries in southern eastern and western Europe [] and is considered a reemerging public health challenge in the European Union EU with annual seasonal outbreaks during the summer months and early autumnMigratory birds are thought to be the source of introductions of new virus strains into previously unaffected areas [] In Europe the virus has been introduced through migratory birds from Africa [] and its circulation is probably greatly influenced by the flying routes of migratory bird species [] WNV overwinters in mosquitoes [] and overwintering in local bird species in Europe cannot be excluded [] WNV lineage was the main lineage circulating in Europe [] from the 1960s whereas lineage spread over central and southern eastern Europe [] since causing major outbreaks [] In the largest number of cases of WNV infection was recorded in Europe with their total number exceeding the cumulative number of all cases recorded in the previous years between and []In Greece human cases of WNV infections have been recorded since [] with seasonal outbreaks from end June to early October on an almost annual basis [] The causative WNV strain Nea SantaGreece2010 belongs to the Central EuropeanHungarian subclade of WNV lineage [] This strain was detected in patients blood donors mosquitoes horses and birds in many seasons from to []Enhanced surveillance of human WNV infection is annually implemented since from May to November by the Hellenic National Public Health anization NPHOformer Hellenic Center for Disease Control Prevention HCDCP The surveillance is undertaken to promptly identify human cases of WNV infection and monitor their temporal and geographical distribution It also has the objective to detect cases and WNVaffected areas in a timely fashion to inform national and local authorities for the implementation of appropriate response measures including blood safety measures intensified vector control and communication campaigns In the long term surveillance aims to quantify the disease burden and identify seasonal geographical and demographic patterns and populations at risk [] Herein we describe the clinical characteristics and laboratory findings of human cases as well as their distribution in time and place during in GreeceMethodsSurveillanceAs part of routine procedures implemented over the last years before the beginning of each WNV season informative material was sent by the NPHOformer HCDCP to all healthcare facilities and medical associations around the country in May to raise awareness of physicians concerning the diagnosis of WNV infection The NPHOformer HCDCP prompted physicians to further conduct laboratory testing of all WNV infection suspected cases who were defined as any person with acute onset of neurological syndromes encephalitis meningitis or myelitis as well as any person with nonneurological illness but unexplained fever NPHOformer HCDCP recommended referral of samples to specific specialised laboratories for testingThe Vectorborne Diseases VBD Office of the NPHOformer HCDCP implemented active laboratorybased surveillance throughout the transmission season with daily communication with three referencespecialised laboratories further described in the ˜laboratory methods™ section and daily reporting of diagnosed cases to NPHOformer HCDCP Laboratory diagnosis was freeofcharge for patients in the referencespecialised laboratories reimbursed by NPHOformer HCDCPBlood safety authorities informed NPHOformer HCDCP about any infections diagnosed among blood donors tested in affected areasThe EU case definition of WNV infection [] was used with slight modifications in that only laboratory “ and not epidemiological “ criteria were used to define probable cases a confirmed case was defined as a person with either PCR detection of WNV nucleic acid in any biological specimen such as blood cerebrospinal fluid CSF or urine or detection of a WNV specific IgM antibody response in CSF or WNV IgM and IgG antibody response and confirmation by neutralisation tests A probable case was defined as a person with positive IgM antibody response only in serum Asymptomatic infections detected during blood screening in affected areas were also included “ for the first time in “ in the case definitionMunicipalities the lowest administrative unit where at least one human laboratorydiagnosed case of WNV infection was exposed during the period were defined as affected areasData collectionThe VBD Office of the NPHOformer HCDCP investigated and recorded all cases with laboratorydiagnosed WNV infection symptomatic or not with or without neuroinvasive disease The disease was classified as WNND encephalitis meningitis meningoencephalitis or myelitisAFP based on the treating physicians™ clinical assessment andor additional laboratory andor imaging findings when availableCases were investigated by the VBD Office with indepth telephone interviews with the patients or if this was not possible with their close relatives ideally within “ hours after diagnosis using a standardised investigation form to obtain detailed travel history during incubation period “ days before symptom wwweurosurveillance 0cTable Numbers of total WNV affected areasa infection cases WNND cases and related deaths and case fatality per year as well as annual incidence of total WNND cases Greece “2018b a0n WNV infection casesCriteriaNumber of affected municipalitiesNumber of affected regional unitscNumber of affected regionsNumber of WNV infection casesNumber of WNND casesPercentage of WNND casesIncidence of WNND cases per populationNumber of fatal cases with WNV infectionCase fatality among cases with WNV infectionNumber of fatal cases with WNNDCase fatality of cases with WNNDNANANANANANA2018b NA not applicable NUTS nomenclature of territorial units for statistics WNND West Nile virus neuroinvasive disease WNV West Nile virusa a0Number of affected municipalities regional unitsb and regions with ‰¥ case of WNV infectionb a0To put the data in a further more recent perspective the numbers for were affected municipalities affected regional units five affected regions WNV infection cases WNND cases corresponding to of WNV infection cases WNND cases per population fatal cases with WNV infection corresponding to a case fatality of among cases with WNV infection and fatal cases with WNND representing a case fatality of in cases with WNNDc a0Regional units NUTS3onset and identify the probable place of exposure In case of complex travel history the most probable place of infection was defined “ after risk assessment “ by a multisectoral working group of the Ministry of Health dedicated to designate the areas affected by VBDThe treating physician was interviewed to record the clinical manifestations and underlying diseases of each patientThe data collected included demographic characteristics clinical manifestations underlying chronic diseases dates of onset of symptoms hospitalisation diagnosis discharge admission and discharge from intensive care unit ICU potential risk factors laboratory results detailed travel history during the maximum incubation period Hospitalised patients were actively followed up on a daily basis by telephone and their outcome at the end of hospitalisation was recordedThe NPHOformer HCDCP informed on a daily basis by emails national regional and local public health authorities about the cases and their probable place of exposure in order to implement responsive preventive measures vector control communication activities blood safetyWeekly surveillance reports were published on the NPHOformer HCDCP websiteLaboratory methodsOn a daily basis the VBD Office of the NPHOformer HCDCP received laboratory data of the diagnosed including cases mainly the National Reference Center for Arboviruses and Haemorrhagic Fever Viruses Department of Microbiology Medical School Aristotle University of laboratories from three Thessaloniki the Department of Microbiology Medical School National and Kapodistrian University of Athens and the Diagnostic Services Laboratory Public Health Laboratories Hellenic Pasteur Institute of Athens in which the vast majority of suspected cases were tested and of cases were diagnosed in Serum and CSF specimens were tested for the presence of WNVspecific IgM and IgG antibodies using commercial ELISA kits Focus Technologies Cypress CA United States Reverse transcription RTPCR tests in house and commercial were performed on RNA extracted from blood CSF or urine samples The National Reference Center for Arboviruses performed an RTnested PCR which amplifies a 520bp fragment of the WNV nonstructural protein NS3 gene on PCRpositive samples [] All PCR products were sequenced in a ABI Genetic Analyzer Applied Biosystems Foster City CA US Nucleotide sequences were aligned with similar sequences of the same genomic region retrieved from the GenBank database and phylogenetic analysis was performed using the molecular evolutionary genetics analysis MEGA7 software [] The National Reference Center for Arboviruses performed also neutralisation tests in PCRnegative IgGpositive serum samples and applied PCR combined with Sanger sequencing for the genetic characterisation of the strains in addition next generation sequencing was performed on selected samplesBlood safety measuresBlood safety measures for the protection of blood donations against WNV infection were implemented nationwide for blood donors residing or having visited affected municipalities These measures included blood donor deferral or screening of donated blood for WNV RNA with targeted individual donation ID wwweurosurveillance 0cFigure Number of laboratory diagnosed West Nile virus neuroinvasive disease cases by week of symptom onset Greece “2018a n 242bsesac fo rebmuNJuneJulyAugustSeptemberOctoberWeek number and month of symptom onseta In and no cases were recorded Each box represents one WNND case diagnosed and reported in b One WNND case with undetermined week of symptom onset is not includednucleic acid amplification testing NAT and haemovigilance ie surveillance of serious adverse or unexpected events or reactions in donors or recipients epidemiological followup of donors as referred to in and Annex III of the Commission Directive 200433EC []Data analysisWe performed descriptive analysis of the surveillance data ie geographical and temporal distribution of human cases with WNV infection demographic characteristics age sex clinical manifestations underlying diseases and clinical outcomeWe assigned week numbers using the International anization for Standardization ISO standard []Ethical statementNo ethical approval was needed for this study as no individual data were identifiable and only aggregated data were analysed and presentedResultsOn week NPHOformer HCDCP was notified of the first six human WNV infection cases in the season in Attica Region with symptom onset on May week and within the first fortnight of June Cases continued to occur throughout the transmission period in several areas of the countryA total of autochthonous cases of WNV infection were recorded in all over Greece including WNND cases WNF cases and six asymptomatic cases blood donors The overall WNND incidence was cases per population this was the highest incidence ever recorded in Greece with a statistical significant increase compared with wwweurosurveillance201020112012201320142017 0cFigure Incidence per population of West Nile virus neuroinvasive disease by probable municipality of exposure Greece n 242a]]]]][]a For one case the suspected municipality of exposure could not be determinedp the previous most intense season Table Two of the reported WNV infection cases were diagnosed abroad in Czech Republic and in Italy respectively One more WNND case was classified as imported from Romania not included in the current analysisEleven cases of WNV infection were blood donors aged “ years old diagnosed in affected areas five of these cases developed symptoms and six remained asymptomaticIn of cases presented with WNND compared with a total of of cases diagnosed in the previous “ period ranging from in to in Table A total of cases of WNV infection or their close relatives were directly interviewed by NPHOformer HCDCP These included the exported case diagnosed in Italy who was also investigated in collaboration with the Italian public health authorities Four cases or their relatives diagnosed in Greece could not be directly contacted and the exported case diagnosed in the Czech Republic was investigated by the Czech public health authorities Close relatives of cases were interviewedFor cases of the interviewed cases the exact date of diagnosis in the laboratory was missing For the remaining cases these were investigated within a median period of day range “ after diagnosis of cases were interviewed within days after diagnosis and within dayAll symptomatic cases n had symptom onset within the month period from May week to October week while the last date of positive blood sample was on November from an asymptomatic blood donor The number of recorded WNND cases peaked in weeks and with and WNND cases per week respectivelyFigure a0 shows the reported WNND cases by week of symptom onset in compared with previous seasons “For three cases the probable place of exposure could not be determined The remaining WNV infection cases were exposed in a total of municipalities regional units and regions indicating a wide geographical distribution of cases compared with previous transmission periods Table The geographical distribution of WNND cases is presented in a0Figure Two major epicentres were recorded in the regions of Attica and Central Macedonia Table a0Figure with the highest number of WNV infection and WNND cases ever recorded in Attica and the second highest number of cases ever recorded in Central Macedonia since Table Among the regional units nomenclature of territorial units for statistics NUTS3 level affected in were affected at least once in previous seasons and of cases occurred in regional units previously affected Cases occurred both in rural and urban areas and large cities were also affected including the capitalIn June cases were recorded mainly from Attica and a couple of cases from the nearby Sterea Ellada region From July cases were also recorded in Central Macedonia and from August in East Macedonia and Thrace region in northeastern Greece In September“October cases were further recorded in Thessaly region A couple of cases were also recorded in Crete and Peloponnese regions Figure For cases of WNV infection with available information on their place of exposure the vast majority were infected in their place of permanent residence the rest mainly in the place of summer vacationThe median age of the WNND cases in was years range “ years significantly higher p than the median age of the total WNND cases diagnosed in the previous “ period years range “ Among WNND cases were aged years or older The incidence of WNND cases increased from per population in the less than yearolds to per population in those who were ‰¥ years old Table The age of WNND cases in median age years wwweurosurveillance 0cTable Number of WNV infection cases and WNND cases per probable region of exposure and year Greece “2018a a0n WNV infection casesProbable region of exposureAtticaCentral MacedoniaWest MacedoniaEast Macedonia and ThraceSterea ElladaThessalyPeloponneseCreteWest GreeceIonian islandsNorth AegeanSouth AegeanIpeirosUnknownundeterminedTotalNumber of recorded WNV infection cases number of WNND cases per yeara WNND West Nile virus neuroinvasive disease WNV West Nile virusa a0As no cases of WNV infection in Greece were recorded in and these years are not shown in the Tablerange “ was significantly higher p than that of the diagnosed symptomatic WNF cases median age years range “Among the WNV infection cases and the WNND cases n and n were male respectively The WNND incidence among males was almost two times higher than that among females p Table The median period from symptom onset to diagnosis for WNND cases with available relevant information was days range “ as well as for nonWNND cases excluding blood donors range “ The median period from admission to hospital to diagnosis for cases of WNV infection was days range “Among the WNND cases had encephalitismeningoencephalitis including patients also presenting AFP cases had meningitis and three cases had AFP signs only Table A total of diagnosed WNV infection cases reported clinical symptoms the most common being fever followed by malaisefatigue confusionconsciousness level deterioration anorexia sleepiness chills headache gastrointestinal symptoms diarrhoea nausea vomiting abdominal pain dizziness myalgiaarthralgia extrapyramidal signs tremor parkinsonism ataxiagait disorders rash limb paralysis numbness retroorbital pain vision deterioration lymph nodes enlargement Table Among WNND cases reported at least one underlying chronic disease including cardiovascular diseases including stroke and heart disease heart disease including hypertension hypertension diabetes mellitus coronary heart disease chronic neuropsychiatric disease arrhythmia cancer stroke valvulopathyheart failure other immunosuppression than diabetes and cancer including autoimmune disorders hepatic cirrhosis an transplant myasthenia corticosteroids treatment thyroid disease chronic renal failure respiratory disease alcohol abuse Among the symptomatic cases n were hospitalised of the WNND cases and of the symptomatic nonWNND casesThe median duration of hospitalisation of hospitalised cases of WNV infection discharged from the hospital was days range “ while among WNND cases hospitalised and discharged from the hospital it was days range “ Fortyeight cases were hospitalised in ICU with WNND while the one with nonWNND had other comorbidities The median duration of hospitalisation in ICU before discharge or fatal outcome was days range “A total of deaths were recorded during hospitalisation with an overall case fatality CF of among all cases diagnosed with WNV infection Among fatal cases had WNND with a CF among cases with WNND similar with the total CF among cases wwweurosurveillance 0cFigure Number of laboratorydiagnosed West Nile virus neuroinvasive disease cases by week of symptom onset and region of exposure Greece n 241asesac fo rebmuNRegionAtticaCentral MacedoniaCreteEast Macedonia ThraceSterea ElladaPeloponneseThessaly JuneSeptemberOctoberJulyAugustWeek number and month of symptom onsetWNND West Nile virus neuroinvasive diseasea One WNND case with undetermined week of symptom onset and one WNND case with undetermined region of exposure with symptom onset at week are not includedwith WNND observed during the “ period Table The clinical presentation of WNND among fatal cases is described in a0Table The CF of the encephalitismeningoencephalitis cases was whereas it was among meningitis cases The median age of fatal cases was years range “ The median period from symptom onset to death during hospitalisation was days range “The vast majority of patients were retired reported ng agricultural or gardening activities and reported having a routine outdoor activity after dawnNational and regional public health authorities were informed about diagnosed cases and their probable place of exposure within a few hours after their investigation and within hours after their diagnosis via confidential emailsLaboratory resultsOf the cases were confirmed either by WNVspecific IgM antibody response in CSF n andor positive PCR in any sample n in blood andor CSF andor urine andor by WNVspecific IgM and IgG antibody response in serum and confirmation by neutralisation n A total of cases were confirmed by IgM antibody response in CSF only cases were confirmed by positive PCR in any sample only cases were confirmed by neutralisation only and cases were confirmed by more than one confirmatory methodsA total of cases were considered as probable since the diagnosis was based only on the detection of WNVspecific IgM antibodies in seruminto In all but one of the sequenced samples the sequences clustered the Central EuropeanHungarian subclade of WNV lineage similar to the strain of the outbreak NeaSantaGreece2010 strain One sequence taken from a case in Thrace region northerneastern Greece clustered within the Eastern EuropeanRussian subclade of WNV lineage Figure [] a0In total among the cases tested had WNVspecific in serum and had WNVspecific IgM antibody response in CSF In cases with positive IgM and IgG antibodies in serum the diagnosis was confirmed by neutralisation A total of cases had positive PCR in blood cases had positive PCR in urine in of which this was the method of confirmation and eight cases had positive PCR in CSF The median time from symptom onset to sampling for the cases with positive PCR in blood was days range to for the cases with positive PCR in urine days range “ and for cases with positive PCR in CSF days range “IgM antibodies wwweurosurveillance 0cTable Age and sex distribution of West Nile virus neuroinvasive disease cases Greece n CharacteristicsNumber of casesIncidence rate per populationaRisk ratio confidence intervalAge group in years ““““““‰¥SexFemaleMaleReference “ “ “ “ “ “ “Reference a0 a0 a0 a0 a0 a0 “a a0Population data from Hellenic Statistical Authority ELSTAT []Table Number and percentage of WNND cases n as well as number and percentage of fatal cases with WNND n by clinical presentation Greece ParameterEncephalitisMeningo encephalitisMeningitisAFP onlyAFP and encephalitis meningitisNumber of WNND casesPercentage of total WNND casesNumber of fatal cases with WNNDPercentage of fatal cases among total WNND fatal casesNAAFP acute flaccid paralysis NA not applicable WNND West Nile virus neuroinvasive diseaseIn each row where numbers of cases are presented the sum of the cases in the respective row can be more than the total number of cases as a given case could have more than one presentationDiscussionWNV infections show a strong seasonal pattern in Europe with the first human cases usually observed in June and most cases recorded from July to October [] Accordingly from to human cases of WNV infection in Greece were recorded from the end of June until the beginning of October In contrast the onset of symptoms in the first recorded human case in was in week end of May making this the earliest WNV transmission season ever noted in the country and in Europe The seasonal outbreak moreover lasted until early November Figure and coincided with the longest season ie more than months in the last years in Europe [] suggesting a possible need to widen the surveillance periodThe number of recorded WNND cases in Greece in peaked in weeks and and remained at over per week from week to week The numbers of total WNV infections and WNND cases were also respectively the highest recorded since the emergence of the virus in the country in Moreover the WNND cases accounted for more than a third of the total number of such cases recorded since This suggests a more intense seasonal virus circulation during In this regard large outbreaks occurred in other central European and Mediterranean countries simultaneous to the WNV seasonal outbreak in Greece []The increased number of cases diagnosed in Greece in compared with previous years cannot be attributed to a higher sensitivity of the surveillance system as enhanced surveillance has been consistently implemented in the country during each transmission season since This has included raising awareness of physicians on an annual basis publishing updated weekly surveillance reports and providing freeofcharge diagnosis The large number of WNF cases diagnosed in of the total symptomatic cases and in previous years Table further indicates the enhanced awareness of physiciansIn the case definition was expanded to include asymptomatic cases detected during blood screening in affected areas however this change did not crucially affect the overall amount of recorded WNV infections given the small number of diagnosed asymptomatic cases n Either way the number of WNND cases which describes the burden more accurately and is wwweurosurveillance 0cTable Symptomsa a0of West Nile virus neuroinvasive disease cases n and West Nile fever cases n Greece 2018bWNND casesWNF casesSymptomFeverMalaisefatigueConfusion consciousness level deteriorationAnorexiaSleepinessChillsHeadacheAt least one gastrointestinal symptom ie diarrhoea nausea vomiting abdominal painDizzinessMyalgiaarthralgiaVomitingExtrapyramidal signs tremor parkinsonismAtaxia gait disordersDiarrhoeaNauseaRashAbdominal painLimb paralysisNumbnessRetroorbital painVision deteriorationLymph nodes enlargementProportionb a0with the symptomPercentageProportionb a0with the symptomPercentageNANANACKP creatine phosphokinase NA not applicable SIADH syndrome of inappropriate antidiuretic hormone secretion WNF West Nile fever WNND West Nile virus neuroinvasive diseasea a0Other symptomssigns reported from the treating physicians included acute respiratory failure n bradypsychismus n seizures n respiratory infection n dysarthriaspeech disorders n acute renal failure n diplopia n myoclonous n aphasiaapraxia n arrhythmia n cough n hepatitisliver dysfunction n rhabdomyolysisincreased CPK n SIADHhyponatraemia n biphasic fever n pulmonary oedema n sore throat n chorea ballistic movements n dysautonomia n hearing disorders n myocarditisincreased troponine n pulmonary embolism n hypokalaemia n acute epiglottitis n aneurysm rupture n blepharoptosis n Guillain“Barré syndrome

Thyroid_Cancer

Is preoperative heart rate variability aprognostic indicator for overall survival andcancer recurrence in patients with primarycolorectal cancerM T A StrousID1 A M Daniels1 F M Zimmermann2 F N van Erning3 Y Gidron4 FJ Vogelaar1 Department of Surgery VieCuri Medical Centre Venlo The Netherlands Department of CardiologyCatharina Hospital Eindhoven The Netherlands Department of Research Netherlands ComprehensiveCancer anisation Utrecht The Netherlands Faculty of Welfare and Health University of Haifa HaifaIsrael maudstrousviecurinlAbstracta1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Strous MTA Daniels AM ZimmermannFM van Erning FN Gidron Y Vogelaar FJ Ispreoperative heart rate variability a prognosticindicator for overall survival and cancer recurrencein patients with primary colorectal cancer PLoSONE e0237244 101371journalpone0237244Editor Louise Emilsson University of OsloNORWAYReceived February Accepted July Published August Copyright Strous This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data cannot beshared publicly because of ethical concernsPatients were included on a no objection base toconduct retrospective data studies and publishfindings but were not asked for permission topublish full encrypted data Data are available fromthe VieCuri Institutional Data Access contact viawetenschapsbureauviecurinl for researcherswho meet the criteria for access to confidentialdataBackgroundHeart Rate Variability HRV represents efferent vagus nerve activity which is suggested tobe inversely related to fundamental mechanisms of tumorigenesis and to be a predictor ofprognosis in various types of cancer HRV is also believed to predict the occurrence andseverity of postoperative complications We aimed to determine the role of preoperativeHRV as a prognostic factor in overall and cancer free survival in patients with colorectalcancerMethodsRetrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between January and December who underwent curative surgical treatment HRV was measured as timedomain parameters SDNN StandardDeviation of NNintervals and RMSSD Root Mean Square of Successive Differencesbased on preoperative second ECGs Groups were created by baseline HRV Low HRVSDNN 20ms or RMSSD 19ms and normal HRV SDNN �20ms or RMSSD �19msPrimary endpoints were overall and cancer free survivalResultsA total of patients were included in this study HRV was not significantly associated withoverall survival SDNN 20ms vs SDNN �20ms244 vs adjusted HR “ p RMSSD 19ms vs RMSSD �19ms270 vs adjustedHR “ p or cancer recurrence SDNN 20ms vs�20ms201 vs adjusted HR “ p RMSSD 19ms vs�19ms vs adjusted HR “ p There was noPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existsignificant association between HRV and CEAlevel at one year followup or between HRVand occurrence of a postoperative complication or the severity of postoperativecomplicationsConclusionsHeart rate variability was not associated with overall or cancer free survival in patients withprimary colorectal cancer who underwent curative surgical treatment These results do notalign with results found in studies including only patients with advanced cancer which suggests that there is only an association in the other direction cancer causing low HRVIntroductionIn there were over million newly diagnosed colorectal cancer patients worldwide andover in the Netherlands alone It is the fourth most common cause of death worldwide[] To improve survival it is of importance to get a better insight into modifiable prognosticfactors Emerging evidence suggests that vagal nerve activity indexed by heart rate variabilityHRV could be one of these prognostic factors [“]HRV is the physiological phenomenon of the fluctuation in time intervals between adjacentheartbeats and represents efferent vagus nerve activity to the heart [“] It has been suggestedthat efferent vagal activity is inversely related with fundamental mechanisms of tumorigenesisas inflammation oxidative stress and excessive sympathetic activity [] These mechanisms arebelieved to be controlled by the vagus nerve via a bidirectional braintoimmune pathwaysthrough the release of neurotransmitters via the cholinergic antiinflammatory pathway[] A higher vagal tone may reflect a more flexible topdown regulation of the immunesystem and physiological activity moderated by the brain [] Absence of vagus activity due tovagotomy has been shown to increase the risk of developing colorectal cancer []In addition to influencing development of cancer vagus nerve activity seems to be a predictor of prognosis in various types of cancer Recent studies show an association betweendecreased activity of the vagus nerve and worse survival in patients with cancer of the gastrointestinal tract liver pancreas lung prostate and breast among others [] Also patientswith normal HRV seem to live longer in different sorts of metastatic cancer independent ofconfounders [] In patients with colorectal cancer a low HRV at baseline has shown to beassociated with higher CEA levels at months after diagnosis which predicts a poorer prognosis []In patients undergoing curative treatment for colorectal cancer HRV does not only seemto influence cancer prognosis A recent study showed that patients with lower HRV have moreintraoperative blood loss and more and more severe postoperative complications []Identifying patients with low HRV is easy and noninvasive When its predictive value forthe prognosis of cancer patients is of satisfactory significance vagus nerve activation prior toor during cancer treatment could theoretically be beneficial in improving prognosis [] Alsoif we could predict the occurrence and severity of postoperative complications based on HRVimproving HRV before surgery could possibly accelerate postoperative recovery and indirectlyaffect patients™ prognosis Recent studies focussing on improving HRV by improving physicalfitness by means of physical exercise show promising results in both older men and woman[] However the only previous study on colon cancer and HRV including patients receiving curative treatment included a small sample and did not examine whether HRV predictsPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancersurvival in these patients [] To clarify the predictive value of HRV in prognosis of patientswith colorectal cancer further exploration is needed Current studies identifying HRV as aprognostic factor did not specifically focus on colorectal cancer have small study populationsdid not correct for confounders and mainly focused on metastatic disease [“]The aim of this study was to determine the role of preoperative HRV as a prognostic factorin overall and cancer free survival in patients with primary colorectal cancer who underwentcurative surgical treatmentMethodsData collectionData from the Netherlands Cancer Registry NCR were used The NCR collects data on allnewly diagnosed cancer patients in the Netherlands Information on patient and tumour characteristics diagnosis and treatment is routinely collected from the medical records by trainedadministrators of the cancer registry Anatomical site of the tumour is registered according tothe International Classification of Diseases Oncology The tumournodemetastasis TNMclassification is used for stage notification of the primary tumour according to the editionvalid at time of cancer diagnosis Quality of the data is high due to thorough training of theregistration team and consistency checks []For the study population additional data were collected from the medical records of thepatients This encompassed information on HRV CEAlevels ASA classification comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroid disease pulmonary disease vascular disease neurological disease and otheroccurrence and severity of postoperative complications and cancer recurrence Groups ofcomorbidities were chosen based on matching features within these groups and their potentialinfluence on HRV or the endpoint being analysed Severity of the postoperative complicationsaccording to the ClavienDindo classification was also documented Medical records wereassessed between January and July and reevaluated for revision of this between the 20th and 25th of April This study was approved by the research committee and the Board of Directors of VieCuriMedical Centre Data was obtained under the law ˜scientific research and statistics in the interest of public health where asking for permission is not possible or appropriate for several reasons™ in the Netherlands unless patients objected to use of their personal medical record forscientific research Data was encrypted with an encryption key provided by the NCR Encryption was shortly lifted to access the patients™ number for accessing hisher medical record Following extraction data were encrypted againStudy populationThe study population included all consecutive patients diagnosed with primary colorectal adenocarcinoma between January and January at VieCuri Medical Centre who underwent curative surgical treatment Patients with metastatic disease at time of surgery orcarcinoma in situ were excluded as their treatment and prognosis differs from those receivingcurative treatment for colorectal cancer Metastasis found within months after surgery wereconsidered present at time of surgery and therefore excluded Other excluded patients werepatients with neuroendocrine tumours because of different tumour characteristics and prognosis patients with cardiac arrhythmias including atrial and ventricular extrasystole pacemakers patients taking betablockers as this enhances HRV indexes or patients withbradycardia heart rate bpm or tachycardia heart rate bpm as this precluded reliable calculation of HRV [] We did not exclude patients taking alphablockers calciumPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerinhibitors diuretics amiodarone ACEinhibitors or ARB™s as these types of medicationreduce central sympathetic functioning rather than peripheral and their influence on HRV istherefore less univocal and sometimes completely absent []Heart rate variabilityHeart rate variability was analysed using a 12lead 10second ECG 150Hz used for preoperative screening In case of multiple ECG™s per patient the most recent ECG before date of surgery was used for HRVanalysis In case of multiple ECG™s per patient on the same date theECG with the best quality was chosen meaning an ECG without motion artefacts In case ofmotion artefacts there was always an ECG without motion artefacts available recorded on thesame date Time between two consecutive Rpeaks was measured in lead II with an accuracy of02ms using MUSEECG HRV was presented using the timedomain HRV parameters SDNNStandard Deviation of NNintervals and RMSSD Root Mean Square of Successive Differences in milliseconds calculated using the following calculations []rSDNN ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRi 00 RRmeanÞ2Pnn 00 RMSSD ¼rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRiþ1 00 RRiÞ2Pn 00 n 00 ð1Þð2ÞSDNN and RMSSD obtained from 10s ECGs were found to correlate with results of ECGsof longer durations Power spectral analysis HRV parameters as LF and HF can only beobtained in longer recording ECGs and were therefore not implementable in this study[]SDNN and RMSSD were both analysed as continuous variables as well as binary variablesusing cutoffs of 20ms versus �20ms and 19ms versus �19ms respectively In case of anSDNN 20ms or RMSSD 19ms HRV was classified as low and in case of SDNN �20ms orRMSSD �19ms as normal These cutoff values were based on cutoff values used in otherstudies showing an association between lowHRV as SDNN 20ms and RMSSD 19ms andcolorectal cancer as there is no standardised definition of low and normal HRV []Endpoints and definitionsThe primary endpoints of this study were overall and cancer free survival Overall survival wasdefined as the time between the date of surgery to the date of death or last followup date inmonths Cancer free survival was defined as the time in months from the date of surgery untilthe date of cancer recurrence defined as the first date of either radiologic or pathologic diagnosis of metastases or tumour recurrence of colorectal cancer Patients dying without cancerrecurrence were censored on day of death Secondary endpoints were elevated CEAlevel ugl at oneyear followup occurrence of postoperative complications within daysafter surgery and severity of postoperative complications according to the ClavienDindoclassificationStatistical analysisIn this retrospective observational cohort study we utilized descriptive statistics to provide anoverview of control variables of the study population patient characteristics as age sex BMIcomorbidities and ASAclassification heart rate and tumour characteristics as TNMstagetumour localisation and tumour differentiation and their association with HRV andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerprognosis Normal distribution of the continuous variables heart rate age and BMI as well asSDNN and RMSSD were tested with a KolmogorovSmirnov test Because of normal distribution heart rate age and BMI were compared between HRVgroups using unpaired ttest Allother variables were categorical and were compared between HRVgroups using Chisquarestatistics as groups were all of sufficient powerDifferences in overall survival and cancer free survival in months according to SDNN andRMSSD were visualized by means of KaplanMeier curves and statistically tested using the logrank test Multivariate coxregression analyses were conducted to calculate the prognosticassociation between HRV and overall and cancer free survival while adjusting for other prognostic variables Multivariate logistic regression was used to assess the independent effect ofSDNN and RMSSD on CEAlevels and the occurrence and severity of postoperative complications Variables included for adjustment were chosen by means of forward stepwise selectionbased on clinical judgment differences at baseline eg differences on any predictor betweenpatients who later died or not and database availability and depended on the analysed endpoint Those included patient demographics age sex bodymassindex comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroiddisease pulmonary disease vascular disease neurological disease other including Crohn™sdisease hepatitis kidney failure disorders anaemia depression arthritis tumour characteristics localisation stage differentiation and the occurrence of postoperative complicationswhen the later was not an outcome Differences in CEAlevel at baseline and one year checkup between and within groups of low HRV and normal HRV were assessed with a repeatedmeasures linear model and tested using the tukey test To test the implication of a longer timebetween ECG and treatment all analyses were repeated after excluding patients with an ECGolder than months A twotailed pvalue � was considered significant in all analysesData were analysed using IBM SPSS Statistics version IBM Corp NY Armonk USAResultsOf colorectal cancer patients that underwent a surgical resection a total of patientswere included in this study Reasons for exclusion are presented in Fig Median SDNN andRMSSD were 204ms interquartile range IQR 115ms351ms and 175ms IQR 99ms299ms respectively Table shows descriptive data of the included patients by HRV groupsBaseline heart rate and age were negatively associated with HRV The group of patients withlow HRV contains more patients with a history of cardiac disease regardless of the HRV defining parameter When defining low HRV by RMSSD 19ms more patients in this group havehypertension as comorbidity This group also contains more patients with an ASA classification greater than oneDuring a median followup of months IQR “ all causedeath occurred in patients Cancer recurrence occurred in patients during a median followup of months IQR “To rule out any distort in results caused by a delay between ECG and treatment all analyseswere repeated after exclusion of ECG™s older than months This did not lead to any new significant results Therefore these results were not displayed in detail in this paperSurvivalIn low HRV groups slightly more patients died compared to normal HRV groups SDNN20ms versus �20ms versus respectively RMSSD 19ms versus�19ms versus respectively These observed differences between HRVgroups in overall survival were not significant Fig A SDNN p B RMSSDPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Flowchart of the study101371journalpone0237244g001p After adjustment for some potential confounders these associations remained notsignificant SDNN 20ms versus �20ms HR “ p and RMSSD19ms versus �19ms HR “ p Tables and Age cardiac disease tumour stage and postoperative complications had a significant influence on overall survival Age also differed at baseline and was identified as a confounder When defining low andnormal HRVgroups by SDNN cardiac disease was identified as confounder When conducting sensitivity analyses with SDNN and RMSS as continuous variables results remained thesame There was no association between HRV and overall survival SDNN HR “ p and RMSSD HR “ p In low HRV groups slightly more patients had recurrence of cancer compared to normalHRV groups SDNN 20ms versus �20ms versus respectivelyRMSSD 19ms versus �19ms versus respectively These observed differences between HRV groups in cancer free survival were not significant Fig A SDNNp B RMSSD p As in overall survival after adjustment for some potentialconfounders these associations remained not significant SDNN 20ms versus �20msHR “ p and RMSSD 19ms versus �19ms HR “ p Tables and In SDNNbased groups baseline heart rate was identified asa confounding variable Sensitivity analyses with SDNN and RMSSD as continuous variablesPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Descriptive data of included patients according to prespecified HRV groupsSDNN 20ms n SDNN �20ms n pRMSSD 19ms n RMSSD �19ms n pHRV and prognosis in colorectal cancerHeart Rate�Age�Age n year� yearSex nMaleFemale [“] [“] [“] [“] [“] [“] [“] [“] Mean BMI SD [“] [“] [“] [“]Comorbidities nCardiac diseaseHypertensionDiabetes MellitusThyroid diseasePulmonary diseaseVascular diseaseNeurological diseaseOtherASA nASA1ASA2ASA34Localisation tumour nRight colonLeft colonRectumTumour stage nIIIIII Differentiation grade nWellmoderate Poorundifferentiated � Non normaldistributed data presented as median with interquartile range101371journalpone0237244t001did not alter these results There was no association between HRV and cancer free survivalSDNN HR “ p and RMSSD HR “p CEAlevelCEAlevel at baseline and one year checkup was registered in patients and elevated in of these patients This was elevated at one year checkup in only patients Low HRV was notsignificantly associated with elevated CEAlevels at one year checkup as shown in Table Sensitivity analyses with SDNN and RMSSD as continuous variables did not alter these resultsSDNN HR “ p and RMSSD HR “PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for overall survival in groups of low HRV and normal HRV presented as A SDNN andB RMSSD101371journalpone0237244g002PLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of SDNN and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerSDNN20ms�20msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ “ “ reference “ “ reference “ “Postoperative complicationNoYes reference reference “ “101371journalpone0237244t002p Adjusting for covariates was not possible because of the small number of patientswith an elevated CEAlevelDifferences between CEAlevel at baseline and one year checkup were compared betweenand within HRVgroups Results were displayed in Fig There were no significant differencesin CEAlevel at baseline and one year checkup between the low HRV group and normal HRVgroup defined by SDNN and RMSSD p and p respectively Also there were nosignificant differences in CEAlevel at baseline and at one year checkup within the low HRVgroup and normal HRV group defined by SDNN and RMSSD p and p respectivelyPostoperative complicationsIn patients one or more postoperative complications occurred within days after surgeryThe occurrence of a postoperative complication was not significantly associated with lowHRV defined as SDNN 20ms or RMSSD 19ms even after adjustment for some potentialconfounders Table Heart rate age cardiac disease and hypertension were identified asconfounding covariates When conducting sensitivity analyses with SDNN and RMSS as continuous variables the association between occurrence of a postoperative complication withPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of RMSSD and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerRMSSD19ms�19msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “Postoperative complicationNoYes reference reference “ “101371journalpone0237244t003baseline HRV remained not significant SDNN HR “ p andRMSSD “ p The same applied when postoperative complicationswere graded according to the ClavienDindo classification and the complication that is gradedthe highest for each patient is compared to the absence of postoperative complicationsTable DiscussionIn this observational cohortstudy we determined the Heart Rate Variability in preoperativeECGs of patients with primary colorectal cancer who underwent curative surgical treatment HRV refers to physiological variations in beattobeat intervals It was presented intimedomain parameters SDNN and RMSSD HRV was not significantly associated with overall survival or cancer free survival independent of some risk factors Also this study showedno significant differences in CEAlevels at one year checkup between patients with low HRVand patients with normal HRV Patients with low HRV did not have more or more severepostoperative complications compared to patients with normal HRVTumorigenesis has three fundamental mechanisms inflammation promoting oxidativestress and stimulating tumour growth oxidative stress causing DNAdamage andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for cancer free survival in groups of low HRV and normal HRV presented as A SDNNand B RMSSD101371journalpone0237244g003PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Univariate analyses of low HRV and risk of elevated CEAlevel at one year checkupCEA μglOR CIpCEA μglOR CIIndependent of baseline CEAIndependent of baseline CEASDNN 20ms n “RMSSD 19ms n “SDNN �20ms n Normal baseline CEA � μglSDNN 20ms n SDNN �20ms n Elevated baseline CEA μglSDNN 20ms n reference “ reference “RMSSD �19ms n Normal baseline CEA � μglRMSSD 19ms n RMSSD �19ms n Elevated baseline CEA μglRMSSD 19ms n reference “ reference “SDNN �20ms n referenceRMSSD �19ms n referencep101371journalpone0237244t004interfering with subsequent repair mechanisms and sympathetic neurotransmitters stimulating tumour metastasis and progression [] It has been suggested that afferent fibres of thevagus nerve inform the brain about peripheral inflammation This is followed by a braintoimmune response via the efferent route of the vagus nerve that modulates the function ofimmuneregulatory cells through the release of neurotransmitters via the cholinergic antiinflammatory pathway Malfunctioning of this route may play part in the onset of cancer []This theory has been supported by studies of patients with gastric ulcers who underwent avagotomy who then showed an increased risk of developing lung and colorectal cancer [“] The vagus nerve is also believed to modulate tumour progression An active vagus nervecan inhibit inflammation oxidative stress and the release of sympathetic neurotransmittersthat stimulate tumour metastasis and progression [“] Absence of this inhibitory effect inturn results in metastasis and tumour progression as shown in a study of Erin showingthat mice who underwent chemical vagotomy developed more metastasis of breastcancer cellsthan controls [] Epidemiologically low HRV has been associated with worse overall survivalover time in patients with recurrent or metastatic cancer of various types even after correctionfor confounders [“] However the results of the present study do not support thesefindingsTo our knowledge this is the first study including only patients with colorectal cancer whoare eligible for curative treatment by partial bowel resection and not those receiving palliativetreatment De Couck studied the relationship between HRV and tumour burden in bothcurative and palliative patients with prostate cancer or nonsmall cell lung cancer Independent of confounders the hypothesised inverse relationship of HRV and the tumour markerPSA at and months after diagnosis was only significant in patients with stage IV prostatecancer not stage II and III [] In colorectal cancer Mouton found that low HRV definedas SDNN ms predicted significantly higher levels of the tumour marker CEA at months after diagnosis Again these results were only found in patients receiving palliativetreatment not curative [] Only one previous study showed a significant inverse relationshipbetween HRV and mortality in cancer in general independent of stage [] This study of Guo had a large study population of patients with various types of cancer Low HRV wasdefined as SDNN 70ms and was significantly associated with shorter survival times Thissuggests that HRV is a predictive indicator of survival time not only in palliative but also incurative patients However results were not controlled for cancer type which could affect bothHRV and survival and should therefore be interpreted with caution [] The fact that thepatients recruited in the present sample were only with less advanced cancer may partlyexplain the lack of prognostic role in HRV in this samplePLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Bar chart for CEAlevel at baseline and one year checkup in groups of low HRV and normal HRV presented as ASDNN and B RMSSD101371journalpone0237244g004PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and risk of occurrence of a postoperative complication within daysPostoperative complicationOR CIUnadjustedSDNN 20ms n “SDNN �20ms n referenceAdjusted�SDNN 20ms n “SDNN �20ms n referenceUnadjustedRMSSD 19ms n “RMSSD �19ms n referenceAdjusted�RMSSD 19ms n “RMSSD �19ms n reference�adjusted for heart rate age cardiac disease and hypertension101371journalpone0237244t005pSome of these previous studies suggest a bidirectional relationship between vagus nerveactivity and cancer [] However based on current evidence on this subject we cannot supportthis hypothesis The positive association between low HRV and worse prognosis found inpatients with colorectal cancer receiving palliative treatment but not in patients receivingcurative treatment suggest that this relation is not bidirectional but that advanced cancer isassociated with low HRV Midlatestage tumours are often accompanied by damage to autonomic nerves resulting in decreased HRV [] A study of De Couck showed that cancerpatients in general have a significantly lower HRV than healthy people [] The same resultswere found in studies of Bijoor where RMSSD was found to be significantly lower inpatients with early and advanced stage cancer compared to a healthy control group []When comparing patients with advanced stage cancer TNM III and IV to patients with anearly stage of cancer TNM I and II RMSSD was found to be significantly lower in patientswith advanced stages of cancer [] Thus though experimental studies in animals showthat vagal nerve functioning can causally slow tumorigenesis the human data suggests that themalignant tumour causes vagal nerve dysfunction and therewith decreased HRV []Besides the proposed influence of low HRV on survival of colorectal cancer patientsthrough development and increased progression of cancer Reimer suggested that lowHRV could influence survival of those undergoing surgical treatment due to more and moresevere postoperative complications [] However the results found in this study were notconcurrent with those of Reimer who included patients with ASA undergoingelective surgery Their analysis of HRV was through powerspectrum parameters based on longer ECGrecordings instead of the timedomain parameters based on 10s ECGs used in thisstudy But the difference in used parameters used in both studies is probably not the explanation for the differences in results between both studies since it has been demonstrated thatRMSSD and SDNN based on ECG recordings of 10s are in substantial agreement with those of45min and a 10s ECG therefore suffices to determine time domain HRV parameters HoweverReimer did not correct for possible confounders In their study patients with low HRVwere more likely to have diabetes a known risk factor for postoperative ileus and wound infections both found to be common postoperative complications in their low HRV group Correcting for this comorbidity may change the significance of their findings [] A study ofPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and severity of postoperative complications according to ClavienDindo classificationUnadjustedMinor grade I and IIOR CIpMajor grade III IV VOR CISDNN 20msn “n “SDNN �20msn referencen referenceAdjusted�SDNN 20msn “SDNN �20msn referenceUnadjustedRMSSD 19msn “RMSSD �19msn referenceAdjusted�n “n referencen “n referenceRMSSD 19msn “n “RMSSD �19msn referencen referencep�adjusted for heart rate age categories vs � and hypertension101371journalpone0237244t006Scheffler did not show any significant preoperative differences in HRV between patientswith or without postoperative complications or between patients with postoperative complications of different severity levels [] Thus also in relation to predicting postoperative complications results are not uniform

Thyroid_Cancer

"Accumulating evidence has revealed the critical role of long noncoding RNAs lncRNAs in cellularprocesses during tumor progression As documented in cancerrelated literatures LINC00992 expression isassociated with cancer progression whereas its function in tumors including prostate cancer has not beencharacterized yetMethods Data from GEPIA database suggested LINC00992 expression in prostate cancer tissues The expressionlevels of RNAs were monitored via qRTPCR Western blot evaluated the levels of proteins The proliferationapoptosis and migration of prostate cancer cells were assessed by CCK8 EdU TUNEL Transwell and woundhealing assays Luciferase reporter RNA pull down and RIP assays were applied to detect the interplays amongLINC00992 miR3935 and GOLM1Results Elevated levels of LINC00992 and GOLM1 were detected in prostate cancer tissues and cells LINC00992exerted facilitating functions in prostate cancer cell proliferation and migration Mechanically LINC00992 interactedwith and negatively regulated miR3935 to elevate GOLM1 expression in prostate cancer cells In addition thein vitro suppressive effect of silenced LINC00992 on prostate cancer cell proliferation and migration was reversed byGOLM1 upregulation Likewise LINC00992 depletion restrained tumor growth in vivo was offset by enhancedGOLM1 expressionConclusions LINC00992 competitively bound with miR3935 to elevate GOLM1 expression and therefore facilitatethe oncogenic phenotypes of prostate cancer cells implying a potential LINC00992targeted therapy for prostatecancerKeywords INC00992 miR3935 GOLM1 Prostate cancer Correspondence engineyangsinacom5Department of Urology the Second Affiliated Hospital of Bengbu MedicalCollege Hongye Road Bengbu Anhui ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Cancer Page of BackgroundClinically prostate cancer manifests as a dominatingcause of malerelated death worldwide and is characterized as the most continually occurred tumor amongmen in the United States [] The biggest challenge isthe detectable bone metastases in roughly advancedprostate cancer [] Virtually all prostate cancer patientsduring years™ androgen deprivation treatment inevitably undergo castrationresistance which contributes tothe poor clinical consequences in prostate cancer []However the mechanism underlaid prostate cancer remains mostly unknownThe widely studied long noncoding RNAs lncRNAsare transcribed from nonproteincoding human genomeand have more than nt in length [] LncRNAs are increasingly functionally identified and experimentally consolidated to be related to tumor neoplasia and progressionin diverse cancers [] Additionally lncRNAs with dysregulation can functionally modulate tumor developmentfrom multiple pathological aspects such as cell proliferation drugresistance and metastasis [“] For examplelncRNA A1BGAS1 inhibits cell proliferation and invasionin hepatocellular carcinoma via targeting miR216a5p []LncRNA LOC730100 sponges miR760 from FOXA1 toaccelerate cell proliferation and invasion in glioma []LncRNA SNHG16 functions as an oncogene in hepatocellular carcinoma [] Long intergenic nonprotein codingRNA LINC00992 is a novel lncRNA that has beenpreviously revealed to be elevated in tumors and substantiated as a master regulator for chemoresistance [] Besides LINC00992 has been uncovered as an elevatedlncRNA in prostate cancer [] which is consistent withthe detection from GEPIA database Despite that no previous study has given a comprehensive explanation aboutthe precise function or detailed mechanism of LINC00992in prostate cancerIn past decades the fact that lncRNAs function in tumors depending on their secondary or tertiary structureshas been reported in many cancerlinked studies For instance in the nucleus lncRNAs are entitled to work asmolecular scaffolds or alternative splicing assistants [] On the contrary lncRNAs dispersing in cytoplasm influence downstream mRNA translation or degradationthrough serving as miRNA sponges [ ] For exampleTNFαinduced lncRNA LOC105374902 promotes themalignant behaviors of cervical cancer cells by acting as asponge of miR12853p [] LncRNA TTNAS1 promotes papillary thyroid cancer tumorigenesis by regulatingmiR1533pZNRF2 axis[] Nevertheless whetherLINC00992 could exert its functions in prostate cancer viaits sponging role of certain miRNA remains unknownWe conducted this research aiming to explore thefunction or probable mechanism of LINC00992 in prostate tumor which might enrich the understanding interms of prostate tumor pathology and contribute to awider applied scopeMethodsTissue samplesThe prostate cancer tissue samples and matched peritumor tissue samples were collected from patientsdiagnosed with prostate cancer under the approval ofthe Ethics Committee of the First Affiliated Hospital ofKunming Medical University Each participant did notreceive radiotherapy and chemotherapy prior to tissuecollection and signed the written informed consentsbefore this study All samples were snapfrozen in liquidnitrogen and then stored at °C until required forfurther analysisCell cultureThe prostate epithelial cell line RWPE1 CRL11609and prostate cancer cellsincluding PC3 CRL1435LNCaP CRL1740 C4“ CRL3314 and DU145HTB81 were all purchased from American TypeCulture Collection ATCC Manassas VA USAinOctober All cells were cultured as recommendedin Dulbecco™s modified Eagle™s medium containing FBS GIBCO MA USA under the condition of a cellincubator with CO2 at °C Before using in thisstudy all cell lines were authenticated by STR profilingand tested for mycoplasma contamination in June Cell transfectionLINC00992 shRNA or negative control shRNA andpcDNA31LINC00992 pcDNA31GOLM1 or its emptycontrol pcDNA31 plasmid were chemically synthesizedand provided by Gene Pharma Shanghai China MiR mimics miR3935 inhibitor and theirrelatednegative controls NCmimics NCinhibitor were allpurchased for upregulating or downregulating miR3935from Ribobio Guangzhou China In line with the directions of LipofectamineTM RNAiMAX TransfectionReagent Thermo Fisher Scientific transfection of theseplasmids into DU145 PC3 and RWPE1 cells wasconducted and qRTPCR checked the transfection efficiency The sequences were as follows shNC ²CCGGTAGTAATTGACAACCATTATACTCGAGTATAATGGTTGTCAATTACTATTTTTG3²shLINC009921²CCGGATTATCCAAGAGTATTAACATCTCGAGATGTTAATACTCTTGGATAATTTTTTG3² shLINC0 ²CCGGTGTTAGATGATCATTGAGGTGCTCGAGCACCTCAATGATCATCTAACATTTTTG3² s²CCGGTTACCTAATCAGTAGAThLINC009923GCAGCTCGAGCTGCATCTACTGATTAGGTAATTTTTG3² NCmimics ²UCAGGUAGGGCUCAAACCAACC3² miR3935 mimics ²UGUAGAUACGAGCACCAGCCAC3² NCinhibitor²CUGGCUUUAG 0cChen BMC Cancer Page of GGUGCCACUUAG3² miR3935 inhibitor ²GUGGCUGGUGCUCGUAUCUACA3²Quantitative realtime PCR qRTPCROn the basis of the instructions of Trizol reagent Invitrogen USA RNA extraction was executed in prostatecancer cells After the examination of RNA purity withspectrophotometry cDNA was obtained from aboveRNA with reverse transcription kit ThermoFisher Scientific shanghai China qRTPCR analysiswas devised with the aid of a BioRad CFX96 system andSYBR green was applied for investigating the RNA levelsThe internal reference for LINC00992 and mRNAs wasGAPDH whereas that for miRNAs expression was U6Relative expression was assessed based on the method ofˆ’ΔΔCtab97779Western blotProtein content in cells was determined by western blotanalysis RIPA lysis buffer Beyotime Shanghai Chinawas adopted for cell lysing followed by the evaluation ofthe protein concentration with BCA Protein Assay KitP0011 Beyotime Tech SDSPAGE gel was applied for separating proteins μg protein per sampleand then proteins were transferred onto μm PVDFmembranes BioRad Hercules CA USA Antibodiesincluding antiGOLM1 ab109628 AbcamCambridge UK antiPCNA ab92552 AbcamantiCDK2 ab32147 Abcam antiCyclin D1ab40754 Abcam antiBax ab32503 Abcam antiBcl2 ab32124 Abcam antiMMP2antiMMP9ab38898 Abcam antipSrc ab40660 Abcam antiSrc ab47405 Abcam antipFAKab81298 Abcam antiFAK ab131435 Abcam antiGAPDH ab8245 Abcam andantiTubulin ab7291 Abcam were applied toprobe the membranes overnight at °C After that themembranes were further incubated for h with HRPconjugated secondary antibody Santa Cruz Co LtdSant Cruz CA USA atroom temperature ECLSubstrates Millipore Billerica MA USA was utilizedfor the visualization of signals followed by exposure toXfilm Kodak Rochester NY USA The quantificationof immunoblots was conducted with the aid of imageJsoftware National Institute of Health Bethesda MDUSA with GAPDH or Tubulin as the normalizer asneeded AbcamLuciferase reporter assayFragments of fulllength LINC00992 with wildtype ormutant binding sites for miR3935 and sequences ofGOLM1 ™UTR containing wildtype or mutated miR binding sites were inserted into the pmirGLOvectors Promega Madison WI USA for the construction of reporters LINC00992WT LINC00992MUTGOLM1WT GOLM1MUT Then the four reportersand miR3935 mimics or miR3935 inhibitor GenePharma were cotransfected into DU145 and PC3 cellsapplying lipofectamine2000 Invitrogen as neededFortyeight hours later DualLuciferase Reporter AssaySystem Promega was employed for the examination ofthe luciferase activity GloMax® Discover Multimode Microplate Reader Promega assessed the ratio of FireflyRenilla luciferase activity and the activity of Renilla wasthe normalized controlRNA immunoprecipitation RIP assayAccording to the direction for usage of Magna RIP„¢RNA Binding Protein Immunoprecipitation Kit “Millipore RIP assay was strictly performed RIP lysisbuffer was firstly applied to treat the transfected DU145and PC3 cells Afterwards the obtained cell lysates wereprocessed with magnetic beads integrated with humanantiAgo2 antibodies ab32381 Abcam MA USA orantiIgG AP162KC Millipore Following the recoveryof antibody by the protein AG beads qRTPCR detected the levels of LINC00992 miR3935 and GOLM1mRNA in the precipitates IgG worked as the negativecontrol for the normalization of RNAIPsRNA isolation of nuclear and cytoplasmic fractionsThe dispersion of LINC00992 in the prostate cancercells was assayed as described previously [] The isolation of cytosolic and nuclear sections was executed followingAM1921Invitrogen RNA levels of U1 nuclear control GAPDHcytoplasmic control and LINC00992 were all estimatedby qRTPCR analysisPARIS„¢ KittheprotocolofFluorescence in situ hybridization FISH assayIn line with the recommendation of Ribo„¢ FISH KitC10910 Ribobio Guangzhou China FISH analysiswas implemented for testing the presence of LINC00992in prostate tumor cells Ribobio Company synthesizedthe LINC00992 probes labeled by Cy3 fluorescent dyeFollowing the fixation by paraformaldehyde and Triton X100 permeabilization DU145 and PC3 cellswere subsequently blocked in prehybridization bufferblocking solution Then incubation of cells with probehybridization buffer was later performed Next day afterrinsing and Hoechst staining the fluorescence was measured under a confocal laser scanning microscope ZeissGermanyCell counting kit8 CCK8 assayFor the viability assessment in DU145 PC3 and RWPE cells CCK8 assay was implemented as described 0cChen BMC Cancer Page of previously [] Cell viability was monitored at and h In short after being seeded onto 96well platesand cultured for indicated times cells were processedwith μl of CCK8 solution Then a microplate readerexamined the absorbance values at the wavelength of nm²ethynyl2²deoxyuridine EdU incorporation assayCell proliferation was examined through EdU assay asdescribed previously [] by using ClickiT EdU AlexaFluor Imaging Kit C10086 Invitrogen After hoftransfection EdU staining was carried out asinstructed The observation and calculation of EdUpositive cells was proceeded under the fluorescencemicroscopyTransferasemediated dUTP nick end labeling TUNELstainingTUNEL assay was carried out as described previously[] for probing DU145 and PC3 cell apoptosis with theassistance of an In Situ Cell Death Detection Kit Roche Mannheim Germany TUNELpositivecells were recorded under a light microscope × from visual fields which were chosen at randomTranswell migration assayThe application of transwell chambers with pore size μm Corning Costar Cambridge MA USA was aimedfor detecting cell migration in strict line with the instructions Cells that were previously suspended inserumfree RPMI1640 media were seeded into theupper chamber RPMI1640 medium containing FBS was supplemented in lower chamber as a chemoattractant Cells in the filters following h incubationwere immobilized in methanol and went through crystal violet staining The images of cells migratedthrough the filters were obtained and counted under themicroscopeWound healing assayThe DU145 PC3 and RWPE1 cells × cellswellwere prepared on glass culture dishes and cultivated at °C for a whole night to allow cells adhered to theplates followed by the straight scratch made with a plastic pipette tip after cell samples reached confluenceLater cells were rinsed in PBS to clear the detachedcells Finally the wounds at and h were imaged viaa light microscopy Olympus Tokyo JapanIn vivo experimentSixteen sixweekold male BALBC athymic nude micewere commercially available from the National Laboratory Animal Center Beijing China and maintained inSPFgrade animal lab All animalrelated protocols wereapproved by the Animal Research Ethics Committee ofthe First Affiliated Hospital of Kunming Medical University The in vivo experiment was undertaken via subcutaneous injection of × DU145 cells into the nudemice while the DU145 cells injected into indicated fourshgroups of mice were transfected with shNCLINC009921shLINC009921 pcDNA31 orshLINC009921 pcDNA31GOLM1 Tumor volume wasmonitored every days 28day after injection nudemice were sacrificed via cervical dislocation and thentumor samples were carefully dissected for weight assessment and hematoxylin and eosin HE stainingImmunohistochemistry IHCThe tumor samples collected from in vivo experimentswere treated with PFA dehydrated and embedded inparaffin Afterwardsthe paraffinembedded sections μm were prepared for IHC assay as described previously [] by use of the antiKi67 and antiPCNA antibodies AbcamStatistical analysisSPSS statistical software SPSS Armonk NY USAwas employed in the processing of data from threebiological replicates and data were expressed as mean ±SD Significance of difference within two groups wasdetermined using Student™s ttest while that among noless than two groups was tested via oneway or twowayANOVA P was considered as the threshold ofsignificanceResultsLINC00992 is overexpressed in prostate cancer andregulates cell proliferation apoptosis and migrationLINC00992 expression pattern in prostate cancer wasacquired from online GEPIA database As a resultLINC00992 was considerably upregulated in PRADprostate adenocarcinomatissues relative to normalones Fig 1a After detecting LINC00992 expression intissue samples obtained from patients with prostate cancer we observed that LINC00992 expression was higherin prostate cancer tissues than that in peritumor tissuesFigure S1A Moreover clinical data showed that higherexpression of LINC00992 in prostate cancer patientswas associated with lower survival rate Figure S1BFurthermore LINC00992 expression in the prostate cancer cells and RWPE1 cells was evaluated by qRTPCRConsequently higher level of LINC00992 was exhibitedin prostate cancer cells than that in RWPE1 cells Fig1b which was completely consistent with the result presented in previous discovery [] Particularly DU145and PC3 cells expressed the highest level of LINC00992and was thereby chosen for the later assays For silencingLINC00992 special shRNAs targeting LINC00992 was 0cChen BMC Cancer Page of Fig LINC00992 was overexpressed in prostate cancer and regulates cell proliferation apoptosis and migration a GEPIA database demonstratedthe overexpression of LINC00992 in tumor tissues in contrast to adjacent normal ones b LINC00992 expression was detected by qRTPCR in fourprostate cancer cell lines and control RWPE1 cells c LINC00992 expression was monitored by qRTPCR in DU145 and PC3 cells after transfectionwith shRNAs targeting LINC00992 shNC was used as the negative control d The viability of DU145 and PC3 cells was estimated through CCK8assay following LINC00992 depletion e The proliferation of DU145 and PC3 cells was investigated after LINC00992 depletion via EdU assay Scalebar μm f The apoptosis of DU145 and PC3 cells transfected with shLINC0099212 or shNC was estimated via TUNEL assay Scale bar μm g Western blot analysis was applied to examine the expression of apoptosisrelated proteins hi The migration of DU145 and PC3 cellswas analyzed via Transwell migration assay scale bar μm and wound healing assay scale bar μm after inhibiting LINC00992expression The fulllength images for blots in Fig 1g were presented in Supplementary figure P p transfected into DU145 and PC3 cells and the efficiencywas corroborated in qRTPCR Fig 1c And then thedata from CCK8 assay revealed that LINC00992 depletion suppressed the proliferation of DU145 and PC3cells Fig 1d As expected a declined proportion ofEdU positive cells was observed after knocking downLINC00992 Fig 1e suggesting the suppressive effect ofLINC00992 deficiency on prostate cancer cell proliferation Additionally the expression levels of proliferationrelated proteins PCNA CDK2 and Cyclin D1 were allreduced by silenced LINC00992 Figure S1C On thecontrary TUNEL assay uncovered that LINC00992knockdown facilitated cell apoptosis Fig 1f Meanwhile western blot analysis revealed that LINC00992knockdown promoted the apoptosis of DU145 and PC3cells as Bax protein level was increased whereas Bcl2protein level was decreased after LINC00992 was silenced in these two cells Figs 1g Figure S1D FurtherTranswell and wound healing assays indicated that themigration of DU145 and PC3 cells was retarded byLINC00992 depletion Fig 1hi Likewise the expression of migrationrelated molecular markers MMP2MMP9 pSrc and pFAK was decreased by LINC00992inhibition Figure S1E To further verify the biological 0cChen BMC Cancer Page of role of LINC00992 in prostate cancer we carried outgainoffunction assays in RWPE1 cells After overexpressing LINC00992 in RWPE1 cells Figure S2A cellproliferation was promoted Figure S2BC As expectedthe expression of PCNA CDK2 and Cyclin D1 wasdecreased by upregulation of LINC00992 Figure S2DSimilarly LINC00992cellIn addition upregulatingmigration Figure S2EFLINC00992 resulted in the elevated protein levels ofMMP2 MMP9 pSrc and pFAK Figure S2G All thesedata elucidated that LINC00992 could facilitate cell proliferation and migration whereas suppress cell apoptosisin prostate cancerupregulationfacilitatedMiR3935 is targeted by LINC00992Given the high correlation of the sublocalization ofLINC00992 with its functional mechanism the predication of LINC00992 presence in cells was performed viaLncLocatorhttpwwwcsbiosjtueducnbioinflncLocator Result predicted that LINC00992 located mainlyin cytoplasm Fig 2a Likewise FISH assay and RNAisolation of nuclear and cytoplasmic fractions furtherverified the abundance of LINC00992 in the cytoplasmof prostate cancer cells Fig 2bc highlighting a posttranscriptional control of LINC00992 in such cellsHence we speculated that LINC00992 might act as aceRNA in prostate cancer regulation According toDIANAlncBase the top three potential miRNAs possessing the binding capacity with LINC00992 were listedFig 2d To targetthe highlymatched miRNA toLINC00992 qRTPCR analysis was conducted to testthe expression changes of these miRNAs following eitherLINC00992 depletion or augmentation The resultsdemonstrated that only miR3935 expression was increased by LINC00992 depletion Fig 2e but reducedby LINC00992 overexpression in the meantime Fig 2fThus miR3935 was chosen for further analysis Afterwards RNA pull down assay was implemented and theresult depicted that LINC00992 was pulled down byBiomiR3935WT Fig 2g which indicated the bindingof LINC00992 and miR3935 Later we observed thesatisfactory efficiency of miR3935 overexpression andmiR3935 inhibition through qRTPCR analysis Fig2h Thereafter RIP assay applying antiAgo2 was executed Results illustrated that LINC00992 and miR3935were highly enriched in antiAgo2 group in comparisonwith control antibody Fig 2i certifying the associationof LINC00992 with miR3935 in the RNAinduced silencing complexes RISCs To further explore the interaction between LINC00992 and miR3935 the bindingsites between LINC00992 and miR3935 were predictedat first and then data from luciferase reporter assayrevealed that miR3935 upregulation decreased theluciferase activity of LINC00992WT reporter whereasmiR3935 inhibition increased the luciferase activity ofLINC00992WT reporter Fig 2j Altogether LINC0 combined with miR3935 to act as a miRNA decoyin prostate cancerLINC00992 regulates the expression of GOLM1 a targetof miR3935Present evidence has suggested that miRNAs can bindwith downstream target genes to inhibit their expressionHerein we searched the miR3935 target genes andeight mRNAs were found out Subsequently we detectedtheir expression in prostate cancer cells and normalcells Interestingly we found that only Golgi membraneprotein GOLM1 was highly expressed in four prostate cancer cell lines relative to normal controls Fig 3aFurther we discovered that GOLM1 expression wasmarkedly upregulated in prostate cancer tissues according to data from GEPIA database Fig 3b SimilarlyGOLM1 expression was much higher in prostate cancertissue samples than in peritumor samples Figure S3AIn addition the mRNA and protein levels of GOLM1were overexpressed in prostate cancer cells in contrastto RWPE1 cells Fig 3c Figure S3B Besides GOLM1has been previously revealed as a prostate cancer facilitator and was metastasisrelated in prostate tumor [“] Thus we hypothesized that GOLM1 might act asthe downstream of LINC00992miR3935 signaling inprostate cancer Through TargetScan httpwwwtargetscanvert_72 the binding site between GOLM1and miR3935 was predicted Fig 3d After conductingluciferase reporter assay in DU145 and PC3 cells weobserved that upregulation of miR3935 specifically decreased the luciferase activity of GOLM1WT reporterFig 3e confirming the interaction between miR3935and GOLM1 relied on the putative binding sites Thenwe unveiled that GOLM1 mRNA and protein levels wereboth reduced by LINC00992 inhibition or miR3935upregulation according to qRTPCR and western blotanalyses Fig 3fg Figure S3C Moreover data fromRIP assay unveiled the binding of miR3935 to GOLM1in the RISCs Fig 3h Further we demonstrated thatthe decreased mRNA and protein levels of GOLM1 induced by LINC00992 depletion could be restored afterinhibiting miR3935 expression Fig 3ij Figure S3DAll the results showed that LINC00992 upregulatedGOLM1 expression via directly binding to miR3935LINC00992 promotes prostate cancer cell proliferationand migration via elevating GOLM1 expressionTo test whether LINC00992 affected prostate cancer cellproliferation apoptosis and migration via regulatingmiR3935targeted GOLM1 we executed the rescue experiments with the upregulation of GOLM1 To beginwiththe efficiency of overexpressing GOLM1 was 0cChen BMC Cancer Page of Fig MiR3935 was targeted by LINC00992 a LncLocator predicted LINC00992 subcellular location b FISH analysis of LINC00992 distribution inprostate cancer cells Scale bar μm c RNA isolation of nuclear and cytoplasmic fractions assayed the subcellular distribution of LIN00992 inprostate cancer cells d Top three miRNAs which might interact with LINC00992 were predicted by DIANAlncBase e After transfection ofLINC00992silencing plasmids the expression of miR31575p miR11783p and miR3935 was examined via qRTPCR f Following LINC00992upregulation qRTPCR tested the levels of miR31575p miR11783p and miR3935 in DU145 and PC3 cells g RNA pull down assay wasimplemented to testify the binding capacity between LINC00992 and miR3935 h miR3935 overexpression efficiency and inhibition efficiencywere examined by qRTPCR i RIP assay disclosed the binding of miR3935 to LINC00992 in the antiAgo2 group j The potential binding sitebetween LINC00992 and miR3935 was shown And the luciferase activity of LINC00992WT or LINC00992MUT reporter was assessed vialuciferase reporter assay in DU145 and PC3 cells after transfection with miR3935mimics miR3935inhibitor NCinhibitor or NCmimics P p p analyzed through qRTPCR and western blot analysesand the outcome turned out to be satisfactory Fig 4abFigure S3E Then we observed that overexpression ofGOLM1 could significantly elevate the mRNA and protein expression of GOLM1 in shLINC009921transfected cells Figure S3F Afterwards data from CCK8revealed that the viability of DU145 cells was firstly hindered due to LINC00992 depletion while subsequentGOLM1 elevation reversed the inhibitory trend onDU145 cell viability Fig 4c Results from EdU assayalso exposed similar trends that GOLM1 upregulationimpactposedthesuppressivecountervailedbyLINC00992 downregulation on DU145 cell proliferationFig 4d Similarly the restraining effect of silencedLINC00992 on the expression of proliferationrelatedproteins could be reversed by GOLM1 upregulationFigure S3G Later TUNEL assay revealed that cellapoptosis rate was elevated by LINC00992 depletion andthen overexpressing GOLM1 reduced the increasedapoptosis rate of LINC00992depleted cells Fig 4eLikewise western blot analysis uncovered that overexpressing GOLM1 could offset the effect of LINC00992 0cChen BMC Cancer Page of Fig LINC00992 regulated the expression of GOLM1 a target of miR3935 a The expression of eight mRNAs in four prostate cancer cell linesand RWPE1 cells was detected by qRTPCR b GOLM1 was overexpressed in prostate cancer tissues according to GEPIA database c The mRNAand protein levels of GOLM1 were evaluated in prostate cancer cell lines and RWPE1 cell line by qRTPCR and western blot respectively d Thebinding sites between GOLM1 and miR3935 were predicted via TargetScan e Luciferase reporter assay presented the inhibited luciferase activityof GOLM1WT reporter in the presence of miR3935 mimics not NCmimics fg GOLM1 expression in transfected cells was tested by qRTPCR andwestern blot analyses h The combination of GOLM1 with miR3935 in the antiAgo2 group was validated by RIP assay ij The mRNA and proteinlevels of GOLM1 in different groups were examined via qRTPCR and western blot The fulllength gels for western blot data in Fig 3c g and jwere presented in Supplementary Figure P p p downregulation on the expression of apoptosisrelatedproteins Fig 4f Figure S3H Moreover Transwellmigration and wound healing assays illuminated thatthe retarding influence of silenced LINC00992 on cellmigration could be rescued by GOLM1 overexpression Fig 4gh As expected the inhibitory effect ofLINC00992 depletion on the expression of migrationrelated molecular markers MMP2 MMP9 pSrc andpFAK could be countervailed by GOLM1 overexpression Figure S3I Collectively GOLM1 was requiredcancercellular processesLINC00992regulatedinprostateLINC00992 contributes to tumor growth via upregulatingGOLM1 expressionAfter the in vitro exploration of LINC00992 performance in prostate cancer we applied the in vivo assays tofurther validate above findings As shown in Fig 5a tumors derived from LINC00992silenced DU145 cellswere smaller with the growth rate quite slower thanthose from control cells more importantly such blockage on tumor growth was obviously countervailed afterGOLM1 overexpression Besides elevating GOLM1 expression could recover the lessened tumor volume anddeclined tumor weight induced by LINC00992 deficiency 0cChen BMC Cancer Page of Fig LINC00992 promoted prostate cancer cell proliferation and migration via elevating GOLM1 expression ab GOLM1 mRNA and proteinlevels in DU145 cells transfected with pcDNA31 or pcDNA31GOLM1 were detected via qRTPCR and western blot pcDNA31 served as thenegative control c The viability of DU145 cells was determined via CCK8 following transfection of different plasmids d The proliferation oftransfected cells was evaluated via EdU assay e The apoptosis of transfected cells was monitored via TUNEL assay Scale bar μm f Theprotein levels of Bax and Bcl2 in different groups were detected via western blot gh The migration of transfected cells was measured viaTranswell migration assay scale bar μm and wound healing assay scale bar μm The fulllength gels for western blot data in Fig 4band f were presented in Supplementary Figure P p Fig 5bc Of note we discovered decreased level ofLINC00992 and enhanced level of miR3935 in tumorsfrom latter three groups compared to control group whilethe lowered expression of GOLM1 in tumors withLINC00992 inhibition was normalized under GOLM1overexpression Fig 5d In addition the inhibitory impactof silenced LINC00992 on the positivity of proliferationassociated proteins PCNA and Ki67 could be reversedby upregulation of GOLM1 Fig 5e Taken togetherLINC00992 promoted the tumorigenesis of prostate cancer through upregulating GOLM1 expressionDiscussionAs documented the aberrant regulation of lncRNAs is afrequent event in diversified tumor types Besides thecorrelation between abnormallncRNA expression andprostate cancer oncogenesis has also been extensivelyexplored For example lncRNA SNHG7 facilitates prostate cancer carcinogenesis via cyclin D1 by spongingmiR503 [] LncRNA SChLAP1 aggravates prostatecancer cell proliferation and metastasis by targetingmiR198 [] LncRNA PCAT1 contributes to prostatecancer tumorigenesis through modulating FSCN1 andsponging miR1455p [] In our work LINC00992 wasrevealed to be highly expressed in prostate cancer tissuesand cells but unlike former investigations our studygave a precise explanation about its role in prostate cancer Our study unveiled that LINC00992 promoted cellproliferation and migration whereas suppressed cellapoptosis in prostate cancer Abovementioned data 0cChen BMC Cancer Page of Fig LINC00992 contributes to tumor growth via upregulating GOLM1 expression a Representative images and the growth curves of tumorsfrom indicated groups bc The volume and weight of tumors from above groups d The expression of LINC00992 miR3935 and GOLM1 intumors from different groups was detected via qRTPCR analysis e The staining of PCNA and Ki67 in different groups was measured via IHCScale bar μm p 0cChen BMC Cancer Page of validated that LINC00992 elicited a tumorpromotingfunction in prostate cancerPresently accumulating evidence has indicated thatcytoplasmic lncRNAs assisted the expression of downstream miRNAtargeted mRNAs via sponging the specific miRNAs Before exploring LINC00992mediatedmechanism in prostate cancer herein we firstly discovered its subcellular distribution in prostate cancer cellswith both aids from online prediction tool LncLocatorand experimental data FISH and RNA isolation of nuclear and cytoplasmic fractions Our study for the firsttime uncovered that LINC00992 located mainly in thecytoplasm of prostate cancer cells Besides our studyalso completed LINC00992modulated mechanism bydisclosing the downstream target miR3935 The directinteraction between LINC00992 and miR3935

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poorest prognoses of all malignancies with little improvement in clinical outcome over the past years Pancreatic ductal adenocarcinoma is responsible for the vast majority of pancreatic cancer cases and is characterised by the presence of a dense stroma that impacts therapeutic efficacy and drives protumorigenic programs More specifically the inflammatory nature of the tumour microenvironment is thoughtto underlie the loss of antitumour immunity and development of resistance to current treatments Inflammatory pathways are largely mediated by the expression of and signallingthrough cytokines chemokines and other cellular messengers In recent years there hasbeen much attention focused on dual targeting of cancer cells and the tumour microenvironment Here we review our current understanding of the role of IL6 and the broader IL6cytokine family in pancreatic cancer including their contribution to pancreatic inflammationand various roles in pancreatic cancer pathogenesis We also summarise potential opportunities for therapeutic targeting of these pathways as an avenue towards combating poorpatient outcomesIntroductionPancreatic cancer is a devastating malignancy with a 5year relative survival rate of only “ dependenton the geographical location surveyed [“] with these statistics exhibiting only modest improvementover the last four decades [“] The median survival postdiagnosis ranges from just “ months forlocally advanced disease and “ months for metastatic disease [] It was estimated by the World Healthanisation that pancreatic cancer is currently the 7th leading cause of cancerrelated death being responsible for over deaths worldwide in [] With incidence increasing pancreatic cancerhas been predicted to be the third leading cause of cancerrelated death in the European Union by [] and the second leading cause of cancerrelated death in the United States of America and Germanyby []Several factors contribute to the poor survival of pancreatic cancer patients A current lack of reliablediagnostic markers that would enable early screening [] coupled with largely nonspecific symptoms ofdisease results in over of patients presenting with metastatic disease at diagnosis [] This subgroupof patients have limited therapeutic options and are thus typically administered palliative chemotherapyaimed at prolonging survival and reducing symptoms during endoflife care [“] Moreover whilstapproximately “ of patients present with localised disease that is eligible for potentially curativesurgery [] disease recurs in over of patients postresection [] Ultimately these factorsculminate in more than of patients diagnosed with pancreatic cancer succumbing to disease []These harrowing statistics highlight that despite research efforts there remains a lack of understandingof the pathogenesis of disease which in turn limits the development of new therapeuticsReceived March Revised July Accepted August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Pancreatic ductal adenocarcinomaPancreatic ductal adenocarcinoma is the predominant pancreaticmalignancyPancreatic cancer can arise from either the endocrine or the exocrine region of the pancreas Tumours arising fromthe exocrine compartment are termed pancreatic ductal adenocarcinoma PDAC and account for over of allpancreatic cancers []PDAC develops via a stepwise progression from normal tissue through to invasive lesions which is associated withdistinct morphological characteristics [“] It has been proposed that this process begins with a phenomenontermed acinartoductal metaplasia ADM which is a normal homeostatic mechanism whereby acinar cells transdifferentiate into a ductal phenotype in response to particular stimuli [] However if compounded by an oncogenic˜hit™ cells are pushed towards a pathogenic phenotype that develops into pancreatic intraepithelial neoplasia PanIN[“] Disease progresses through preinvasive stages termed PanIN1A PanIN1B PanIN2 and PanIN3 priorto invasive PDAC [] This progression is associated with increasing nuclear atypia whereby the nuclei are no longerpositioned basally and loss of normal architecture as cells become more papillary in nature with PanIN3 lesionsdemonstrating increased mitoses [] As disease progresses to PDAC cells become invasive and breach the basement membrane growing through the extracellular matrix and metastasising to distant ans Figure Less common precursor lesions include intraductal papillary mucinous neoplasms IPMNs and mucinous cysticneoplasms MCNs that also develop through multistep processes [] Whilst they share some common featureseach lesion is morphologically and genetically distinct In contrast with PanINs that form within small ducts IPMNsdevelop within the primary or secondary branches of the main pancreatic duct whilst MCNs lack ductal involvement[]An ‚ammatory tumour microenvironment contributes to PDACpathogenesisAn archetypal feature of PDAC is the development of extensive stromal networks within the tumour microenvironment TME that can account for up to of the total tumour volume [“] This unique characteristic drives theinflammatory nature of PDAC that contributes to its aggressive phenotype [] Desmoplasia is driven by pancreaticstellate cells PSCs and cancerassociated fibroblasts CAFs that upon activation produce a range of extracellularmatrix ECM components such as collagen laminin and fibronectin which in turn form a physical barrier preventing the penetration of therapeutics [“] Though PSCs and CAFs have been shown to support cancer metastasis and drug resistance they interact with cancer cells in a bidirectional manner with each promoting the survivalgrowth and proliferation of the other [“] Quiescent fibroblasts are able to differentiate into two unique subtypes termed myofibroblastic CAFs myCAFs and inflammatory CAFs iCAFs [] These two subtypes are distinct whereby myCAFs express high levels of αsmooth muscle actin αSMA and are located adjacent to cancercells while iCAFs express low levels of αSMA and instead secrete high levels of inflammatory mediators including IL6 and are distributed distant from cancer cells within desmoplastic tumour regions [] Broadly myCAFsappear to have roles in epithelialtomesenchymal transition EMT and ECM remodelling whilst iCAFs appear tobe involved in inflammation and ECM deposition [] A third less abundant subtype termed antigenpresentingCAFs apCAFs has more recently been defined [] These cells express low levels of both αSMA and IL6 andinstead express high levels of major histocompatibility complex class II MHCII and related genes [] As such allthree subtypes are transcriptionally and functionally distinctThe wider TME contains a plethora of other cell types including endothelial cells tumourassociated macrophagesTAMs and neutrophils TANs mast cells regulatory Tcells myeloidderived suppressor cells MDSCs dendriticcells natural killer NK cells and nerve cells [] Interactions between various cells within the TME can driveeither proor antitumorigenic functions of others for example cancer cells can induce PSCs to secrete inflammatorycytokines that drive immune cells towards an immunosuppressive phenotype and also form a positive feedback loopby increasing the tumorigenic potential of cancer cells [] The ECM itself has also been suggested to modifyPSC behaviour in particular that ECM stiffness promotes the myCAF phenotype indicated by increased αSMAexpression [] This results in substantial complexity that ultimately determines tumour phenotype []The components of the microenvironment modify tumour behaviour through the production of cytokines growthfactors and other signalling molecules that predominantly drive a proinflammatory and immunosuppressive program that enhances PDAC tumour growth and progression [“] Figure The ability of the TME toinhibit therapeutic efficacy through both molecular mechanisms and physical fibrotic barriers contributes to the The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Figure Our current understanding of the contribution of IL6 family cytokines to PanIN and PDAC developmentPreinvasive PanIN lesions develop from normal ductal epithelia through PanIN stages 1A 1B and to stage invasive andormetastatic PDAC This process is associated with acinartoductal metaplasia ADM early in disease combined with an accumulation of oncogenic mutations most common mutations are indicated A number of cells within the tumour microenvironment havebeen shown to secrete IL6 family cytokines which in turn results in the activation of a protumorigenic signalling cascade A betterunderstanding of the relationship between each of these cells within the tumour microenvironment may reveal new therapeuticopportunities to manage cancer progressionintrinsic resistance of disease [] Thus dual targeting of cancer cells and the TME may be required to induce afavourable therapeutic response although this poses a signficant scientific and clinical challenge [“]Molecular basis of pathogenesisPDAC development is associated with accumulation of mutationsThe progression of tumorigenesis through PanIN and PDAC stages is associated with the stepwise accumulation ofspecific genetic mutations that drive malignant transformation The most frequent genetic alteration is an activatingKRAS point mutation codon that occurs early on in tumour development [] and is detected in over ofPDAC tumours [“] Mutations in KRAS have been shown to drive development of precursor PanIN lesions andwhen combined with an appropriate tumour suppressor mutation these lesions progress to invasive or metastaticPDAC [] Figure Patient tumours harbouring wildtype WT KRAS often carry activating mutations indownstream effector molecules such as BRAF or PIK3CA [] The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Inactivation of a range of tumour suppressor proteins is also common including mutations in TP53 CDKN2Aand SMAD4 in approximately and of tumours respectively [] Whilst each mutation has uniquemechanistic outcomes all three proteins are either directly or indirectly involved in the regulation of the G1S cellcycle checkpoint Analysis of patient tumours indicates that two or more of these mutations often occur together withCDKN2A mutation being combined with either TP53 SMAD4 or both usually in the background of KRAS mutation [] This suggests that by disrupting this checkpoint cancer cells are able to overcome inhibitory mechanismsallowing continued progression to invasive diseaseUnbiased sequencing efforts have also enabled identification of low prevalence PDAC mutations observed in lessthan of cases [] These include mutations in genes involved in chromatin modification KDM6A DNAdamage repair ATM and other tumourrelated processes such as growth TGFBR1 or TGFBR2 []Furthermore it is important to note that technical advances are continuously uncovering epigenetic mechanisms thatfurther modulate the PDAC transcriptional landscape and ultimately influence disease heterogeneity and tumourprogression []Molecular subtypesThe PDAC epithelial compartment is typically divided into two subtypes including a classical subtype exhibiting anepitheliallike expression profile and a squamous or mesenchymallike subtype [] An additional third exocrinesubtype is outlined in some analyses and is characterised by a gene expression profile related to digestive enzymeproduction [] The classical or epithelial subtype has also been further divided into a pancreatic progenitor andan immunogenic subtype whereby the immunogenic subtype is distinguished by significant immune infiltration andassociated gene programmes [] Though there is no consensus on which classification system will allow the mostvaluable stratification of patients the mesenchymal subtype is invariably associated with a poor prognosis []The stromal compartment has also been classified into either normal or activated subtypes reflecting the proandantitumorigenic capabilities of the TME with the activated subtype associated with reduced survival [] This isparticularly valuable as the extensive heterogeneity of PDAC complicates clinically relevant stratification of patientsThus the identification of molecularly unique subtypes may enable development of tailored therapeutic regimensthat will provide improved clinical outcomesCurrent treatment optionsRegardless of disease stage at time of diagnosis patients have relatively limited treatment options For the majorityof patients disease will be locally advanced or metastatic disqualifying them from undergoing potentially curativesurgery [] In these cases patients are typically offered chemotherapy with palliative intent [“]Surgery provides the only potentially curative treatmentSurgical resection remains the only potentially curative treatment option due to minimal efficacy of standardofcarechemotherapy and radiotherapy Due to its aggressive nature the majority of patients present to clinic with locallyadvanced or metastatic disease with only “ of patients presenting with localised tumours that are eligiblefor surgical resection [] Even for those able to undergo surgical intervention over of patients relapsepostresection [] with median survival improving to months and 5year relative survival rate increasingmodestly to “ [] The use of neoadjuvant therapy is generally reserved for borderline resectable disease inan effort to enable patients to become eligible for surgery [] However a range of recent trials have shown improved clinical outcomes including overall survival for neoadjuvant treatment of patients with resectable tumours[] Following surgical resection patients are typically treated with adjuvant gemcitabinebased chemotherapy []although a recent study showed improved diseasefree survival and overall survival with a modified FOLFIRINOXtherapy combination of oxaliplatin irinotecan leucovorin and fluorouracil []Radiotherapy provides variable clinical outcomeWhilst the use of radiotherapy and chemoradiotherapy combination chemo and radiotherapy in the neoadjuvantand adjuvant settings have been investigated there remains a lack of consensus regarding therapeutic benefit []This is due to issues such as insufficient radiation dose and low participant numbers as well as low uptake of moderntechniques [] In the neoadjuvant setting preliminary studies reported reduced lymph node positivity and rates oflocal recurrence for chemoradiotherapy compared to surgery with adjuvant chemotherapy [] However the useof radiotherapy in the palliative setting was reported to modestly reduce overall survival [] More recent studiesusing ablative doses of radiation have shown a survival benefit highlighting that technological advancements mayprovide an avenue for improved clinical outcomes following radiotherapy [] These contrasting results highlight the The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211need to determine which subset of patients may benefit from the inclusion of radiotherapy approaches in standardtreatment regimensChemotherapy remains the cornerstone of treatmentDespite modest improvements in overall survival palliative chemotherapy remains the standard treatment optionfor patients with locally advanced or metastatic disease Gemcitabine monotherapy has been the mainstay treatmentfor pancreatic cancer since when it was demonstrated to improve median survival by just over month compared with fluorouracil [] Within the last decade there have been some further improvements in clinical outcomewith combination chemotherapies gemcitabinenabpaclitaxel and FOLFIRINOX providing median overall survivalbenefits of and months respectively compared with gemcitabine alone [] Although FOLFIRINOX treatment resulted in a lower percentage of patients experiencing reduced quality of life it also had increased toxicity andadverse events thus preventing its administration to patients with multiple comorbidities []Therapeutic resistance remains a significant barrier to patient survivalDespite advances in chemotherapeutic options treatment efficacy and patient prognosis remain poor due to the inherent therapeutic resistance of pancreatic cancer It has been proposed that this drug resistance may be driven by theTME including changes to cytokine signalling and metabolic pathways [“] This intrinsic resistance is demonstrated by patients experiencing similar overall survival for chemotherapy treatment “ months compared withbest supportive care “ months which encompasses the use of palliative surgery psychological support painmanagement and other methods of symptom control [] Whilst a range of targeted treatments such as EGFR orcheckpoint inhibitors have been trialled with or without chemotherapy they have shown limited success [“]Emerging roles for the IL6 family of cytokines in PDACCytokines are soluble molecular messengers that enable efficient communication between a range of cell types andhave been recognised to be major contributors to the growth and metastasis of cancers [“] In pancreatic cancer cytokines mediate signalling between cancer cells and the cells of the TME including PSCs CAFs endothelialcells and a range of immune cells including macrophages mast cells neutrophils and regulatory Tcells [“]It is the specific signalling pathways active within this community of cells that dictates the balance of pro andantitumorigenic functions []The IL6 family of cytokinesThe interleukin IL6 family of cytokines includes IL6 IL11 leukaemia inhibitory factor LIF oncostatin MOSM ciliary neurotrophic factor CNTF cardiotrophin1 CT1 cardiotrophinlike cytokine CLC neuropoietin NP IL27 and IL31 [“] These cytokines are grouped as they share structural similarity forming a fourαhelical bundle termed helices AD with an upupdowndown topology []IL6 and IL11 utilise a hexameric signalling complex consisting of two molecules each of the cytokine αreceptoreither IL6R or IL11R respectively and βreceptor glycoprotein gp130 [“] IL6 and IL11 are ableto signal via two distinct mechanisms termed classic and transsignalling Classic signalling involves the formation of a complex including membranebound IL6R or IL11R with gp130 and the respective cytokine Converselytranssignalling utilises soluble IL6R or IL11R molecules which are able to form a signalling complex with gp130and the respective cytokine [“] In this way classic signalling relies on the responding cell™s intrinsic expressionof IL6R or IL11R whilst transsignalling is able to activate any cell expressing gp130 []LIF OSM IL27 and IL31 signal through trimeric complexes with a single cytokine molecule engagingthe respective receptor LIFR OSMR IL27R WSX1 or IL31R and either gp130 or OSMR for IL31[“] CNTF CT1 CLC and NP form tetrameric signalling complexes composed of one cytokinemolecule one LIFR one CNTFR and one gp130 receptor [] In each case the active signalling complex consists of two chains that are signalling competent with a combination of either gp130 LIFR OSMR IL27R or IL31R[] The requirement for multiple receptor subunits means that although gp130 is ubiquitously expressed the expression of other receptor subunits dictates the ability for any given cell to respond to cytokine as signalling initiationrequires the presence of cytokine and a compatible receptor complex [] Figure 2ASignalling complex assembly leads to transphosphorylation and activation of receptorassociated Janus tyrosinekinases JAKs largely JAK1 and to a lesser extent JAK2 and TYK2 [] In the case of gp130mediated signalling this results in phosphorylation of the cytoplasmic domain of gp130 at tyrosine Y and [] Phosphotyrosine pY and of gp130 provide docking sites for signal transducer and The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Figure IL6 family cytokine signalling pathwayA Schematic representation of the stepwise binding process for the IL6 family members with IL6 as an example The site interaction involves cytokine binding to the respective receptor with the site interaction generally between the cytokine and thecommon gp130 receptor chain finally site interactions involve formation of the final active signalling complex in this case formation of the IL6IL6Rgp130 hexameric complex B General outline of the IL6 family cytokine signalling pathway Formation ofan active hexameric complex leads to activation of JAKs with subsequent activation of the STAT3 MAPK and PI3K pathways leftThis results in upregulation of the negative regulator SOCS3 as well as a range of inflammatory and protumorigenic moleculesThe pathway is inhibited by SOCS3 PIAS3 and PTPs via dephosphorylation ubiquitinmediated proteasomal degradation andSUMOylation right The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211activator of transcription STAT molecules leading to their subsequent phosphorylation by JAK1 and formation ofactive STAT dimers [“] Phosphorylated STAT pSTAT dimers then translocate to the nucleus and modulatetarget gene expression including upregulation of a range of genes involved in inflammatory and protumorigenicpathways [“] Figure 2B Broadly these STAT3regulated genes can be categorised into pathways associatedwith inhibition of apoptosis increased cell proliferation modulation of immunity and inflammation increased angiogenesis and increased invasive and metastatic potential [“]Although JAKSTAT signalling is the predominant pathway activated downstream of IL6 family cytokines themitogenactivated protein kinase MAPK and phosphoinositide 3kinase PI3K pathways can also be activated[] The MAPK pathway has been suggested to be activated by a Src homology domain 2containing phosphatase SHP2mediated mechanism whereby SHP2 is recruited to pY759 on gp130 allowing JAKmediated phosphorylation of SHP2 [] This promotes association with the adaptor protein growth factor receptor bound protein Grb2 leading to activation of the Gprotein Ras via son of sevenless SOS with a subsequent phosphorylationcascade including Raf MEK and ERK12 activity [] Following this a MAPKdependent phosphorylationevent leads to the recruitment of Grb2associated binding protein Gab1 to the plasma membrane where Gab1 issuggested to act as a scaffold or adaptor protein to allow binding of PI3K and SHP2 leading to activation of the PI3Kand MAPK pathways respectively [] Figure 2BThe suppressor of cytokine signalling SOCS3 is largely responsible for regulation of signalling and is directlyupregulated by STAT3 [] SOCS3 contains an SH2 domain allowing it to bind to pY residues within the gp130receptor [] with preferential binding to Y759 [] Once bound SOCS3 recruits an E3 ubiquitin ligasecomplex containing Cullin5 Rbx2 and adaptors Elongin B and C via its SOCS box domain [] This complexubiquitinates the gp130 receptor inducing its internalisation and targeting it for proteasomal degradation [“]and is also able to ubiquitinate JAK2 in vitro [] SOCS3 also mediates direct inhibition of the kinase activityof JAK12 via its kinase inhibitory region [“] Thus SOCS3 is able to downregulate IL6 family cytokinesignalling pathways through two distinct mechanismsThe phosphotyrosine phosphatases PTPs and protein inhibitors of activated STATs PIASs also limit the strengthand duration of cytokine signalling [] A range of PTPs including SHP2 are responsible for dephosphorylatingtyrosine phosphorylated substrates including JAKs STATs and other SHP2 molecules [] PIAS3 preferentially binds pSTAT3 and inhibits activity either by preventing STAT3 interaction with DNA by recruiting transcriptional repressors to STAT3 target genes or by SUMOylating STAT3 to prevent its activity [] Figure 2BInterleukin in PDACElevation of serum IL6 is a negative prognostic marker in human PDACSerum IL6 levels were increased in PDAC patients compared with healthy patients [“] or those withchronic pancreatitis [] and were also increased in patients with metastatic PDAC compared to thosewith locally advanced disease [“] Moreover elevated serum IL6 positively correlated with increased diseaseburden weight losscachexia and metastasis [““] however there are conflicting observations inthe literature regarding IL6 and cachexia [] Although increased serum IL6 levels correlate with increased disease stage and in metastatic patients correlates with poor overall survival [] As such it has been suggestedthat IL6 may be a superior marker for diagnostic and prognostic purposes compared with the standard Creactiveprotein CRP carcinoembryonic antigen CEA and carbohydrate antigen CA199 markers []IL6 is expressed within the TMElL6IL6 was overexpressed in human PDAC tumours in comparison with adjacent normal tissue [] Whilstthis tumourspecific elevation has been correlated with reduced survival in some studies [] othersshowed no significant correlation with survival [] similar to the data available in The Cancer Genome AtlasTCGA dataset for both IL6 and IL6R Figure 3AB The TCGA comprise aggregate sequencing data which doeshave limitations regarding interpretation of contributions of individual cell populations to disease outcome howeverit remains a widely used resource for exploratory investigations However overexpression of IL6 has been observedat the mRNA and protein level in the pancreata of PDAC mice [] with Il6 expression increasing with agewhich is indicative of disease stage in these models []Despite the presence of IL6 in tumours primary human and commercial pancreatic cancer cell lines have been reported to exhibit variable expression levels of IL6 and secreted cytokine albeit consistently higher than normal pancreatic ductal epithelial cells [] In an anoid model minimal IL6 was expressed by pancreaticcancer cells PCCs or PSCs in monoculture however in coculture PCCs expressed only Il6ra whilst iCAFs expressedhigh levels of IL6 with this activating STAT3 within PCCs [] iCAFs also demonstrate an upregulation of The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Figure IL6 family cytokine expression in PDAC patientsOverall survival for patients with high top quartile and low bottom quartile level expression of A IL6 B IL6R C IL11 D IL11RE LIF F OSM G CNTF H CTF1 CT1 I CLCF1 CLC and J IL27 n per group Data and graphs obtained fromOncoLnc [] using data from The Cancer Genome Atlas TCGA Statistical significance determined by MantelCox Logranktest The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211the JAKSTAT pathway with expression of IL6 being dramatically increased in vitro when incubated with PCC conditioned media indicating that soluble factors trigger IL6 production [] More recently PCCderived IL1αhas been shown to induce autocrine LIF secretion and thereby promote the iCAF phenotype including activation ofthe JAKSTAT signalling pathway and IL6 production []In addition TAMs have been identified as producers of IL6 in pancreatic cancer by correlative immunohistochemistry and expression analysis of isolated cell populations [] Production of IL6 by TAMs was shownto influence tumour development via bonemarrow chimeras as mice reconstituted with IL6 knockout KO Il6myeloid cells developed lowgrade PanINs whilst those reconstituted with IL6 WT cells developed PanIN3 lesions[]IL6 is a driver of PDAC pathogenesisBoth in vitro and in vivo studies suggest that the presence of IL6 in the TME can drive activation of STAT3 []with IL6 inhibition reducing STAT3 phosphorylation [] This IL6STAT3 program has been proposed tobe a driver of PDAC pathogenesis by enhancing tumour initiation and progression angiogenesis regulation of cytokine expression and immune cell behaviour resistance to apoptosis and promotion of metastasis [“] In aninducible KRASdriven mouse model genetic deletion of Il6 resulted in a reduction of ADM and PanIN formationwhen KRAS mutation was initiated embryonically compared with controls suggesting a role for IL6 in tumour initiation [] This was also observed in a constitutive KRAS mutant model where genetic deletion of IL6 preventedtumour initiation in vivo with a reduction in the number of PanIN and lesions [] Interestingly oncogenicKRAS and hypoxic conditions both features of PDAC tumours [] were shown to induce IL6 production[] perhaps representing a feedforward pathway enhancing tumorigenesis [] However IL6 is notabsolutely required for PanIN formation as induction of KRAS mutation at weeks of age in conjunction with anexperimental pancreatitis model drove formation of PanIN lesions that were not significantly different between IL6WT and KO mice []Il6 mice exhibited reduced tumour progression with decreased proliferative capacity of both cancer and stromal cells enabling regression of precursor lesions [] Furthermore this inhibition of tumour progression by IL6deletion was due at least in part to the reversal of ADM with ductal cells reverting to an acinarlike phenotype[] Increased apoptosis of cancer and stromal cells was also shown to contribute to this reduced tumour progression as demonstrated by appropriate immunohistochemical analyses with upregulation of proapoptotic anddownregulation of antiapoptotic BCL2 family members [] This is mirrored by in vitro data whereby IL6 stimulation increased the expression of antiapoptotic BCL2BCL2 and BCL2L1BCLXL [] with blockade of IL6signalling or STAT3 activation inducing apoptosis [] Collectively these data suggest that whilst IL6 contributes it is not required for PDAC initiation and progressionThe process of angiogenesis supports tumour growth and progression by enabling adequate blood supply whichis enhanced by IL6 signalling Upon IL6 stimulation PDAC cell lines upregulate key angiogenic factors such asvascular endothelial growth factor VEGFVEGF and neurophilin1 NRP1NRP1 [] with significant correlation observed between the expression of IL6R and VEGF on human PDAC sections [] IL6inducedupregulation of VEGF correlated with a growth advantage in PCCs with both features inhibited by treatment witha JAK2 inhibitor []Another facet of the protumorigenic effects of IL6 is the regulation of cytokine expression that enables modulationof the immune system [] In particular it has been shown that IL6 is able to upregulate a type cytokine profile invitro that may inhibit antitumour immunity in disease [] IL6 suppressed the differentiation of human CD14cells into dendritic cells DCs in vitro whilst combination treatment with IL6 and granulocyte colonystimulatingfactor GCSF inhibited the ability of DCs to respond to alloantigen a process that is required for DC maturationand antigen presentation where these effects were reversed by blockade of IL6 andor GCSF [] IL6 has alsobeen implicated in driving increased apoptosis of type I conventional DCs cDC1s leading to cDC1 dysfunctionea

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SurveillanceWest Nile virus in humans Greece the largest seasonal number of cases years after its emergence in the countryDanai Pervanidou¹ Annita Vakali¹ Theano Geakopoulou¹ Takis Panagiotopoulos2 Eleni Patsoula² Gee Koliopoulos Constantina Politis¹ Kostas Stamoulis´ Elpida Gavanaµ Styliani Pappaµ Maria Mavrouli¶ Maria Emmanouil· Gee Sourvinos¸ Andreas Mentis· Athanassios Tsakris¶ Christos Hadjichristodoulou¹ Sotirios Tsiodras110 Anna Papaµ Hellenic National Public Health anizationformer Hellenic Center for Disease Control Prevention Athens Greece School of Public Health Faculty of Public Health Policy University of West Attica Athens Greece Benaki Phytopathological Institute Athens Greece Hellenic National Blood Transfusion Center Athens Greece National Reference Center for Arboviruses and Haemorrhagic Fever Viruses Department of Microbiology Medical School Aristotle University of Thessaloniki Thessaloniki Greece Department of Microbiology Medical School National and Kapodistrian University of Athens Athens Greece Diagnostic Services Laboratory Public Health Laboratories Hellenic Pasteur Institute Athens Greece Laboratory of Clinical Virology Medical School University of Crete Heraklion Crete Greece Laboratory of Hygiene and Epidemiology Medical School University of Thessaly Larisa Greece National and Kapodistrian University of Athens Athens GreeceCorrespondence Danai Pervanidou dpervanidoueodygovgrCitation style for this Pervanidou Danai Vakali Annita Geakopoulou Theano Panagiotopoulos Takis Patsoula Eleni Koliopoulos Gee Politis Constantina Stamoulis Kostas Gavana Elpida Pappa Styliani Mavrouli Maria Emmanouil Maria Sourvinos Gee Mentis Andreas Tsakris Athanassios Hadjichristodoulou Christos Tsiodras Sotirios Papa Anna West Nile virus in humans Greece the largest seasonal number of cases years after its emergence in the country Euro Surveill 20202532pii1900543 10280715607917ES202025321900543 submitted on Aug accepted on Feb published on Aug Background Human cases of West Nile virus WNV infection are recorded since in Greece with seasonal outbreaks occurring almost annually Enhanced surveillance has been implemented since to promptly characterise cases™ temporal and geographical distribution and inform authorities for implementation of appropriate measures mosquito control health education blood safety Aim We describe the epidemiology of WNV human infections in Greece focusing on the season Methods The National Public Health anization advised physicians to test all suspect WNV infection cases and refer samples to reference laboratories Laboratories notified diagnosed cases on a daily basis Treating physicians patients and infected blood donors were interviewed within hours after diagnosis and the probable infection location was identified Hospitalised cases were followed up until discharge Results A total of autochthonous WNV infection cases were diagnosed in Among them cases had neuroinvasive disease WNND representing a increase of WNND cases compared with the previous most intense season There were deaths Cases started occurring from week earlier than usual Both rural and urban areas were affected with of the total municipalities belonging to seven of the total regions recording cases Two major epicentres were identified in Attica and Central Macedonia regions Conclusions The largest number of human cases of WNV infection ever recorded in Greece occurred in with a wide geographical distribution suggesting intense virus circulation Enhanced surveillance is vital for the early detection of human cases and the prompt implementation of response measuresIntroductionWest Nile virus WNV is a flavivirus primarily transmitted to humans equids and other mammals through the bites of infected mosquitoes mainly of the a0Culex a0genus [] WNV lineages and have been associated with significant outbreaks in humans [] Birds serve as reservoir hosts [] whereas humans and equids are considered deadend hosts [] Transmission through blood transfusion and an transplantation can occasionally occur and other rare modes of transmission have been also recorded such as transmission from mother to child during pregnancy delivery or breastfeeding and in laboratory settings []Most humans infected with WNV remain asymptomatic ca develop a disease with fever andor other influenzalike symptoms known as West Nile fever WNF and less than develop neuroinvasive disease WNND such as encephalitis meningitis or “ more rarely “ acute flaccid paralysis AFP [] Elderly and immunocompromised persons are at higher risk of developing severe disease and having a fatal outcome [] No specific treatment or vaccine exists to cure or prevent the disease in humanswwweurosurveillance 0cWNV infection is considered endemoepidemic in parts of Europe affecting countries in southern eastern and western Europe [] and is considered a reemerging public health challenge in the European Union EU with annual seasonal outbreaks during the summer months and early autumnMigratory birds are thought to be the source of introductions of new virus strains into previously unaffected areas [] In Europe the virus has been introduced through migratory birds from Africa [] and its circulation is probably greatly influenced by the flying routes of migratory bird species [] WNV overwinters in mosquitoes [] and overwintering in local bird species in Europe cannot be excluded [] WNV lineage was the main lineage circulating in Europe [] from the 1960s whereas lineage spread over central and southern eastern Europe [] since causing major outbreaks [] In the largest number of cases of WNV infection was recorded in Europe with their total number exceeding the cumulative number of all cases recorded in the previous years between and []In Greece human cases of WNV infections have been recorded since [] with seasonal outbreaks from end June to early October on an almost annual basis [] The causative WNV strain Nea SantaGreece2010 belongs to the Central EuropeanHungarian subclade of WNV lineage [] This strain was detected in patients blood donors mosquitoes horses and birds in many seasons from to []Enhanced surveillance of human WNV infection is annually implemented since from May to November by the Hellenic National Public Health anization NPHOformer Hellenic Center for Disease Control Prevention HCDCP The surveillance is undertaken to promptly identify human cases of WNV infection and monitor their temporal and geographical distribution It also has the objective to detect cases and WNVaffected areas in a timely fashion to inform national and local authorities for the implementation of appropriate response measures including blood safety measures intensified vector control and communication campaigns In the long term surveillance aims to quantify the disease burden and identify seasonal geographical and demographic patterns and populations at risk [] Herein we describe the clinical characteristics and laboratory findings of human cases as well as their distribution in time and place during in GreeceMethodsSurveillanceAs part of routine procedures implemented over the last years before the beginning of each WNV season informative material was sent by the NPHOformer HCDCP to all healthcare facilities and medical associations around the country in May to raise awareness of physicians concerning the diagnosis of WNV infection The NPHOformer HCDCP prompted physicians to further conduct laboratory testing of all WNV infection suspected cases who were defined as any person with acute onset of neurological syndromes encephalitis meningitis or myelitis as well as any person with nonneurological illness but unexplained fever NPHOformer HCDCP recommended referral of samples to specific specialised laboratories for testingThe Vectorborne Diseases VBD Office of the NPHOformer HCDCP implemented active laboratorybased surveillance throughout the transmission season with daily communication with three referencespecialised laboratories further described in the ˜laboratory methods™ section and daily reporting of diagnosed cases to NPHOformer HCDCP Laboratory diagnosis was freeofcharge for patients in the referencespecialised laboratories reimbursed by NPHOformer HCDCPBlood safety authorities informed NPHOformer HCDCP about any infections diagnosed among blood donors tested in affected areasThe EU case definition of WNV infection [] was used with slight modifications in that only laboratory “ and not epidemiological “ criteria were used to define probable cases a confirmed case was defined as a person with either PCR detection of WNV nucleic acid in any biological specimen such as blood cerebrospinal fluid CSF or urine or detection of a WNV specific IgM antibody response in CSF or WNV IgM and IgG antibody response and confirmation by neutralisation tests A probable case was defined as a person with positive IgM antibody response only in serum Asymptomatic infections detected during blood screening in affected areas were also included “ for the first time in “ in the case definitionMunicipalities the lowest administrative unit where at least one human laboratorydiagnosed case of WNV infection was exposed during the period were defined as affected areasData collectionThe VBD Office of the NPHOformer HCDCP investigated and recorded all cases with laboratorydiagnosed WNV infection symptomatic or not with or without neuroinvasive disease The disease was classified as WNND encephalitis meningitis meningoencephalitis or myelitisAFP based on the treating physicians™ clinical assessment andor additional laboratory andor imaging findings when availableCases were investigated by the VBD Office with indepth telephone interviews with the patients or if this was not possible with their close relatives ideally within “ hours after diagnosis using a standardised investigation form to obtain detailed travel history during incubation period “ days before symptom wwweurosurveillance 0cTable Numbers of total WNV affected areasa infection cases WNND cases and related deaths and case fatality per year as well as annual incidence of total WNND cases Greece “2018b a0n WNV infection casesCriteriaNumber of affected municipalitiesNumber of affected regional unitscNumber of affected regionsNumber of WNV infection casesNumber of WNND casesPercentage of WNND casesIncidence of WNND cases per populationNumber of fatal cases with WNV infectionCase fatality among cases with WNV infectionNumber of fatal cases with WNNDCase fatality of cases with WNNDNANANANANANA2018b NA not applicable NUTS nomenclature of territorial units for statistics WNND West Nile virus neuroinvasive disease WNV West Nile virusa a0Number of affected municipalities regional unitsb and regions with ‰¥ case of WNV infectionb a0To put the data in a further more recent perspective the numbers for were affected municipalities affected regional units five affected regions WNV infection cases WNND cases corresponding to of WNV infection cases WNND cases per population fatal cases with WNV infection corresponding to a case fatality of among cases with WNV infection and fatal cases with WNND representing a case fatality of in cases with WNNDc a0Regional units NUTS3onset and identify the probable place of exposure In case of complex travel history the most probable place of infection was defined “ after risk assessment “ by a multisectoral working group of the Ministry of Health dedicated to designate the areas affected by VBDThe treating physician was interviewed to record the clinical manifestations and underlying diseases of each patientThe data collected included demographic characteristics clinical manifestations underlying chronic diseases dates of onset of symptoms hospitalisation diagnosis discharge admission and discharge from intensive care unit ICU potential risk factors laboratory results detailed travel history during the maximum incubation period Hospitalised patients were actively followed up on a daily basis by telephone and their outcome at the end of hospitalisation was recordedThe NPHOformer HCDCP informed on a daily basis by emails national regional and local public health authorities about the cases and their probable place of exposure in order to implement responsive preventive measures vector control communication activities blood safetyWeekly surveillance reports were published on the NPHOformer HCDCP websiteLaboratory methodsOn a daily basis the VBD Office of the NPHOformer HCDCP received laboratory data of the diagnosed including cases mainly the National Reference Center for Arboviruses and Haemorrhagic Fever Viruses Department of Microbiology Medical School Aristotle University of laboratories from three Thessaloniki the Department of Microbiology Medical School National and Kapodistrian University of Athens and the Diagnostic Services Laboratory Public Health Laboratories Hellenic Pasteur Institute of Athens in which the vast majority of suspected cases were tested and of cases were diagnosed in Serum and CSF specimens were tested for the presence of WNVspecific IgM and IgG antibodies using commercial ELISA kits Focus Technologies Cypress CA United States Reverse transcription RTPCR tests in house and commercial were performed on RNA extracted from blood CSF or urine samples The National Reference Center for Arboviruses performed an RTnested PCR which amplifies a 520bp fragment of the WNV nonstructural protein NS3 gene on PCRpositive samples [] All PCR products were sequenced in a ABI Genetic Analyzer Applied Biosystems Foster City CA US Nucleotide sequences were aligned with similar sequences of the same genomic region retrieved from the GenBank database and phylogenetic analysis was performed using the molecular evolutionary genetics analysis MEGA7 software [] The National Reference Center for Arboviruses performed also neutralisation tests in PCRnegative IgGpositive serum samples and applied PCR combined with Sanger sequencing for the genetic characterisation of the strains in addition next generation sequencing was performed on selected samplesBlood safety measuresBlood safety measures for the protection of blood donations against WNV infection were implemented nationwide for blood donors residing or having visited affected municipalities These measures included blood donor deferral or screening of donated blood for WNV RNA with targeted individual donation ID wwweurosurveillance 0cFigure Number of laboratory diagnosed West Nile virus neuroinvasive disease cases by week of symptom onset Greece “2018a n 242bsesac fo rebmuNJuneJulyAugustSeptemberOctoberWeek number and month of symptom onseta In and no cases were recorded Each box represents one WNND case diagnosed and reported in b One WNND case with undetermined week of symptom onset is not includednucleic acid amplification testing NAT and haemovigilance ie surveillance of serious adverse or unexpected events or reactions in donors or recipients epidemiological followup of donors as referred to in and Annex III of the Commission Directive 200433EC []Data analysisWe performed descriptive analysis of the surveillance data ie geographical and temporal distribution of human cases with WNV infection demographic characteristics age sex clinical manifestations underlying diseases and clinical outcomeWe assigned week numbers using the International anization for Standardization ISO standard []Ethical statementNo ethical approval was needed for this study as no individual data were identifiable and only aggregated data were analysed and presentedResultsOn week NPHOformer HCDCP was notified of the first six human WNV infection cases in the season in Attica Region with symptom onset on May week and within the first fortnight of June Cases continued to occur throughout the transmission period in several areas of the countryA total of autochthonous cases of WNV infection were recorded in all over Greece including WNND cases WNF cases and six asymptomatic cases blood donors The overall WNND incidence was cases per population this was the highest incidence ever recorded in Greece with a statistical significant increase compared with wwweurosurveillance201020112012201320142017 0cFigure Incidence per population of West Nile virus neuroinvasive disease by probable municipality of exposure Greece n 242a]]]]][]a For one case the suspected municipality of exposure could not be determinedp the previous most intense season Table Two of the reported WNV infection cases were diagnosed abroad in Czech Republic and in Italy respectively One more WNND case was classified as imported from Romania not included in the current analysisEleven cases of WNV infection were blood donors aged “ years old diagnosed in affected areas five of these cases developed symptoms and six remained asymptomaticIn of cases presented with WNND compared with a total of of cases diagnosed in the previous “ period ranging from in to in Table A total of cases of WNV infection or their close relatives were directly interviewed by NPHOformer HCDCP These included the exported case diagnosed in Italy who was also investigated in collaboration with the Italian public health authorities Four cases or their relatives diagnosed in Greece could not be directly contacted and the exported case diagnosed in the Czech Republic was investigated by the Czech public health authorities Close relatives of cases were interviewedFor cases of the interviewed cases the exact date of diagnosis in the laboratory was missing For the remaining cases these were investigated within a median period of day range “ after diagnosis of cases were interviewed within days after diagnosis and within dayAll symptomatic cases n had symptom onset within the month period from May week to October week while the last date of positive blood sample was on November from an asymptomatic blood donor The number of recorded WNND cases peaked in weeks and with and WNND cases per week respectivelyFigure a0 shows the reported WNND cases by week of symptom onset in compared with previous seasons “For three cases the probable place of exposure could not be determined The remaining WNV infection cases were exposed in a total of municipalities regional units and regions indicating a wide geographical distribution of cases compared with previous transmission periods Table The geographical distribution of WNND cases is presented in a0Figure Two major epicentres were recorded in the regions of Attica and Central Macedonia Table a0Figure with the highest number of WNV infection and WNND cases ever recorded in Attica and the second highest number of cases ever recorded in Central Macedonia since Table Among the regional units nomenclature of territorial units for statistics NUTS3 level affected in were affected at least once in previous seasons and of cases occurred in regional units previously affected Cases occurred both in rural and urban areas and large cities were also affected including the capitalIn June cases were recorded mainly from Attica and a couple of cases from the nearby Sterea Ellada region From July cases were also recorded in Central Macedonia and from August in East Macedonia and Thrace region in northeastern Greece In September“October cases were further recorded in Thessaly region A couple of cases were also recorded in Crete and Peloponnese regions Figure For cases of WNV infection with available information on their place of exposure the vast majority were infected in their place of permanent residence the rest mainly in the place of summer vacationThe median age of the WNND cases in was years range “ years significantly higher p than the median age of the total WNND cases diagnosed in the previous “ period years range “ Among WNND cases were aged years or older The incidence of WNND cases increased from per population in the less than yearolds to per population in those who were ‰¥ years old Table The age of WNND cases in median age years wwweurosurveillance 0cTable Number of WNV infection cases and WNND cases per probable region of exposure and year Greece “2018a a0n WNV infection casesProbable region of exposureAtticaCentral MacedoniaWest MacedoniaEast Macedonia and ThraceSterea ElladaThessalyPeloponneseCreteWest GreeceIonian islandsNorth AegeanSouth AegeanIpeirosUnknownundeterminedTotalNumber of recorded WNV infection cases number of WNND cases per yeara WNND West Nile virus neuroinvasive disease WNV West Nile virusa a0As no cases of WNV infection in Greece were recorded in and these years are not shown in the Tablerange “ was significantly higher p than that of the diagnosed symptomatic WNF cases median age years range “Among the WNV infection cases and the WNND cases n and n were male respectively The WNND incidence among males was almost two times higher than that among females p Table The median period from symptom onset to diagnosis for WNND cases with available relevant information was days range “ as well as for nonWNND cases excluding blood donors range “ The median period from admission to hospital to diagnosis for cases of WNV infection was days range “Among the WNND cases had encephalitismeningoencephalitis including patients also presenting AFP cases had meningitis and three cases had AFP signs only Table A total of diagnosed WNV infection cases reported clinical symptoms the most common being fever followed by malaisefatigue confusionconsciousness level deterioration anorexia sleepiness chills headache gastrointestinal symptoms diarrhoea nausea vomiting abdominal pain dizziness myalgiaarthralgia extrapyramidal signs tremor parkinsonism ataxiagait disorders rash limb paralysis numbness retroorbital pain vision deterioration lymph nodes enlargement Table Among WNND cases reported at least one underlying chronic disease including cardiovascular diseases including stroke and heart disease heart disease including hypertension hypertension diabetes mellitus coronary heart disease chronic neuropsychiatric disease arrhythmia cancer stroke valvulopathyheart failure other immunosuppression than diabetes and cancer including autoimmune disorders hepatic cirrhosis an transplant myasthenia corticosteroids treatment thyroid disease chronic renal failure respiratory disease alcohol abuse Among the symptomatic cases n were hospitalised of the WNND cases and of the symptomatic nonWNND casesThe median duration of hospitalisation of hospitalised cases of WNV infection discharged from the hospital was days range “ while among WNND cases hospitalised and discharged from the hospital it was days range “ Fortyeight cases were hospitalised in ICU with WNND while the one with nonWNND had other comorbidities The median duration of hospitalisation in ICU before discharge or fatal outcome was days range “A total of deaths were recorded during hospitalisation with an overall case fatality CF of among all cases diagnosed with WNV infection Among fatal cases had WNND with a CF among cases with WNND similar with the total CF among cases wwweurosurveillance 0cFigure Number of laboratorydiagnosed West Nile virus neuroinvasive disease cases by week of symptom onset and region of exposure Greece n 241asesac fo rebmuNRegionAtticaCentral MacedoniaCreteEast Macedonia ThraceSterea ElladaPeloponneseThessaly JuneSeptemberOctoberJulyAugustWeek number and month of symptom onsetWNND West Nile virus neuroinvasive diseasea One WNND case with undetermined week of symptom onset and one WNND case with undetermined region of exposure with symptom onset at week are not includedwith WNND observed during the “ period Table The clinical presentation of WNND among fatal cases is described in a0Table The CF of the encephalitismeningoencephalitis cases was whereas it was among meningitis cases The median age of fatal cases was years range “ The median period from symptom onset to death during hospitalisation was days range “The vast majority of patients were retired reported ng agricultural or gardening activities and reported having a routine outdoor activity after dawnNational and regional public health authorities were informed about diagnosed cases and their probable place of exposure within a few hours after their investigation and within hours after their diagnosis via confidential emailsLaboratory resultsOf the cases were confirmed either by WNVspecific IgM antibody response in CSF n andor positive PCR in any sample n in blood andor CSF andor urine andor by WNVspecific IgM and IgG antibody response in serum and confirmation by neutralisation n A total of cases were confirmed by IgM antibody response in CSF only cases were confirmed by positive PCR in any sample only cases were confirmed by neutralisation only and cases were confirmed by more than one confirmatory methodsA total of cases were considered as probable since the diagnosis was based only on the detection of WNVspecific IgM antibodies in seruminto In all but one of the sequenced samples the sequences clustered the Central EuropeanHungarian subclade of WNV lineage similar to the strain of the outbreak NeaSantaGreece2010 strain One sequence taken from a case in Thrace region northerneastern Greece clustered within the Eastern EuropeanRussian subclade of WNV lineage Figure [] a0In total among the cases tested had WNVspecific in serum and had WNVspecific IgM antibody response in CSF In cases with positive IgM and IgG antibodies in serum the diagnosis was confirmed by neutralisation A total of cases had positive PCR in blood cases had positive PCR in urine in of which this was the method of confirmation and eight cases had positive PCR in CSF The median time from symptom onset to sampling for the cases with positive PCR in blood was days range to for the cases with positive PCR in urine days range “ and for cases with positive PCR in CSF days range “IgM antibodies wwweurosurveillance 0cTable Age and sex distribution of West Nile virus neuroinvasive disease cases Greece n CharacteristicsNumber of casesIncidence rate per populationaRisk ratio confidence intervalAge group in years ““““““‰¥SexFemaleMaleReference “ “ “ “ “ “ “Reference a0 a0 a0 a0 a0 a0 “a a0Population data from Hellenic Statistical Authority ELSTAT []Table Number and percentage of WNND cases n as well as number and percentage of fatal cases with WNND n by clinical presentation Greece ParameterEncephalitisMeningo encephalitisMeningitisAFP onlyAFP and encephalitis meningitisNumber of WNND casesPercentage of total WNND casesNumber of fatal cases with WNNDPercentage of fatal cases among total WNND fatal casesNAAFP acute flaccid paralysis NA not applicable WNND West Nile virus neuroinvasive diseaseIn each row where numbers of cases are presented the sum of the cases in the respective row can be more than the total number of cases as a given case could have more than one presentationDiscussionWNV infections show a strong seasonal pattern in Europe with the first human cases usually observed in June and most cases recorded from July to October [] Accordingly from to human cases of WNV infection in Greece were recorded from the end of June until the beginning of October In contrast the onset of symptoms in the first recorded human case in was in week end of May making this the earliest WNV transmission season ever noted in the country and in Europe The seasonal outbreak moreover lasted until early November Figure and coincided with the longest season ie more than months in the last years in Europe [] suggesting a possible need to widen the surveillance periodThe number of recorded WNND cases in Greece in peaked in weeks and and remained at over per week from week to week The numbers of total WNV infections and WNND cases were also respectively the highest recorded since the emergence of the virus in the country in Moreover the WNND cases accounted for more than a third of the total number of such cases recorded since This suggests a more intense seasonal virus circulation during In this regard large outbreaks occurred in other central European and Mediterranean countries simultaneous to the WNV seasonal outbreak in Greece []The increased number of cases diagnosed in Greece in compared with previous years cannot be attributed to a higher sensitivity of the surveillance system as enhanced surveillance has been consistently implemented in the country during each transmission season since This has included raising awareness of physicians on an annual basis publishing updated weekly surveillance reports and providing freeofcharge diagnosis The large number of WNF cases diagnosed in of the total symptomatic cases and in previous years Table further indicates the enhanced awareness of physiciansIn the case definition was expanded to include asymptomatic cases detected during blood screening in affected areas however this change did not crucially affect the overall amount of recorded WNV infections given the small number of diagnosed asymptomatic cases n Either way the number of WNND cases which describes the burden more accurately and is wwweurosurveillance 0cTable Symptomsa a0of West Nile virus neuroinvasive disease cases n and West Nile fever cases n Greece 2018bWNND casesWNF casesSymptomFeverMalaisefatigueConfusion consciousness level deteriorationAnorexiaSleepinessChillsHeadacheAt least one gastrointestinal symptom ie diarrhoea nausea vomiting abdominal painDizzinessMyalgiaarthralgiaVomitingExtrapyramidal signs tremor parkinsonismAtaxia gait disordersDiarrhoeaNauseaRashAbdominal painLimb paralysisNumbnessRetroorbital painVision deteriorationLymph nodes enlargementProportionb a0with the symptomPercentageProportionb a0with the symptomPercentageNANANACKP creatine phosphokinase NA not applicable SIADH syndrome of inappropriate antidiuretic hormone secretion WNF West Nile fever WNND West Nile virus neuroinvasive diseasea a0Other symptomssigns reported from the treating physicians included acute respiratory failure n bradypsychismus n seizures n respiratory infection n dysarthriaspeech disorders n acute renal failure n diplopia n myoclonous n aphasiaapraxia n arrhythmia n cough n hepatitisliver dysfunction n rhabdomyolysisincreased CPK n SIADHhyponatraemia n biphasic fever n pulmonary oedema n sore throat n chorea ballistic movements n dysautonomia n hearing disorders n myocarditisincreased troponine n pulmonary embolism n hypokalaemia n acute epiglottitis n aneurysm rupture n blepharoptosis n Guillain“Barré syndrome

Thyroid_Cancer

Relevance Function of LincROR inthe Pathogenesis of CancerWenjian Chen1  Junfa Yang23  Hui Fang4  Lei Li5 and Jun Sun1 Anhui Provincial Children™s Hospital Affiliated to Anhui Medical University Hefei China Key Laboratoryof Anti‚ammatory and Immune Medicine Ministry of Education Institute of Clinical Pharmacology Anhui MedicalUniversity Hefei China School of Pharmacy Anhui Medical University Hefei China Department of PharmacologyThe Affiliated Hospital of Hangzhou Normal University Hangzhou China The Affiliated Hospital of Anhui Medical UniversityHefei ChinaLong noncoding RNAs lncRNAs are the key components of noncoding RNAsncRNAs with a length of nucleotides They are transcribed from the socalledœdark matter of the genome Increasing evidence have shown that lncRNAs play animportant role in the pathophysiology of human diseases particularly in the developmentand progression of tumors LincROR as a new intergenic nonprotein coding RNAhas been considered to be a pivotal regulatory factor that affects the occurrence anddevelopment of human tumors including breast cancer BC colorectal cancer CRCpancreatic cancer PC hepatocellular carcinoma HCC and so on Dysregulation ofLincROR has been closely related to advanced clinicopathological factors predicting apoor prognosis Because lincROR can regulate cell proliferation apoptosis migrationand invasion it can thus be used as a potential biomarker for patients with tumorsand has potential clinical significance as a therapeutic target This reviewed therole of lincROR in the development of tumors its related molecular mechanisms andclinical valuesKeywords lncRNAs ncRNAs lincROR cancers biomarkerEdited bySridhar MuthusamiKarpagam Academy of HigherEducation IndiaReviewed byAnca Maria CimpeanVictor Babes University of Medicineand Pharmacy RomaniaOmar TorresQuesadaUniversity of Innsbruck AustriaCorrespondenceJun Sunsunjun500aliyuncomsunjun14190163com These authors have contributedequally to this workINTRODUCTIONSpecialty sectionThis was submitted toMolecular and Cellular Oncologya section of the journalFrontiers in Cell and DevelopmentalBiologyReceived April Accepted July Published August CitationChen W Yang J Fang H Li L andSun J Relevance Functionof LincROR in the Pathogenesisof CancerFront Cell Dev Biol 103389fcell202000696Cancer is a serious disease that aï¬ects human health being one of the main causes of death all overthe worldwide According to research in of new tumor cases and of the cancerassociated deaths occurred in lowincome and developing countries Kumar and Sharawat Noorolyai Owing to a shortage in eï¬ective screening methods and lack of identificationof early symptoms most patients were already in advanced stages when they were diagnosedwith cancer Bray Koo Additionally some clinical studies have shownthat polarity and adhesion of cancer cells was decreased leading to heir increased mobility andinvasion which is a key step in the development of cancer Yan Therefore studies haveshown that the high mobility of cancer cells is the main factor leading to high mortality rates inpatients with cancer Currently there are many ways employed in the treatment of cancer includingsurgery radiotherapy chemotherapy biotherapy and targeted therapy Nie Howeverin the past years the survival rate of patients with cancers remains dismal Nakashima Abbreviations BC breast cancer ceNAs competing endogenous RNA EMT epithelial“mesenchymal transition HCChepatocellular cancer iPSCs induced pluripotent stem cells lincROR Long intergenic nonprotein coding RNA regulatorof reprogramming lncRNAs Long noncoding RNAs PC pancreatic cancerFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORin the process of developing human antitumorThereforestrategiesto find new earlyitbiomarkers and thus identify potential regulatory mechanisms toimprove the survival rate of patients with cancersis particularly importantOver the past decades ncRNAs constitute more than of the RNAs made from the human genome but mostof the known noncoding RNAs ncRNAs havebeen discovered and remain largely unstudied Bhan Slack and Chinnaiyan Transfer RNA tRNA and ribosomal RNA rRNA constitute themajority of ncRNAsfollowed in abundance by messengerRNAs mRNAs Thus the remaining ncRNAs includingcircular RNA circRNA small nuclear RNA snRNA smallnucleolar RNA snoRNA microRNA miRNA and long nonfor ˆ¼ ofcoding RNA lncRNA together accounttotalncRNA Despite their low abundancethese ncRNAs havebeen reported to play critical roles in transcription posttranscriptional processing and translation such as epigeneticsposttranscriptional regulation chromatin modification andregulation of the cell cycle Huarte Kondo Peng 2017b Additionally because ncRNAs can be packagedinto extracellular vesicles EV including exosomes Meldolesi they have been shown to provide a mechanism forintercellular communication through the transfer of miRNAand lncRNA to recipient cells both locally and systemicallySun It is important to note that the expressionof ncRNAs their posttranscriptional modification particularlylncRNAs and their subcellular distribution have been shownto be important to when assigning their potential functionPalazzo and Lee Recently nextgeneration sequencingand bioinformatics technology have revealed that circRNAsplay crucial role in diagnosis and prognosis of various diseasesPamudurti Briefly circRNAs are singlestrandedtranscripts generated by backsplicing Jeck and Sharpless with covalently linked headtotail closed loop structures withneither 5cid48“3cid48 polarity nor a polyadenylated tail Memczak that range in length from a few hundred to thousandsof nucleotides and are widely expressed in mammals therebyshowing higher stability compared to that in linear RNAs ChenJ and exhibiting a celltype or developmentalstagespecific expression pattern Barrett and Salzman Wang J J Many functions of circRNAs have alsobeen identified including their role as miRNA sponges bindingto RNAbinding proteins and protein decoys and functioningas regulators of transcription Hansen Du Yang Y Interestingly many circRNAs havebeen shown to be dysregulated in pathophysiological processesand circRNAs are known to regulate the expression of geneby acting as miRNA sponges in a mechanism that is termedas competitive endogenous RNA ceRNA mechanism Zheng Wang J J For example circMTO1have been demonstrated to harbor conventional miRNA bindingsites and has been identified as an inhibitor of miRNA9 inhepatocellular carcinoma HCC Han Additionallyour previous study has demonstrated that miRNA plays arole in limiting the development of liver fibrosis by markedlyblocking the activation and proliferation of hepatic stellatecells HSCs suggesting that miRNAs might be involved inthe development and progression of several forms of cancersYang J Yang Of notelncRNAswhich are mainly transcribed by RNA polymerase II are anew kind of ncRNA that are longer than nucleotidesMa Owing to the lack of reading frameslncRNAs have extremely limited or no protein coding capacityRuan Li J These new regulatorswere initially regarded as transcriptional noise with no specificbiologicalfunctions Kim and Sung Recently ourlaboratory found that epigenetic silencing of lncRNA ANRILpromoted the progression of liver fibrosis thereby indicating thatlncRNAs were associated with the progression of cancers Yang Interestingly increasing evidence have shown thatcellular events including diï¬erentiation proliferation invasionapoptosis and migration have all been associated with lncRNAsGuttman Additionally there has been new evidencesuggesting that lncRNAs may regulate a variety of biologicaland disease processes from gene transcription and translationto posttranslational modifications Davalos and Esteller Pang More importantlylncRNAs have beenreported to be used as tumor suppressor genes or oncogenesthus aï¬ecting the proliferation and metastasis of various typesof tumors during tumorigenesis Chen Q N Lu Subsequent studies have demonstrated thatlncRNAs may serve as ceRNAs for miRNAs and in chromatinremodeling during the development of cancers Huang Wang C J Figure illustrates the functionsof lncRNAs at the molecular level Regarding certain cancerassociated human lncRNAsit was demonstrated that lincROR was demonstrated to be predominantly upregulated intumors Peng 2017a The abnormal expression of lincROR in tumors has been suggested to be one of the mainleading factors driving the development The main ways torevert this eï¬ect would be to aï¬ect cell growth migrationand invasionthus leading to the inhibition of epithelialmesenchymal transition EMT enhancement of the sensitivityto chemotherapy etc Chen 2016a Zhao Forexample the expression level of lincROR in HCC tissues wasinhibited compared with the adjacent tissues At the same timethe downregulation of lincROR was linked to the aggressiveprocess of the disease in patients with HCC Furthermore theability of migration and invasion of HCC cells may be delayedby the low expression level of lincROR In this review weattempted to introduce the latest research on the biologicaleï¬ects potential clinical applications and molecular mechanismsof lincROR in human tumors and discuss its prognostic andtherapeutic valuesOVERVIEW OF LincRORlincROR isAmong lncRNAsand importantcarcinogenic kb lncRNA located in chromosome which was initially identified as a highly expressed transcriptof pluripotent and embryonic stem cells Chen 2016bStudies found that the octamerbinding transcription factor a novelFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORFIGURE Paradigms for the function of long ncRNAs Recent studies have identified a variety of regulatory paradigms for the mechanism by which long ncRNAsfunction many of which are highlighted here Transcription from an upstream noncoding promoter pink can negatively or positively affect the expression ofthe downstream gene purple by inhibiting the recruitment of RNA polymerase II or inducing chromatin remodeling respectively An antisense transcript orangeis able to hybridize to the overlapping sense transcript purple and block the recognition of the splice sites by the spliceosome thus resulting in an alternativelyspliced transcript Alternatively hybridization of the sense and antisense transcripts can allow Dicer to generate endogenous siRNAs By binding to specificprotein partners a noncoding transcript blue can modulate the activity of the protein serve as a structural component that allows the formation of a largerRNAprotein complex or alter where the protein localizes in the cell Long ncRNAs green can be processed to yield small RNAs such as miRNAspiRNAs and other less wellcharacterized classes of small transcriptsOCT4 SRYbox transcription factor SOX2 and Nanoghomeobox Nanog key pluripotency factors could regulatelincROR Wang HoweverlincROR was alsofound to be expressed in several ans including lungliver breast and colon Since its discovery research in thisfield has been extensively expanded during the past yearsrevealing the important role of lincROR in tumorigenesislincROR has been suggestedAdditionally upregulation ofto mediate the reexpression offetal and cardiomyocytehypertrophyrelated genes Wang Li inMany reportsrecent years have revealed thatlincROR is positively correlated with the clinicopathologicalcharacteristics and poor prognosis of tumorsincluding thestages of advanced tumor node metastasis TNM positivelymph node metastasis LNM and lower survival rate buthigher recurrence rateregarding lincROR and tumorigenesisthe upregulation ofCurrent evidence have strongly indicated thatlincRORmay exert an impact on a variety of cancers Pan Furthermore both tumorigenesis and metastasis havebeen shown to be induced by lincROR via activation of theEMT in various cancers Hou Huang Zhan For example lincROR was demonstratedto be upregulated thereby promoting EMT in HCC Li J Besides it was also reported that selfrenewal anddiï¬erentiation of glioma stem cells was significantly aï¬ectedby lincROR Zhang Feng Moreimportantly the chemoresistance of pancreatic cancer PCand breast cancer BC Li as well as radioresistance of colorectal cancer CRC cells were observed to beelevated by lincROR Yang P Moreover lincROR has also been shown to exert a significantly eï¬ect onthe stem celllike characteristics and tumorigenic potential ofPC Recentlyit was also reported that lincROR could beused as a biomarker in the field of diagnosis and prognosisof BC and oral cancer Arunkumar Zhao Notably increasing studies showed that lincROR couldbe used as a ceRNA thus exerting its impact in the posttranscriptional network of tumor pathogenesis For examplein triplenegative BC lincROR has been shown to serve asa ceRNA therefore promoting the migration and invasion ofBC cells Signal Overall lincROR is a typicallncRNA that plays important regulatory roles in interactionwith miRNAs and maintenance of stem cell pluripotencytriggering the EMT as well Furthermore lincROR has alsobeen involved in various key roles under hypoxia and in thepromotion of tumorigenesis Figure Therefore lincRORmay be considered as an oncogene aï¬ecting the progressionof tumor and a promising predictor for the poor prognosis inpatients with cancer The transition of lincROR from basicresearch to clinical application requires further investigations asearly as possibleFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORFIGURE LincROR is a typical lncRNA that plays important regulatory roles in interacting with miRNAs and maintaining stem cell pluripotency as well astriggering the EMT as well LincROR is also involved in various key roles under various stresses and in epigenetic regulationREGULATORY ROLE OF LincROR INVARIOUS TYPES OF CANCERIncreasing evidence has shown that the lincROR was abnormalexpression in many cancers and its dysregulation was associatedwith cellular functions Fu Spinelli Additionally studies found that the expression level of lincROR was substantially upregulated in samples of papillarythyroid carcinomas PTCs and PTCs cell lines as well as inmetastatic PTCs samples and PTCs cell lines Zhang Simultaneously cell migration and invasion could be regulatedby lincROR via aï¬ecting EMT Pastushenko and Blanpain More importantly studies demonstrated that lincRORwas abnormally expressed in several cancers and led to elevatedthe invasion and metastasis of cancer cells to promoting theprogression of tumors Hashemian Li 2020bcThis review summarizes the status of lincROR research invarious human cancers and discusses its mechanism and clinicalsignificance in the development and progression of tumor Theexpression pattern functional role and regulatory mechanism oflincROR are summarized in Table and depicted in Figure Breast CancerBC which accounts for a quarter of all female cancer casesis the most commonly diagnosed cancer and the leading causeof cancerassociated deaths among women worldwide Li Z In it was estimated that there would be million or so newly diagnosed cases of female BC Bray Hannafon The main risk factors forBC which is the difficult to change due to prolonged exposureto endogenous hormones is difficult to control Rudel Bray However comprehensive treatmentapproaches have resulted in relatively good clinical outcomesfor some patients with BC Rudel Goel Kumler Nevertheless it has been reported that aboutonethird of the patients with BC have the potential for cellmetastasis chemotherapy resistance and even recurrence Goel Kumler Hence there is an urgent need todevelop new therapies targeting various molecular mechanismsof tumorigenesis for the treatment of BCthe level ofthe level ofHou et althe expression ofinvestigated the expression level of lincRORin patients Hou Their results revealedlincROR was increased in BC tissuesthatMoreoverlincROR in theperipheral blood ofthe patients with BC was shown tobe closely related to TNM phase and LNM In additionthe woundhealing assay showed that overexpression of lincROR increased BC cells MCF10A mobility Transwell assayrevealed that lincROR overexpression remarkably increased themigration ability Hou More importantly theyfound that ectopic expression of lincROR induced an EMTprogram in MCF10A cells Fluorescence activated cell sorteranalysis demonstrated that the subpopulation of the stem cellphenotype CD44highCD24low was elevated in MCF10A cellstransfected with lincROR plasmid Mechanistically the resultsof bioinformatic analysis and RNA immunoprecipitation analysisfrom Hou demonstrated thatlincROR functions asa ceRNA to regulate miR205 activity toward prevention ofthe degradation of transcripts of miR205 target genes suchas ZEB1 and ZEB2 from degradation Additionallyit wasshown that the expression levels of miR205 members weredecreased upon lincROR overexpression in MCF10A cellsHou More importantly Zhao recentlydemonstrated that CRISPRCas9generated BRCA1knockdownadiposederived stem cells stimulated a more aggressive behaviorFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORlateiLlateoaGlatenahZlatenehClateCiLlateihZlateiLnuSdnanaYlatenaPalategnahZlateuoHsecnerefeRBEZRmRORcniilMKPPBTPRmRORcniilgonaNRmRORcniilnirehdacEHZERORcnilSOSFDMNLegatsMNTTMEinosavniTMEinosavninoitargminoitarefiloPicnegocnOldetaugerpUamoncraciraulllecotapeHllseuceomyrotaugeRllnoitaerroclacniilCleorlanoitcnuFleoRinosserpxEsepytrecnaCsrecnacnamuhniRORcnLiELBATiRmRORcnilevrucCORMNLegatsMNTnoitargmiissotpopanoitarefilorPicnegocnOldetaugerpUrecnactsaerBTMEinosavnipBEZRORcnilliavvrusroopMNLegatsMNTnoitargminoitarefilorphtwicnegocnOldetaugerpUrecnaccitaercnaPMXOFpRmRORcniilNCSFRmRORcnipRmRORcniililiRmRORcnilecnatsserigurdTMEinosavniTMElecycllecinosavniliavvrusroopMNLegatsMNTnoitargmiissotpopanoitarefilorPicnegocnOldetaugerpUrecnacdoryhTiSOSFDMNLegatsMNTinoitargmecyclllecissotpopAicnegocnOldetaugerpUrecnacgnuLecnatsseriyparehtodariTMEinosavniSOSFDMNLegatsMNTnoitargmiissotpopanoitarefilorPicnegocnOldetaugerpUrecnaclatcerooClin BC cells than wildtype adiposederived stem cells Zhao Therefore we believe that CRISPRCas9 may beused to in the treatment of BC by inhibiting the expressionof lincROR during the progression of BC Conclusively thelincRORmiRNAs axis has been reported to closely aï¬ect theoccurrence and development of BCPancreatic CancerPC is the fourth most common cause of cancerrelated mortalityworldwide leading to approximately deaths annuallySiegel Sabater The ˆ’year relativesurvival of patients with PC remained at approximately for“ Siegel Hence PC has been proposedto be one of the top two cancers in terms of fatalities in thenext decade Rahib Surgical resection remainsthe exclusive potential curative treatment Xiong However approximately half ofthe patients present withmetastasis at the time of diagnosis missing the opportunityfor an eï¬ective treatment Vincent Xiong A growing body of literature has demonstrated that bothmetastasis and limited eï¬ective biomarker for the diagnosis andtreatment are the main obstacles for the efficient medical therapyof PC Vincent Boj Basuroy Thus it is an absolute necessity to identify potential biomarkersand therapeutic targets in PCZhan have highlighted the oncogenic eï¬ectsof lincROR in the initiation and progression of PC Theirstudy demonstrated that the level of lincROR was significantlyelevated in PC tissues Zhan Moreoverthewoundhealing assay and Boyden™s chamber assay results showedthat lincROR silencing reduced the migratory capability andmetastasis of PC cells Zhan Another study byChen showed that the proliferation rates of shRORcellsin which the level of lincROR was suppressed were evidentlylower than those of shNCcells This result was confirmed bycolony formation assay suggesting that lincROR accelerated thegrowth of PC cells Chen 2016a Interestingly silencingof lincROR was shown to result in increased levels of theepithelial markers Ecadherin and αcatenin and decreased levelsof mesenchymal markers Ncadherin and vimentin indicatingthat lincROR plays an important role in the regulation ofEMT in PC cells Zhan Chen Moreimportantly microarray analysis identified ZEB1 as potentialtarget gene of lincROR Further the expression of lincRORand ZEB1 were observed to be negatively correlated with thatof p53 suggesting that lincROR might mediate migration andmetastasis in PC cells may partly via activation of ZEB1 throughthe inhibition of the expression of p53 Zhan Interestingly the fluorescence in situ hybridization and luciferasereporter assay results showed that the expression of lincRORwas demonstrated to be negatively correlated to that of miR145MiR145 can induce posttranscriptional silencing of its targetedgenes by binding to the mRNA ™UTR or lincROR specificsites indicating that lincROR can act as a ceRNA to decreasemiR145 in PC cells thereby activating expression of Nanogthus leading to the proliferation of pancreatic cancer stem cellsPCSCs Gao Additionally Li furtherFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORFIGURE Underlying molecular mechanisms of lincROR in multiple cancers A LincROR binds to miR205 to upregulate ZEB2 while it also regulated theexpression of EMT markers B LincROR could interact with miR145 to inhibit FSCN1 and upregulated EMTassociated proteins while it decreases G0G1 arrestand facilitated drug resistance C LincROR facilitated EMT through upregulate EZHZ and regulated ZEB2 by competitively binding to miR145 and ZEB2overexpression leads to increased EMT In addition lincROR binds to miR8765p to upregulate FOXM1 D LincROR downregulated through EMT production andrepress the expression of miR145 E LincROR binds to miR68333p while also regulating the process of EMT F LincROR upregulated ZEB1 to attenuate theexpression of p53 while also decreasing the expression of miR145 to increase the level of Nanog and reduce that of miR124 to suppress PKM2 Detailedmechanisms of lincROR in other cancers are provided in the reviewproved that the impact of lincROR can be partly reversed byoverexpression of miR124 Consistently lincROR was observedto exhibited a negative correlation with the expression of miR Li Hence a lincRORmiR124PTBP1PKM2axis was identified in PC shedding new light on the lncRNAbased diagnosis and therapeutic approaches in PC Li 2020b Notably recent studies showed that PC cellderivedEVs could be used as eï¬ective carriers of paclitaxel to theirparental cells thereby bringing the drug into cells through anendocytic pathway and increasing its cytotoxicity Saari Additionally it was demonstrated that vesiclecontainingncRNAs could serve as EVassociated PC detection markersWorst Thus the presence of lincRORs in EVs frompatients with PC could serve as a potential diagnostic biomarkerand a novel target for the therapy of patients with PC this isworthy of further and wider research attentionHepatocellular CarcinomaAs the sixth most international commonly occurring cancer in HCC has become the fourth cause of cancerassociateddeaths worldwide It has been estimated that new casesand deaths will occur each year Bray BrieflyHCC has been reported to account for “ of all the livercancer cases half of which have been detected in China Omata Bray As such HCC poses a huge threatto the worldwide health especially that of the Chinese peopleOmata About of the patients is expected torecrudescent within years after hepatectomy and of thepatients will die from this tumor Vigano Thereforeon the basis of studying the pathogenesis of HCC it is apparent tolook for more eï¬ective molecular markers and therapeutic targetsfor the management of HCCLi C and Chen reported that theexpression level of lincROR was obviously elevated in HCCtissues and four cell lines compared to the corresponding nontumor tissues and normal liver cell lines respectively suggestingthat lincROR might be critical regulator in the progression ofHCC Furthermore biological function assay demonstrated thatlincROR could play promoting role in regulating migration andinvasion of HCC Chen Moreover downregulationof lincROR could result in a significant increase in G1G0phase and an obvious decrease in S phase Li C More importantly silencing of lincROR could lead to theincreased expression of Eˆ’cadherin and decreased expressionlevel of Nˆ’cadherin in HCC cell lines Li C furtherconfirmed that lincROR could bind to the zeste homolog EZH2 thereby aï¬ecting the expression of Ecadherin furtherindicating that lincROR could regulate the progression of EMTMoreover lincROR was further determined to be associatedwith DNA repair Currently mounting studies have identifiedreliable indicators of DNA damage such as phosphorylatedhistone H2AX γH2AX Chen uncover thatFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORlincROR could obviously decrease theoverexpression ofexpression level of γH2AX illuminating the suppressive eï¬ectsof the overexpression of lincROR on DNA repair in HCCFurther research demonstrated that lincROR could interactwith miRˆ’ and dramatically downregulate the expression ofmiRˆ’ in HCC cells Li C The abovementionedresults revealed that lincROR might play a promoting role inthe proliferation migration invasion and EMT of the HCC cellwhich was contrary to the ‚uence of miRˆ’ enrichmentLi C It suggested that overexpressed miRˆ’could eï¬ectively reverse the promotion of HCC tumorigenesisinduced by the overexpression of lincROR Li C Liand his colleagues proposed a mechanistic model that lincRORpromotes HCC tumorigenesis and autophagy partly throughnegatively regulating the expression of miRˆ’ Aside fromthat it has been reported that miR145 represses EMT tumormigration and invasion by directly targeting the ™UTRs ofZEB2 in the tumor The decrease in miRˆ’ and increase inZEB2 can obviously reversed the inhibition of cell migrationand invasion mediated by the lincROR knockdown Thereforeit was suggested that targeting the lincRORmiRˆ’145ZEB2axis might represent a novel therapeutic application in HCCLi C Similarly Zhi similarly showedthat the migration and invasion of cells was reduced by theknockdown of lincROR Moreover they further confirmedthat FOXM1mediated activation of lincROR contributes tothe poor sensitivity of HCC cells to sorafenib via partiallyregulating the miR8765pFOXM1 axis which forms a positivefeedback loop Further evaluation of the regulatory mechanisminvolving this axis may provide new insights for exploringa potential therapeutic strategy for the management of HCCZhi Consequently these studies may oï¬er newinsights regarding the pathology of HCC and provide potentialstrategies for lncRNAdirected treatment However both thein vivo ‚uence and other underlying mechanisms of lincRORstill remain to be determined and clarified in the future researchthe prognosisColorectal CancerThere are approximately million new CRC cases and CRCrelated deaths worldwide each year thus making CRC thethird most common cancer in the world Torre Although the treatment of CRC has significantly improvedin recent decadesremains unsatisfactoryespecially in case of advanced tumors with distant metastasesBogousslavsky Torre Current studiesresults showed that approximately of cases with CRChave synchronous liver metastases during the time of diagnosisKawaguchi These patients have inherently lowsurvival rates of less than within years with an even worseprognosis Hu Kawaguchi Thus there isan urgent need to better understand the progression of CRC andto identify novel and sensitive biomarkers for the diagnosis andtreatment of patients with CRCYang detected the expression of lincROR in CRCtissues compared to normal tissues by using qRTPCR Theyfound that the expression of lincROR was remarkably increasedin CRC tissues compared with normaltissues SimilarlylincROR was shown to be overexpressed in five CRC cell linesYan and Sun Li 2020a Then they also performeda series of functional assays to clarify the biological eï¬ects ofthe aberrant expression of lincROR on proliferation viabilityapoptosis migration and invasion of CRC cells Knockdownof lincROR was shown to eï¬ectively inhibit the proliferationof CRC cells whereas its overexpression obviously increasedthe proliferative capacity of CRC cells Accordingly silencing oflincROR strongly inhibited the migratory and invasive abilitiesof CRC cells compared with that in the control cells Li et al2020a In contrast the migratory and invasive ability of cells wasactivated following the overexpression of lincROR The MTS345dimethylthiazol2yl53carboxymethoxyphenyl24sulfophenyl2Htetrazolium assay results showed thatthelincROR could enhance the viability ofoverexpression ofCRC cells Furthermore the flow cytometric analysis resultsrevealed that the percentage of apoptotic cells in lincRORoverexpression group was reduced by ± indicatingthat the overexpression of lincROR could inhibit apoptosisin the CRC cell lines Li 2020a More importantly arecent study revealed the role of lincROR in the EMT It wasrevealed that the upregulation of lincROR could increase theexpression of Ncadherin and Vimentin as well as decrease thelevel of Ecadherin leading to the promotion of the progressionof EMT Zhou Yan and Sun Meanwhilethe high expression of lincROR in CRC was also confirmedby Hu Mechanistically Li 2020a provedthat that lincROR could bind to miR68333p which wasdetermined to be significantly downregulated in CRC tissuesAdditionally a negative correlation was exhibited between theexpression of lincROR and miR68333p in BC tissues AntiAGO2 RNA immunoprecipitation assay further confirmed theseresults Li 2020a Besides rescue assays demonstratedthat downregulation of miR68333p could partly reversed theinhibition of tumorigenesis induced by lincROR knockdown inBC cells Li and his colleagues uncovered that lincROR exertedits oncogenic role through negatively regulating the expressionof miR68333p during the progression of CRC which mightgive new insights into molecular diagnosis and treatment Li 2020a In addition Li 2020a further uncoveredthat lincROR could mediate the expression level of SMC bysponging miR68333p in CRC cells thus promoting CRCprogression As for the eï¬ects of lincROR on radiotherapyresistance Yan and Sun showed that overexpression oflincROR increased the ability of CRC cells for radiotherapyresistance Collectively these findings indicated that lincRORmight be engaged in the metastatic process of CRC cells andcould promote the development of CRC through a variety ofmolecular mechanismsLung CancerLung cancer is the leading cause of cancerrelated deathsworldwide Bray Nonsmall celllung cancerNSCLC accounts for about of the lung cancer typesincluding squamous cell carcinomalung cancerand lung adenocarcinoma Herbertz Bray Brainard and Farver Although there are variousapproaches for its diagnosis and treatments the 5year overallsurvival OS rate for patients with advanced lung cancer is lesslarge cellFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORthan Zhou Therefore in order to carry outan early diagnosis and treatment of lung cancer the search forvaluable and efficient tumor markers is very urgentIn recent yearslincROR has appeared as an importantregulator oflung cancer Research from Qu demonstrated that the expression of lincROR was increasedin NSCLC tissues compared to that in the normal tissuesThe overexpression of lincROR was shown to be closely relatedto the poor prognosis of LNM histological grade and stageof TNM Pan Qu Another s

Thyroid_Cancer

"assess the antioxidative activity of seleniumenriched ChrysomyiaMegacephala Fabricius C megacephala larvae powder SCML and its impact on the diversity and structure ofintestinal microflora in a mouse model of Dgalactose Dgalinduced oxidative damageMethods Sixty male ICR mice were equally randomized to a normal control NC group a model group a positivegroup a lowdose SCML LSCML group a middose SCML MSCML group and a highdose SCML HSCMLgroup Animals in NC and model groups received water animals in the positive group received mgKg vitamin EVE and those in the three SCML groups received SCML which include and μgKg selenium Serespectively An oxidative damage model induced by subcutaneous injection of Dgal for weeks via the neck wasestablished Serum oxidative stress levels and tissue appearance were evaluated Tissues oxidative stress levels weredetected by commercially available kit Nuclear erythroid 2related factor Nrf2 and gut microbiota weredetermined by western blot and high throughput sequencing 16S rRNA gene respectivelyResults An oxidative damage model was established successfully as represented by a significant elevation ofmalondialdehyde MDA and protein carbonylation and inhibition of the antioxidants including superoxide dismutaseSOD glutathione peroxidase GSHPx total antioxidant capacity TAOC and glutathione GSH It was found thatoxidative damage and histological alterations were attenuated the expression of Kelchlike ECHassociated proteinKeap1 was decreased and the expression of Nrf2 and hemeoxygenase1 HO1 was increased after SCML treatmentIn addition significant changes were observed in the gut microbiota including Proteobacteria and the ratio ofBacteroidetes to Firmicutes at the phylum level as well as Helicobacter Clostridium and Lactobacillus at the genus levelContinued on next page Correspondence jiangzcmueducn Dandan Xie and Liqin Jiang contributed equally to this work1College of Pharmaceutical Science Zhejiang Chinese Medical University Binwen Road Hangzhou ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cXie BMC Complementary Medicine and Therapies Page of Continued from previous pageConclusion SCML exerted an antioxidative effect in vivo probably by increasing the antioxidant activity and reducingthe production of oxidation products via the Nrf2 signaling pathway SCML could also redress the intestinal floraimbalance induced by oxidative stress All these findings suggest that SCML could serve as a functional food andnatural drug additive to protect the human body against oxidative damageKeywords Seleniumenriched Chrysomyia Megacephala Fabricius larvae powder SCML Antioxidant activity Nrf2Intestinal microbiota In vivoBackgroundAging is a natural process that involves the gradual loss ofphysiological functions causing enhanced morbidity andmortality due to various diseases This process is closelyrelated to oxidative stress [“] One prevalent theory toexplain aging is the theory of the oxygen free radical []This theory posits that the macromolecules such as nucleic acids lipids sugars and proteins that make up cellsand tissues are subjected to oxidative stress induced bysuperoxide and other free radicals These macromoleculesthen undergo different degrees of oxidation which initiates oxidative damages and ultimately leads to an function impairment and aging [ ] Changes in the level ofoxidative stress affect the microbial environment in the intestine and lead to intestinal flora disorder [] Disorderedintestinal flora may affect the antioxidant activity and lipidmetabolism [] Hence it may be possible to inhibit oxidative stress by regulating the composition and structure ofthe gut flora To prevent oxidative stressassociated cellular damage it is therefore important to keep prooxidantantioxidant balance by supplementation or induction ofcellular antioxidants A high dose of dgalactose is converted to aldose and hydrogen peroxide by dgalactoseoxidase The products then generate reactive oxygen species through oxidative metabolism and glycosylation leading to oxidative stress The accumulation of oxidationproducts further exacerbates the oxidative damage to tissues and cells which then accelerates the aging process[] Therefore dgalactose overload has been used to establish animal models used to conduct aging related metabolic dysfunction and oxidative stress [ ]Selenium Se is an essential trace element for humanbody and other animals The role of Se is reported to beclosely associated with antioxidant activityimmune response and chemoprevention [“] Se is mainly presentin the active site of enzymes in the form of selenocysteineMultiple Secontaining proteins such as GSHPx and thioredoxin reductase play important roles in preventing oxidativeimportance of Sesupplementation in boosting up the internal antioxidativedefense has been highlighted in recent years Studies haveshown that anic Se supplements can improve tissue Sedeposition antioxidant level and gene expression whereasSe deficiency may result in cardiac muscular osseous and[] Thereforeinjurytheimmune disturbances [ ] Therefore the healthrelatedbenefits of Se including the type of selenium supplementsand optimal dosage remain to be exploredThe importance of Se has inspired researchers to usebioenrichment to prepare high Se compounds [ ] Cmegacephala larvae is a traditional Chinese medicine witha wide range of pharmacological actions including antioxidant antibacterial and antiinflammatory activitieswhich has been widely applied in agriculture and medicine[“] Seenriched C megacephala larvae SCML isgenerated from C megacephala larvae by biological transformation and enrichment of Se Our previous workshowed that SCML was an effective anic Se sourcewith low toxicity and high Se content [] Yet no studyhas reported the antioxidant activity of SCML in vivo andits impact on the gut microbiota which is susceptible toundergo alterations under oxidative stressThe objective of the present study was to evaluate theantioxidant activity of SCML in vivo explore the underlying mechanism as well as evaluate its impact on thegut microbial diversity and structure hoping that the results could provide a scientific basis for a comprehensiveutilization of SCMLMethodsMaterials and chemicalsSCML was provided by Beijing Ershang Biological Technology Co Ltd Beijing China Vitamin E was purchasedfrom Archer Daniels Midland Dictor USA DgalactoseDgal of ‰¥ purity was purchased from Aladdin Industrial Corporation Shanghai China GSHPx SOD TAOC GSH MDA and protein carbonyl assay kits werepurchased from Nanjin Jiancheng Bioengineering InstituteNanjin China RNA trizol reagent and FastStart Universal SYBR Green Master Rox were purchased from Servicebio Wuhan China The primers for Nrf2 SOD1GSHPx and GAPDH were synthesized and purified byWuhan Servicebio Technology Co LTD Wuhan ChinaThe kits for Revert Aid First Strand cDNA synthesis andHyPure„¢Molecular Biology Grade Water were purchasedfrom Thermo Waltham USA and HyClone LoganUSA respectively Keap1 Nrf2 and HO1 polyclonal antibodies were obtained from Proteintech Chicago USARIPA Actin bicinchoninic acid BCA assay kit 0cXie BMC Complementary Medicine and Therapies Page of Western Lightening„¢ PlusECL Enhanced chemiluminescence substrate assay kit and the secondary goat antimouse horseradish peroxides HRP were from ServicebioWuhan China All other chemicals and reagents used inthe study were of analytical grade Water used in the experiments was ultrapureDetermination of the compositions of SCMLCompositions of SCML including protein crude fat andmoisture content were analyzed according to methodGB5009“ of China National Food Safety StandardSe content was detected by Inductively Coupled PlasmaICP according to Vu with minor modifications[] The results are shown in Table Animal experimentsSixty ICR male mice aged weeks and weighing ± gwere purchased from SinoBritish SIPPRBK Lab Animal Ltd Approval No SCXK HU “ Theanimal experiments were performed in accordance withthe guidelines of the Laboratory Animal Center of Zhejiang Chinese Medical University Permit No SYSKZHE “ Allthe experimental procedureswere strictly conducted according to the internationalstandards and nationallegislation on animal care anduse The mice were kept under controlled light conditions h lightdark cycle with free access to food andwater normal light circadian rhythm and 7day adaptivefeeding in a quiet environmentAfter oneweek acclimatization mice were equallyrandomized to six groups normal controlNCgroup model group positive group receiving mgKg·d vitamin E VE group lowdose SCML LSCML group receiving SCML μgKg·d Se middose SCML MSCML group receiving SCML μgKg·d Se and highdose SCML HSCML group receiving SCML μgKg·d Se Except for the mice inNC group animals in the other five groups were givensubcutaneous injection of mgKg·d Dgal for weeksinto the neck to prepare oxidative stress model Animalsin NC and model groups received water and animals inthe other groups as previously described received VE orSCML by intragastric gavage for weeks The experiments were conducted at “ A certain amountof SCML and gellan gum were weighed precisely anddissolved in purified water heated slightly to a suspension There were three different concentrations and μgmL Se Meanwhile VE was dissolved in purified water containing gellan gum which became aTable Compositions of SCMLsuspension mgmL Dgal was dissolved in physiological saline mgmLThe mice were weighed throughout the experimentThe appearance appetite mental condition and behavioral activity of the mice during the experiment werealso observed and recorded Stool samples were collected at weeks after treatment Blood samples wereobtained from the retrobulbar venous plexus at weeksafter treatment The mice were sacrificed by cervical dislocation and the liver kidney heart brain and caecumwere stripped The dissected ans were divided twoparts one for histological analysis and the other for biochemistry analysis Samples for analysis were thawed onice homogenized with mL cold buffer mM potassium phosphate with mM EDTA pH per gram oftissue and centrifuged at ×g for min at °CThe supernatants were collected for analysisAnalysis of serum oxidative stress indexesSerum oxidative stress indexes GSHPx SOD and MDAwere determined by using the respective commercial kitsaccording to the manufacturer™s instructionsAnalysis of tissue oxidative stress indexesThe oxidative stress indexes were determined by measuring GSHPx SOD TAOC GSH MDA and proteincarbonylation ofthe tissue homogenate supernatantusing the commercial kits according to the manufacturer™s instructionsHistological analysisFor histological analysis the animal tissues were fixed in paraformaldehyde for h dehydrated in alcoholparaffin embedded sliced into μm thick sectionsstained with hematoxylineosin HE and finally photographed under a microscope × objective lensRNA extraction and realtime quantitative PCRexperimentsTotal RNA was extracted from the liver and kidney tissues using Trizol reagent RNA was reverse transcribedinto cDNA using RevertAid First Strand cDNA SynthesisKit Realtime quantitative PCR qRTPCR was performed using FastStart Universal SYBR Green MasterRox and the ABI7900Faxt Sequence Detection systemThe thermal cycle condition was cycle at °C for min followed by cycles of amplification at °C for s and then °C for 30s And the dissolution curvestarted from °C then ascending to °C at °C15ContentProtein g100 gCrude Fat g100 gMoisture g100 gSe μggSCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of s All samples were run in triplicate in each experimentValues were normalized to that for GAPDH The sequences of the primers used are shown in Table Theresults were calculated by using the 2ΔΔCT methodliverandtissueskidneyWestern blot analysisTotal protein and nuclear protein were extracted from mgusing RadioImmunoprecipitation Assay RIPA lysis solution and anuclearcytoplasm protein extraction kit The concentrations of protein lysates were quantified using a BCA protein kit Samples containing an equal amount of protein μg were mixed with the loading buffer containing 2mercaptoethanol heated for min at °C andloaded onto a SDSPAGE gel The proteins fromthe electrophoresing gel were then transferred ontopolyvinylidenethenblocked with milk and Tween in Trisbuffered saline and incubated overnight at °C withantiKeap1 antiNrf2 antiHO1 and actin Then the appropriate horseradish peroxideconjugated secondary antibody wasadded to the membranes at room temperature Finallythe proteins were detected with chemiluminescent substrate Gray semiquantitative analysis was performed byImage J The protein bands were quantified using densitometry Values are expressed as the fold change withrespect to betaactindifluoride membranes whichIntestinal microbiota analysisThe stool samples were sent to BGI Co Ltd WuhanChina for sequencing of the 16S rRNA gene Total genomic DNA of the gut microbiome was extracted and theV3V4 region of the 16S rRNA gene from the sample wassubjected to PCR amplification After normalization of thegenome DNA to ng per PCR reaction V3V4 dualindex fusion PCR primer cocktail and PCR master mixwere added and then a PCR was performed The PCRproducts were purified with Agencourt AMPure XP beadsto remove the unspecific products Pairedend sequencingTable Primers for realtime PCR analysesAccession NoGeneNrf2NM_0109023SOD1GSHPxGAPDHNM_0114341NM_0081606NM_0080842was performed on the Illumina Hiseq platform and theobtained data were subjected to bioinformatics analysisTo obtain clean reads the clean pairedend reads withoverlap were merged to tags using FLASH fast lengthadjustment of short reads v1211 Then the tags wereclustered to operational taxonomic units OTUs at sequence similarity by scripts of software USEARCHv701090 The RDP classifier v22 was used to compare OTUs with the database to comment on the OTUsspecies Finally intestinal microbial diversity and structure were analyzed based on OTUs and taxonomic ranksusing software R v311Statistical analysisAll data are expressed as the means ± SD or means ± SEand analyzed using Statistical Analysis Software SPSS The experimental values were analyzed by oneway ANOVA followed by the Duncan™s multiplerangetests and Pvalue were considered to be statistically significantResultsEffects of SCML on daily behavior and weight gain inmiceUsual performance of the mice was observed and recorded and no abnormal phenomenon found duringthe experimentincluding antifeeding and vomitingSymptoms such as slow movement and listlessnesswere obviously observed in model groupindicatingthat the oxidative stress model induced by subcutaneous injection of Dgal was successfully establishedHowever the above symptoms receded in varying degrees in VE and SCML groups The weight gain ofthe mice is exhibited in Fig Compared with NCgroup body weight of the mice in model group significantly increased slowly P and increasedsteadily in drug treatment groups MSCML μgKg Se group showed a significant difference compared to the model group P Primer Sequences CTGGCTGATACTACCGCTGTTCAGGTGGGATTTGAGTCTAAGGAGATGTGACTGCTGGAAAGGACGCGCAATCCCAATCACTCCACCCAGGAGAATGGCAAGAATGAGGAAGGTAAAGAGCGGGTGACCTCGTCCCGTAGACAAAATGTGAGGTCAATGAAGGGGTCGTProduct Sizebp bp bp bp bp 0cXie BMC Complementary Medicine and Therapies Page of tissues were decreased significantly compared to NCgroup P except for SOD in the heart as well asGSHPx in the liver and brain After administration ofVE or SCML the activity of GSHPx and SOD as wellas the content of TAOC and GSH were increased gradually As shown in Fig 3a the activity of GSHPx in thekidney and heart was increased significantly comparedto model group P except for the heart in VEgroup and LSCML μgKg Se group The activityof GSHPx in the liver and brain remained unchangedsignificantly compared to model group except for VEgroup in the liver As shown in Fig 3b the activity ofSOD in the kidney and brain was increased significantlycompared to model group P except for the brainin LSCML μgKg Se group However the activityof SOD in the liver and heart remained unchanged significantly compared to model group except for the liverin VE group As seen in Fig 3c the content of TAOCin the liver kidney and heart was increased significantlycompared to the model group P except for theliver in LSCML μgKg Se group The content ofTAOC in the brain was not significantly altered compared to model group except for VE group As shownin Fig 3d the content of GSH in liver kidney and brainof VE group and in the kidney of HSCML μgKgSe and MSCML μgKg Se groups was increasedsignificantly compared to model group P As shown in Fig 3e the MDA level in model group wasincreased significantly compared to NC group P and decreased significantly after VE or SCML treatmentcompared to model group P except for LSCML μgKg Se group in the kidney In Fig 3f the proteincarbonylation level in the liver kidney and brain of modelgroup was increased significantly compared to NC groupP However the level decreased significantly inthe mice treated with SCML or VE except for in the liverand brain of LSCML μgKg Se group compared tomodel group P And compared with model groupFig Percentage of weight gain in mice at weeks Valuesrepresent means ± SD n and evaluated by oneway ANOVAfollowed by the Duncan™s multiplerange tests Compared with NCP Compared with Model P SCML SeleniumenrichedC megacephala larvae powderEffects of SCML on serum oxidative stress indexes in miceAs shown in Fig the serum antioxidative enzyme activities in model group were decreased significantly andthe MDA content was increased significantly comparedto NC group P As shown in Fig 2a the GSHPxactivities in animals treated with SCML or VE were increased significantly P As shown in Fig 2b theactivities of SOD in MSCML μgKg Se and VEgroups were significantly increased compared to modelgroup P As shown in Fig 2c the MDA levels inanimals treated with SCML or VE were decreased significantly P Effects of SCML on tissue oxidative stress indexes in miceAs illustrated in Fig after 6week subcutaneous injection of Dgal the activity of the antioxidative enzymesand the content of the antioxidants in different miceFig Oxidative stress level indexes of the mice serum a GSHPx activity in the mice serum b SOD activity in the mice serum c MDA content inthe mice serum Values represent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by theDuncan™s multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig Oxidative stress indexes of the mice tissue a GSHPx activity in the mice tissue b SOD activity in the mice tissue c TAOC content in themice tissue d GSH content in the mice tissue e MDA content in the mice tissue f protein carbonylation content in the mice tissue Valuesrepresent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by the Duncan™s multiplerangetests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powderFig Optical micrographs of mice tissue sections HE staining × Black arrow derangement of hepatic cord cells Red arrow infiltration ofinflammatory cells White arrow pyknosis Blue arrow cavitation and deformation Orange arrow atrophy and breakage of the villus Yellow arrowthinning of the intestinal wall Scale bar50 μm SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of HSCML μgKg Se group in heart also significantlydecreased in protein carbonylation levelEffects of SCML on histopathological changes in miceThe histopathological results are shown in Fig Normal histological architectures were observed in the tissuesections in NC group However the liver tissue sectionsin model group showed that the number of double nuclei was increased the hepatic cords were disarrangedliver cells expanded widely and infiltration oflargenumbers of inflammatory cells was observed Comparedto NC group kidney histopathology in model groupshowed that the glomeruli became atrophic or even disappeared the number of epithelial cells was reduced therenal proximal tubules were dilated Histologically theheart tissue was seen abnormally structured in modelgroupincluding cavitation and deformation in somemyocardial cells nuclear pyknosis and inflammatory cellinfiltration In model group the brain tissue was alsoseen abnormally structured including nuclear pyknosisand incomplete dissolution of nerve fibers The caecallesions including atrophy and breakage of the villus irregular cell arrangement and thinning of the intestinalwall were observed in model group SCML or VE treatment significantly attenuated these abnormal histologicalchanges of the tissues induced by DgalEffects of SCML on oxidative stress gene expression inmiceThe Nrf2 pathway maintains the redox homeostasis exerts antioxidant activity by regulating its multiple downstream cytoprotective genes thereby plays a vital role incell survival The effect of SCML on oxidative stressgene expression is shown in Fig As shown in Fig 5athe Nrf2 expression in model group in liver was lowerthan that of NC group P Except for LSCML μgKg Se group in the liver the Nrf2 expression in liverwas increased all other drug treatment groups comparedwith model group P The Nrf2 expression was increased in the kidney of in HSCML μgKg Se groupcompared to model group P As shown in Fig 5bthe expression of GSHPx mRNA in the liver of modelgroup was decreased P After SCML treatment theGSHPx mRNA expression in the liver was significantly increased compare to model group P and MSCML μgKg group and HSCML μgKg Se group inkidney was increased significantly compared to model groupP As shown in Fig 5c the expression of SOD1mRNA was decreased in model group especially in the kidney compared to NC group P However the expression was obviously increased in the liver of HSCML group μgKg Se and MSCML group μgKg Se compared to model group P Significant change was alsoobserved in SOD1 mRNA expression in the kidney of HSCML group μgKg Se and MSCML μgKgSe group compared to model group P Effects of SCML on oxidative stress protein expression inmiceTo determine whether Nrf2 activation played a role inSCML protection against Dgal induced oxidative stressthe expression of Keap1 Nrf2 and HO1 in the mouseliver and kidney was detected As shown in Fig compared with NC group the western blot results showedthat the Nrf2 and HO1 protein expression in modelgroup was significantly decreased P while theKeap1 protein expression was increased in model groupAfter SCML or VE treatment the Keap1 expression inthe treatment groups was decreased though the difference was not statistically significant Compared withmodel groupthe Nrf2 expression in the treatmentgroups was increased significantly P except forLSCML μgKg Se group in liver Compared withmodel group the HO1 expression in SCML groups wasincreased especially in the liver of HSCML μgKgSe group P Sequencing depth and diversityA total of sequences from all intestinal microbiota samples were produced averaging sequencesFig The effect of SCML on the expression of Nrf2 SOD1 and GSHPx mRNA in the liver and kidney of the mice a Nrf2 mRNA relativeexpression in the liver and kidney b GSHPx mRNA relative expression in the liver and kidney c SOD1 mRNA relative expression in the liver andkidney Values represent means ± SD from three independent replicates and evaluated by oneway ANOVA followed by the Duncan™s multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig The effect of SCML on the protein expression of Keap1 Nrf2 and HO1 in the liver and kidney tissue of the mice a Protein strip b Keap1actin relative density c Nrf2actin relative density d HO1actin relative density Values represent means ± SD from three independentreplicates and evaluated by oneway ANOVA followed by the Duncan™s multiplerange tests Compared with NC P Compared with ModelP SCML Seleniumenriched C megacephala larvae powderclusterper sample These sequences resulted in a mean sequencelength of approximately bp Based on the Clean Tagstheanalysis was processed by USEARCHv701090 The sequences were delineated into operational taxonomic units OTUs at similarity Thevalue of coverage for the observed OTUs was above The species accumulation curves showed clear asymptotes and the curve tended to be flat or reached theplateau stage Fig 7a indicating a nearcomplete sampling of intestinal microbial communities of mice Theboxplot of Shannon index showed that the diversity of theintestinal microbiota was decreased in model group compared to NC group and the diversity of VE group and HSCML μgKg Se group was increased compared tomodel group Fig 7b As shown in Fig 7c the contribution value of PC1 and PC2 for the sample difference was and respectively All intestinal microbiotasamples were presented as three distinct groups Thesefindings indicate that the main components of the intestinal microbiota in model group were different from thoseFig Alpha diversity of the gut microbiota and principal component analysis PCA plots based on abundance of operational taxonomic unitsOTUs a Species accumulation curves b Bacterial diversity estimated by the Shannon index c PCA plots SCML Seleniumenriched Cmegacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of in NC group After VE and SCML treatment the components of the intestinal microbiota were different fromthose in model group while there was an insignificant difference between HSCML μgKg Se group and NCgroup“ BacteroidetesEffects of SCML on species structures in miceThe species profiling histogram was obtained to knowthe community structural composition of differentgroups at phylum and genus levels Fig As shown inFig 8a the most prevalent phyla in all samples were Fir“micutesand Proteobacteria “ There were otherphyla level bacteria with low abundance in the intestinaltract of mice As shown in Fig 8b species were usedto describe the relative abundance ofthe intestinalmicrobiota at the genus level showing that Prevotella“ Helicobacter “ and Clostridium “ were the most abundant followed byOscillospira “ Bacteroides “ andLactobacillus “Effects of SCML on intestinal bacteria of differentclassification levels in miceAs shown in Table Proteobacteria were increased significantly at the phylum level in model group comparedto NC group P Proteobacteria were decreasedsignificantly and Bacteroidetes were increased significantly in VE and SCML groups compared to modelgroup P In addition VE group MSCML μgKg Se group and HSCML μgKg Segroup showed significant differences in Firmicutes compared to model group P and MSCML μgKg Se group showed a significant difference in Actinobacteria compared to model group P As shown in Table there was not significant alteration in Bacteroides Lactobacillus Oscillospira Prevotella and Sutterella at the genus level in model groupcompared to NC group Compared with NC group Helicobacter and Clostridium were increased significantlyand Ruminococcus was decreased significantly in modelgroup P Clostridium Helicobacter and Oscillospira were decreased significantly in VE and SCMLgroups compared to model group P while VEgroup and LSCML μgKg Se group showed a significant difference in Bacteroides P In additionLactobacillus in MSCML μgKg Se group and HSCML μgKg Se group Prevotella in VE groupand Sutterella in MSCML μgKg Se group wereall increased significantly compared to model group P There were not significant alterations in Ruminococcus in VE and all SCML groups compared to modelgroupCorrelation analysis of changes in flora abundance andserum biochemical indexesIn order to explain the relationship between the intestinal flora abundance changes of mice and serum biochemicalindexes Spearman correlation analysis wasperformed to analyze correlation between serum biochemical indexes and Clostridium and Helicobacter theabundance of which were significant difference in eachgroup The change of Clostridium abundance was foundto be negatively correlated with GSHPx and SOD andpositively correlated with MDA There was not significant correlation in Helicobacter and serum biochemicalindexes The specific correlation analysis results wereshown in the Table DiscussionThe results of the present study showed that the dailybehaviors of the mice in model group were differentfrom those of the mice in NC group In addition the tissues of the modeled mice underwent significant pathological changes The antioxidant system parametersincluding GSHPx SOD TAOC and GSH in the antissues or serum were decreased while the MDA andcarbonylated protein levels were increased All these results indicated that the Dgalinduced oxidation mousemodel was successfully established in the present studyVE the monomer of which is often used as the positivecontrol for the studies of aging in mice induced by DgalFig Taxonomic composition of the gut microbiome in the mice a Phylumlevel b Specieslevel SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Table Oneway ANOVA test of species differences at the phylum and species level BacteriaNCModelVEPhylumBacteroidetes ± Proteobacteria ± Firmicutes ± GenusActinobacteriaTenericutesBacteroidesClostridiumHelicobacterLactobacillusOscillospiraPrevotellaRuminococcus ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± SCML μgKg Se ± ± ± ± ± ± ± ± ± ± ± ± Sutterella ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Values represent means ± SE n and evaluated by oneway ANOVA followed by the Duncan™s multiplerange tests Compared with NC P Comparedwith Model P SCML Seleniumenriched C megacephala larvae powder ± ± ± ± [ ] Studiesshowed that mice subcutaneouslyinjected with Dgal in the neck exhibited a significantbody weight declined [] In this study Dgal was foundto significantly inhibit weight gain in mice howeverSCML and VE could increase the body mass in varyingdegrees indicating that SCML and VE could effectivelyenhance the constitution of aging mice Oxidative damage appears in body ans to a large extent Our resultsshowed that Dgalinjection for weeks for mice resulted in severe histopathological changes in the antissues However SCML and VE could alleviate these Dgalinduced pathological damages in an tissues ofmice Recent research work has demonstrated that senescent cells accumulated in various tissues of age anddisease [] Cellular senescence is associated with agerelated phenotypes causally and decreasing senescentcells can retard tissue dysfunction and extend healthspan[] The results suggested SCML c

Thyroid_Cancer

progression of breast cancerare greatly affected by the immune environment Neutrophils are the most abundantleucocytes in circulation and act as the spearhead in ‚ammationincluding inbreast cancer Circulating neutrophils are closely related to the prognosis of breastcancer patients and tumorltrating neutrophils have varied functions at differentstages of breast cancer such as antitumor or tumorpromoting neutrophils which aretermed N1 and N2 neutrophils respectively In this review we will discuss the utilityof circulating neutrophils for predicting prognosis and therapeutic efficacy and theunderlying mechanisms of their chemotaxis the dynamic regulation of their antitumoror protumor functions and their different spatial distributions in tumor microenvironmentFinally we also discuss the possibility of targeting neutrophils as a therapeutic strategyin breast cancerKeywords breast cancer immunotherapy neutrophils neutrophiltolymphocyte ratio tumor microenvironmentSpecialty sectionThis was submitted toCancer Immunity and ImmunotherapyINTRODUCTIONa section of the journalFrontiers in ImmunologyReceived May Accepted July Published August CitationZhang W Shen Y Huang H Pan SJiang J Chen W Zhang T Zhang Cand Ni C A Rosetta Stone forBreast Cancer Prognostic Value andDynamic Regulation of Neutrophil inTumor MicroenvironmentFront Immunol 103389fimmu202001779Breast cancer BC is the most common malignancy in women worldwide Although BCis classified as a malignant disease with low immunogenicity recent evidence has revealeda promising outcome of therapies with blocking immune checkpoints in both early andadvanced stages “ The efficacy of immunotherapy is closely related to the tumor immunemicroenvironment especially to ltrating immune cells To date macrophages and Tcells are the most wellstudied immune cells in BC whereas increasing evidence has indicatedthat neutrophils are also key in the oncogenesis and metastasis of BC in addition circulatingneutrophils have been reported to have great prognostic prediction value Neutrophils are themost abundant leucocytes in blood and usually act as the first line of host defense against pathogens However due to their short life span an average of “ h in blood it is difficult to employthis subset of cells for experiments which has resulted in a poor understanding of their role in solidtumors In addition some contradictory results reported in vitro studies or animal experimentshave suggested a dual eï¬ect of neutrophils in tumor developmentFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerNeutrophils can present both antitumorigenic œN1 andprotumorigenic œN2 phenotypesin various cancers orspecific circumstances The term neutrophil in several studiesalso includes both mature neutrophils and myeloidderivedsuppressor cells MDSCs MDSCs are described as a subsetof neutrophils with immunosuppressive functions that expressCD11b and Gr1 and can be divided into monocyticM CD11bLy6C MDSCs and GPMN CD11bLy6GMDSCs and GPMN MDSCs usually share a commonset of markers and similar morphologicalfeatures withneutrophils To avoid confusion we mainly focus on the biologicalfunction of mature neutrophils and related therapeutic strategiesfor targeting them in BC We provide a comprehensive reviewofthe prognostic value of circulating neutrophils and themechanisms of how tumorassociated neutrophils TANs exertantitumor or tumorpromoting functions in BC and in theend we also discuss the potential of targeting neutrophils as atherapeutic strategy in cancerPROGNOSTIC VALUE OF THENEUTROPHILTOLYMPHOCYTE RATIONLRTumors can be thought of as wounds that will not heal andare characterized by chronic ‚ammation Neutrophils are themost rapidly responding immune cells to ‚ammation andmany studies have found that the NLR is closely related to theprognosis and treatment response in patients bearing BC A recent metaanalysis of studies including patientswith both early and advanced BC revealed that patients witha higher NLR before treatment had poorer diseasefree survivalDFS than those with a lower NLR before treatment but theNLR was not related to overall survival OS the subgroupanalysis found that the NLR was associated with prognosisonly in earlystage patients but not in patients with metastasisAbbreviations BC Breast cancer MDSCs Myeloidderived suppressor cellsTANs Tumorassociated neutrophils NLR Neutrophiltolymphocyte ratio DFSDiseasefree survival OS Overall survival ALC Absolute lymphocyte count NCTNeoadjuvant chemotherapy pCR Pathological complete response PLR Platelettolymphocyte ratio TAMs Tumorassociated macrophages CTCs Circulatingtumor cells NETs Neutrophil extracellular traps MPO MyeloperoxidaseGCSF Granulocyte colonystimulating factor ECs Endothelial cells PMNsPolymorphonuclear neutrophils ICAM1 Intercellular adhesion molecule MMP9 Matrix metalloproteinases9 ROS Reactive oxygen species HMGB1Highmobility group box TLR4 Tolllike receptor TNBC Triplenegativebreast cancer MES Macrophageenriched subtype NES Neutrophilenrichedsubtype H2O2 Hydrogen peroxide TNFα Tumor necrosis factorα HOCIHypochlorous acid TRPM2Transientcation channelsubfamily M member ADCC Antibodydependent cellular cytotoxicityNK Natural killer NE Neutrophil elastase NRP1 Neuropilin1 IRS1 Insulinreceptorsubstrate1 PI3K Phosphatidylinositol 3kinase VEGF Vascularendothelial growth factor TIMP1 Tissue inhibitor of matrix metalloproteaseTGF Transforming growth factor 27HC 27hydroxycholesterol PAD4Peptidyl arginine deiminase TINs Tumorltrating neutrophils CRTConventionalradiotherapy MRT Microbeam radiation therapy DAMPsDamageassociated molecular patterns ICB Immune checkpoint blockadeLDNs Lowdensity neutrophils HDNs Highdensity neutrophils NAMPTNicotinamideadeninedinucleotide GTX granulocyte transfusiontransferase NAD Nicotinamidereceptor potentialphosphoribosyl Since similar metaanalyses were not based on individualpatient data which may cause significant bias we reviewed andcompared the individual reports and found some issues worthdiscussing here Widmann first reported the correlationbetween the NLR and BC prognosis in patients and itwas found that a higher NLR ‰¥ before treatment was anadverse factor for both short and longterm mortality Themajority of retrospective studies thereafter have drawn similars “ and the NLR was found to be consistentamong diï¬erent BC subtypes at baseline However aprospective substudy of GEICAM9906 which comprised patients did not find any prognostic value of the NLR afteradjustment for clinicopathological factors in addition a highNLR was independently associated with worse DFS in only highrisk patients the hormone receptornegativeHER2 populationand in patients with ‰¥ lymph node metastases Anotherstudy with early BC patients also found that the NLR beforesurgery was not associated with DFS indicating that thepresurgery NLR may be valuable only in patients with a hightumor burdenseveralIn addition to the above studiesstudies alsoexplored the prognostic value of the NLR posttreatment or withcontinuous assessment A retrospective study comparing theabsolute lymphocyte count ALC and the NLR eight consecutivetimes before and after chemotherapy found that patients whodied had lower ALC and higher NLR values than patients whoremained alive throughout the treatment course additionallyamong the patients who died a steady increase in the NLRover the baseline measurement was observed at subsequenttime points Another retrospective study included BCpatients with DFS values of more than years and it interestinglyfound that NLR sampled during followup rather than beforeany treatment was an independent prognostic factor for laterecurrence However there is still no compelling explanationfor the abovementioned inconsistent results In addition sincelymphocytes are critical in cancer immune surveillance andneutrophils have been reported to play a protumor role in moststudies low lymphocytes and high neutrophils in circulationmay also suggest immunosuppression status and studiesfocused on the relationship between neoadjuvant chemotherapyNCT and the NLR might support the above hypothesis Acomprehensive review of the existing reports shows that moststudies have found that a low NLR indicates a higher NCTresponse and pathological complete response pCR rate “in addition the NLR has showed predictive value not only inall molecular types of BC but also in both operable and locallyadvanced BC Interestingly although Suppan et aldid not find a significant correlation between the initial NLR andprognosis the same cohort revealed a low NLR as a significantparameter for predicting chemotherapy response p A low NLR was also reported to be associated with a higherresponse rate to primary endocrine therapy for locally advancedor metastatic BC Although increasing evidence suggests a close associationbetween the NLR and prognosis in BC several issues remain thatmake clinical application difficult One of the most importantreasons is the lack of a consensus cutoï¬ value As we listhere Table the cutoï¬ values for the NLR in the publishedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerTABLE Characteristics of the studies related to neutrophiltolymphocyte ratioReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSignificance of NLRNR ysHigh NLR indicates lowersurvival rate p Surgery NCT moSurgery ysNR moUnivariate analysis indicateshigh NLR related to lower RFSp and OS p High NLR indicates lower5year OSMultivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Adjuvanttranstuzumab moHigh pretreatment NLRindicates shorter DFSNoh Korea“Koh Korea“Yao China“Luminal ABHER2enrichedTNBCERPRpositiveHER2enrichedLuminal ABERPRpositiveHER2enrichedTNBCPistelli Italy“TNBCUlas Turkey“HER2enrichedJia China“ER PRpositiveHER2enrichedTNBCBozkurt Turkey“TNBCAsano Japan“TNBCn NLR n ‰¤ NLR ‰¥ n n NLR ‰¤ n NLR n n NLR NLR n NLR ‰¤ n NLR n n NLR n NLR n n NLR n NLR ‰¤ n n NLR ‰¤ n NLR n n NLR n NLR n NCT surgery mo moSurgeryadjuvantchemotherapyandradiotherapyNCT ysMultivariate analysis indicateslow NLR is related to superiorDFS p and p Multivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Univariate analysis indicateslow NLR is related to favorableprognosis in TNBC patientswho achieved pCR p hazard ratio High pretreatment NLRindicates poor allcausemortality with a multivariableHR of CI“High NLR upon recurrenceindicates shorter OSrecurrence rates p Low NLR indicates higher OSp Rimando USA“Nonmetastatic BC n NLR ‰¤ n NLR n Radiotherapychemotherapy moIwase Japan“TNBCn NRL n NLR ChemotherapyNRNCT surgery moHernandez et alSpain“Miyagawa Japan“Ferroni Italy“Luminal ABERPRpositiveHER2enrichedTNBCLocally Advanced orMetastatic BCLuminal ABHER2enrichedTNBCn NLR n NLR ‰¥ n n NLR ‰¤ n NLR n Qiu China“NonmetastaticTNBCn NLR n NLR ‰¥ n EribulinNRLow NLR indicates better PFSp NCTchemotherapyendocrinetherapytrastuzumabregimensSurgery NCTchemotherapy moHigh pretreatment NLRindicates worse DFS HR and OS HR moLow NLR indicates higher OSp and DFS p ContinuedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alTABLE ContinuedNeutrophils in Breast CancerReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSignificance of NLRIimori Japan“Luminal ABHER2enrichedTNBCMando Argentina “Early stage BCLee Korea“TNBCXuan China“TNBCFujmoto Japan“With high counts oflymphocytesImamura Japan“HER2enrichedn NLR n NLR ‰¥ n n NRL n NLR ‰¤ n NLR n n NLR n NLR ‰¥ 293n n NLR n NLR n n NLR n NLR ‰¥ n Endocrinetherapy moSurgery moNCTNRSurgeryNRNRSurgeryadjuvantchemotherapiesendocrinetherapiesTrastuzumabemtansineNRLow NLR indicates aprolongation of PFS p and OS p High NLR indicates lower DFSp Low NLR indicates superiorOS p and DFS p Low NLR indicates longer DFSp Low NLR indicates better RFSp Low NLR at baseline indicatesbetter PFS p andOS p NLR Neutrophiltolymphocyte ratio ER Estrogen receptor PR Progesterone receptor HER2 Human epidermal growth factor receptor Mo Months Ys Years DFS Diseasefreesurvival OS Overall survival PFS Progressionfree survival RFS Relapse free survival pCR Pathological complete response TNBC Triplenegative breast cancer NCT Neoadjuvantchemotherapy NR Not recordedstudies were between and In addition based on individualstudies the sensitivity of the NLR fluctuates greatly “and the specificity is much lower “ Therefore some researchers have tried to determine a betteralternative parameter In addition to the NLR the platelettolymphocyte PLR ratio has also been investigated and comparedwith the NLR in BC A single central retrospective study with hormone receptornegative nonmetastatic BC patients reportedthat both elevated NLR and PLR were associated with poor OShowever the multivariate analysis revealed that only the NLR p but not the PLR p was a significant indicatorfor both DFS and OS Additionally since the absolutelymphocyte count has also been reported as a prognostic factorthe predictive values of the PLR and NLR were evaluated afteradjusting for the total lymphocyte count The results showed thatthe PLR was no longer a significant predictor for 5year mortalityand the NLR remained a significant predictor irrespective ofthe lymphocyte count Furthermore it was revealed thatthe combination of the NLR and PLR could further improvethe predictive value Two retrospective studies found that thehighest rate of pCR was in the group of patients withan NLRlowPLRlow profile and the lowest rate was inthe group with an NLRhighPLRhigh profile in additionwhen the cutoï¬ values for the NLR and PLR were applied thespecificity of predicting a pCR increased from to Howeverthe causal relationship between the NLR andpoor prognosis in malignant disease has yet to be illuminatedAccording to an assessment with paired peripheral bloodand pancreatic cancer specimens Takakura found thata high NLR was associated with increased tumorassociatedTAMsanditseemsdecreased Thereforetumorassociatedmacrophageslymphocytes but was not significantly related to CD66bltrating neutrophilsthat anincrease in neutrophils in peripheral blood is not necessarilyrelated to the number of TANs Several basic studies havesuggested a unique mechanism ofthe protumor functionof circulating neutrophils protecting circulating tumor cellsCTCs Circulating neutrophils can cluster around tumor cellsand induce tumor cell aggregation aiding tumor cell survivalby hiding them from immune surveillance Neutrophilextracellular traps NETs are webs of decondensed chromatbers conjugated together with histones myeloperoxidaseMPO elastase and other cytoplasmic proteins Recentstudies also found that neutrophils could form many NETs bothin circulation and in tumor lesions and could coordinate withplatelets to capture CTCs and facilitate cancer metastasis In addition neutropenia is very common in cancer patientsundergoing chemotherapy and supportive treatment withgranulocyte colonystimulating factor GCSF can inducea neutrophilic response as a consequence neutrophils areprimed toward a proNETotic phenotype and may suppress thecytotoxic activity of T cells as well as impair immune surveillance On the other hand lymphocytes have the propensityto mount an adaptive antitumor response in malignant disease and decreased lymphocyte numbers are considered to berelated to an insufficient immunologic reaction which mayincrease the risk of tumor relapse or metastasis Clearlya general association between prognosis and the NLR exists inBC but large prospective studies and rigorous research are stillrequired to determine its clinical significanceFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerMECHANISM OF NEUTROPHILCHEMOTAXIS TO THE TUMORMICROENVIRONMENTNeutrophils are considered the main immune cells that provideprotection against invading pathogens which can be induced bytrauma infection and malignant disease The recruitmentof neutrophils is greatly dependent on certain chemokinesincluding interleukin IL8 also known as CXCL8 CXCL and CXCL2 IL8 is a pro‚ammatory cytokineand acknowledged as the most important chemoattractant forneutrophils in the tumor microenvironment IL8 mainlycomes from endothelial cells ECs and monocytes in the tumormicroenvironment upon certain stimulation such as physicalinjury hypoxia chemotherapy or radiotherapy and other celltypes including fibroblasts and keratinocytes can secrete IL8 aswell In addition to its chemotactic eï¬ect it was revealedthat IL8 could provoke neutrophils to release NETs to assistcancer cell migration By livecell fluorescence microscopyGupta confirmed that activated ECs could induceNETosis characterized by typical extracellular DNA latticeswhen cocultured with polymorphonuclear neutrophils PMNsand activated ECs In addition activated ECs produceother ‚ammatory cytokines such as Pselectin Eselectinand intercellular adhesion molecule ICAM1 to facilitateneutrophil adhesion to ECs and migration Furthermoretumorpromoting neutrophils in BC cells are also characterizedby high expression of matrix metalloproteinases9 MMP9 which was found to cleave CXCL5 potentiating itsaction in neutrophil recruitment as a positive feedback functionin tumors IL17 was also found to control neutrophilrecruitmentin lung metastasis of BC in a mouse modelCD3CD4 and γδ T cells were the major sources of IL17 and it was interesting to find that the absence of γδT cells or neutrophils markedly reduced pulmonary and lymphnode metastases without ‚uencing primary tumor progressionwhich suggested a collaborative relationship between γδ T cellsand neutrophils in promoting BC lung metastasis Howeverin an orthotopic hepatocellular carcinoma model Sofia et alreported that TANs exert an overt antitumor role by suppressingγδ T17 cells via reactive oxygen species ROS contraryto the phenomenon that within the 4T1derived BC modelCD11bLy6G neutrophils that ltrate and surround livermetastases were found to be tumor promoting Thesecontroversial results suggest both promoting and suppressiveroles of TANs in diï¬erent circumstancesHighmobility group box HMGB1 usually acts as adamageassociated molecular pattern that is released by dyingcells or stressed cells to initiate ‚ammation and was laterfound to be an important chemoattractant for neutrophils Epithelial cellderived HMGB1 was found to recruit neutrophilsto the necrotic site through its receptor RAGE Enrichmentof platelets has been reported in the microenvironment ofmultiple cancers including BC and ltrating plateletscould be activated by the large amounts of adenosine phosphatereleased by necrotic cells as a result of chemotherapy Activated plateletderived HMGB1 known as the major mediatorof injuryinduced thrombosis in vivo can also stimulateNETosis through Tolllike receptor TLR4 and RAGE onneutrophils and as a positive feedback mechanism releasedNETs strongly induce a prothrombotic state and activate platelets Meanwhile tumor cellderived exosomal HMGB1 wasalso found to activate neutrophils through the TLR4NFκB pathway which promotes its survival by increasing theautophagic response and polarizing TANs to a protumor type It is noteworthy that various reports imply the coreposition of the NFκB pathway in the activation and recruitmentof neutrophils In addition to HMGB1 tumor cellsincluding BC cells have been reported to secrete other peptidessuch as a2 isoform VATPase a2V to activate the NFκBpathway in neutrophils thereby promoting their recruitment andinhibiting their apoptosis Additionally breast involutionafter weaning is characterized by acute ‚ammation and anincrease in estrogen It was found that estrogen could inducethe mammary ltration of neutrophils and upregulate theexpression of protumor cytokineschemokines such as COX2and MMPs in mammary ltrating neutrophils similarIn additionto lymphocytes and macrophagesneutrophils are more likely to localize in tumors of triplenegativebreast cancer TNBC than to tumors of other BC subtypes Recently Zhang identified neutrophils and macrophages asthe most frequent ltrating immune cells in various BC murinemodels and BC could be classified into a macrophageenrichedsubtype MES and a neutrophilenriched subtype NES It wasinteresting to find that there were only a few neutrophils inthe MES but a large number of macrophages in the NES This mutual repelling phenomenon in the MES and NES mayresult in spatial segregation within the same tumor The authorsspeculated that a possible mechanism could be the factors derivedfrom macrophages that inhibit the IL8dependent chemotaxis ofneutrophils ANTITUMOR FUNCTION OF TANs IN BCThe polarization of neutrophils can be diï¬erentially regulatedin the tumor microenvironment In a mouse model Fridlender found that TANs from the early tumor stage were liketumorkilling cells which produce high levels of hydrogenperoxide H2O2 tumor necrosis factor TNFα and NOand that TANs are more likely to obtain a protumorigenicphenotype with tumor progression Although few studieshave directly compared the phenotype and function of TANsbetween early and latestage tumors there are still some clues tosupport this hypothesis A phenotypical and functional analysisof TANs in earlystage lung cancer found an activated phenotypeCD62lowCD54high that was able to stimulate T cell proliferationand IFNγ release which suggested a pro‚ammatory ratherthan immunosuppressive state of TANs in earlystage lungcancer MPO is an enzyme characteristic of mature œN1type neutrophils which are able to convert H2O2 to cytotoxichypochlorous acid HOCI Recently a retrospectivestudy of BC cases revealed that MPOpositive neutrophilsFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerin additiondefined as ‰¥ cellstissue punch were found in of evaluablecases while the luminal ERPR and Her2 Her2enriched andtriplenegative types had positive rates of and respectivelyltrationby MPOpositive neutrophils was a significant independentfavorable indicator for both OS and DFS Notably almost all ofthe patients included in this study had earlystage disease T1 N01 and the data suggested that MPOpositiveneutrophils were much more abundant in BC cases with low Tand N stages than in advanced cases in univariate analysesIn addition a direct tumor killing function of neutrophils hasalso been reported One of the classical factors working againsttumor cells is ROS Recent research in mouse BC models revealedthat ROSmediated cell lysis was dependent on Ca2 channelsand mediated by transient receptor potential cation channelsubfamily M member TRPM2 expression on tumor cells Although TCGA analysis revealed a high expression ofTRPM2 in BC cells httpgepia2cancerpkucnindex activeNOX1 catalase and SOD were also increased in the membraneof cancer cells forming a complex mechanism by which tumorcell apoptosis induced by ROS is prevented In additiontumor cells are characterized by enhanced metabolic activity andhigh levels of intracellular ROS which indicates that directcytotoxic eï¬ects of neutrophilproduced ROS are not sufficientIn addition to the direct cytotoxic eï¬ect TANs containing ROShave been found to strongly suppress IL17producing γδ Tcells which are critical for shaping the immune suppressivemicroenvironment in various solid tumors “ and have alsobeen reported to promote BC cell extravasation and metastasis In addition neutrophils could also express Fc receptors andexert antibodydependent cellular cytotoxicity ADCC eï¬ectssimilar to those of T cells and macrophagesleading to atrogocytosis eï¬ect to destroy cancer cells However somestudies have indicated that neutrophils are more likely to bedistributed at the periphery of tumors at the initiation stage which may make controlling tumor growth with thesecellcell contactdependent mechanisms ineï¬ectivePROTUMOR EFFECTS OF TANsMore studies suggest that neutrophils facilitate tumor promotionand metastasis in BC than antitumor eï¬ect Overexpression ofthe chemokines CCL2 and CCL17 is a recognized feature ofN2 neutrophils Richmond found that exogenousCCL2 enhances the killing eï¬ect of neutrophils against BCcells in vitro while this antitumor activity was not observedin vivo Instead intranasal delivery of CCL2 to BALBc micemarkedly enhanced lung metastasis of BC cells and increasedthe recruitment of CD4 T cells and CD8 central memory Tcells CCL17 secretion from TANs was found to support tumrowth by recruiting CD4 Treg cells and macrophages In addition to recruiting immunesuppressive cells TANs werereported to promote the accumulation of BC cells in the lungand directly inhibit natural killer NK cellmediated clearanceof tumor cells Human NK cells can be divided intoCD56dim antitumor and CD56bright protumor subsets andCD56bright NK cells are enriched in the tumor microenvironmentand draining lymph nodes Early reports revealed thatROS and arginase1 from neutrophils impair the maturation andcytotoxic function of NK cells but CD56brightCD16ˆ’ NKcell are resistant to neutrophilderived ROS perhaps due to theirhigh antioxidative capacity Meanwhile NK cells couldbe recruited by TANs via CCL2 and CCL5 which may explainthe preferential accumulation of CD56bright NK cells in tumormicroenvironments with high ROS levels Extracellular arginine is crucial to signal local CD8 cellsand increase their CD3ζ expression which is key for T cellsto survey antigens presented on MHC class I molecules andit was also found to be necessary for T cell activation andsurvival Tumor cellderived IL8 could lead to TANdegranulation resulting in arginase1 release and conversion ofextracellular arginine to ornithine and urea thereby dampeningthe survival and cytotoxic eï¬ect of CD8 T cells Neutrophil elastase NE is also released by TANs and canbe endocytosed by tumor cells via neuropilin1 NRP1 thisresults in the crosspresentation of PR1 which is an NEderivedHLAA2restricted peptide that may be an immunotherapeutictarget Besides upon endocytosis NE is to bind insulinreceptor substrate1 IRS1 which removes the inhibitory eï¬ectof IRS1 on phosphatidylinositol 3kinase PI3K to enhance theproliferation of cancer cells Recentleukocytesreports highlighted thethe important characteristics of malignantespeciallyneutrophils preferentially uptake tumor derived extracellularvesicles or named exosomes Hypercoagulability is oneoftumors andhas been reported associated with NETs Breast cancer cell4T1derived exosomes induced NETs formation in neutrophilsbesides tumorderived exosomes also interacted with NETsto significantly accelerate venous thrombosis in vivo Furthermore several reports also indicated the cancer derivedexosomes prolonged lifespan of neutrophils and also polarizedneutrophils toward protumor type increasingevidence hasIn addition to direct modulation ofthe protumormicroenvironmentfound thatneutrophils promote tumor cell migration and the formation ofa metastatic niche Tumor angiogenesis is regardedas a prerequisite for tumor metastasis and TANs have beenrecognized as an important source of vascular endothelialgrowth factor VEGF upon specific stimulation in the tumormicroenvironment Neutrophils were also found to beone of the main sources of MMP9 and the link betweenMMP9 and VEGF has been reported previously The absenceof MMP9 has been reported to have a similar function as theinhibition of VEGF signaling indicating that MMP9 serves asan angiogenic switch during tumorigenesis by inducing VEGFrelease from the matrix “ In addition Gabriele et alalso found that MMP9 was expressed by a small number ofcells in close proximity to the vasculature such as ltrating‚ammatory cells rather than tumor cells In additionseveral serine proteases are also produced by TANs such asNE cathepsin G and proteinase3 which have been reported toactivate MMP2 to promote tumor invasion and proliferation In addition although neutrophils were reportedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerto produce little tissue inhibitor of matrix metalloproteaseTIMP1 Wang observed that BC cells with CD90positiveexpression could induce the TIMP1 secretion by TANs and asa reciprocal eï¬ect TIMP1 induced EMT and metastasis in BC Other neutrophilderived cytokines such as IL1 IL6and IL17α have been reported to initiate EMT of cancer cells byactivating JAK2STAT3 and ERK signaling Inadditiontheprimaryto modulatingtumormicroenvironment neutrophils can also assist the formationof the cancer premetastatic niche in distant ans CTCsare precursors for metastatic lesion formationintravascularNETs were found to protect CTCs from attack by circulatingimmune cells and dysregulated NETs were found to induce‚ammatory vascular injury EC shrinkage and tissue damage “ Moreover in vitro and in vivo experiments foundthat activated neutrophils promote the adherence of CTCs toECs and facilitate their lung and liver metastasis RecentlyAceto provided strong evidence that neutrophils escortCTCs in BC to assist metastasis With detection of cellsurface markers and Wright Giemsa staining they identified thatmost CTCassociated white blood cells were N2like neutrophilsIn addition singlecell RNA sequencing revealed higher Ki67expression in disseminated tumor cells from CTC neutrophilclusters than in standalone CTCs In the same study TNFαoncostatin M IL1 and IL6 were frequently expressed byCTCassociated neutrophils and matched by the receptors oncorresponding CTCs on the other hand CTCs from the CTCneutrophil clusters expressed high gene levels encoding GCSFtransforming growth factor TGF3 and IL15 which havebeen reported to activate neutrophils “ illuminating amechanism of neutrophilCTC cluster formationIn addition to escorting CTCs in circulation several studieshave found that neutrophil accumulation is a prerequisitefor cancer metastasis For both orthotopic transplantationand spontaneous BC models neutrophils were suggestedto accumulate in the distant an before cancer cellsltration Obesity and elevated cholesterol arerisk factors for BC development and poor prognosis Interestingly 27hydroxycholesterol 27HC increased thenumber of polymorphonuclearneutrophils and γδ T cells atdistal metastatic sites and neutrophils were required for themetastatic eï¬ects of 27HC Egeblad developeda confocal intravital lung imaging system and found that NETswere formed early in the lung and continued to form forthe next few days after tail vein injection of BC cells Inaddition based on immunofluorescence staining of humanprimary BC and matched metastatic lung lesions they foundthat the abundance of NETs was highest in TNBC but NETswere absent or very rare in luminal BC samples which mayexplain the higher metastatic ability of TNBCs than luminalBCs In ovarian cancer an ‚ux of neutrophils in the omentumwas also observed before metastasis and blockade of NETformation with peptidyl arginine deiminase PAD4 an enzymethat is essentia

Thyroid_Cancer

"Older patients with cancer require specific and individualized management The 3groupMultidimensional Prognostic Index MPI based on the Comprehensive Geriatric Assessment CGA has shown apredictive interest in terms of mortality The objective of our study was to assess the prognostic value of MPI for year mortality in an external prospective French cohort of elderly patients with cancerMethods From March to March a prospective singlecenter cohort study enrolled all patients withcancer aged years and older referred to the geriatric oncology clinic We used a proportional hazard model for1year mortality adjusted for age sex tumor sites and metastatic status Cstatistics were used to assess theincremental predictive value of MPI index to these risk factorsResults overall patients underwent CGA with MPI women mean age ± years The most commontumor sites were prostate skin colorectum and breast of patients had a metastaticdisease patients belonged to the œMPI1 group to the œMPI2 group and patients wereclassified in the œMPI3 group Oneyear mortality rate was in MPI1 in MPI2 and in MPI3 p All domains of MPI except cognition and living status were significantly associated with mortality at oneyear aswell as tumor sites and metastatic status Higher MPI was associated with a higher mortality risk adjusted HR [95CI “] and [“] for MPI groups and compared to p Conclusions In addition to established risk factors MPI improves risk prediction of 1year mortality This practicalprognostic tool may help to optimize management of these vulnerable patientsKeywords Aged Neoplasms Mortality Comprehensive geriatric assessment Correspondence Evelyneliuuchupoitiersfr1Department of Geriatrics Poitiers University Hospital Poitiers France2Clinical Investigation Centre CIC1402 CHU Poitiers University of PoitiersINSERM Poitiers FranceFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLiuu BMC Geriatrics Page of BackgroundIndividuals over years old are the fastest growing segment of the population and by will represent about of Americans and of Europeans [] The incidence of cancer continues to increase worldwide it is estimated at millionyear by representing anincrease of in cases compared with [] Theincidence of cancer is times higher in people over years old and people aged and older have a higherrisk of developing invasive cancer []The older population is characterized by a very heterogeneous profile especially in terms of frailty geriatriccharacteristics and comorbidities which explains theneed for specific and adapted care [ ] Neverthelessscientific data are scarce because older subjects are oftenunderrepresented in oncological clinical trials that setthe standards of antineoplastic treatment [ ]Over the last three decades the fiveyear survival ratefor all types of cancer has increased particularly in individuals aged to [ ] Still older patients are atmore risk of toxicity in anticancer therapies such aschemotherapy and require a benefitrisk assessmentprior to treatment [] A comprehensive geriatric assessment CGA is consequently recommended in these patients to diagnose comorbidities and optimize geriatricinterventions and to improve the functional state andpossibly the survival rate by ensuring better tolerance totreatment [ ] CGA has also shown predictive valuein identifying elderly patients with cancer who are exposed to a poor prognosisincluding a higher risk ofdeath during hospitalization [] Among the CGAbased assessment tools the Multidimensional PrognosticIndex MPI has shown a predictive interest in mortalityat months and months in Italian patients aged years and older with advanced cancers [“]The main objective of our study was to validate theprognostic value of the MPI for 1year mortality in an external French cohort of older patients with cancer Thesecondary objective was to assess the major risk factors associated with 12month mortality in these patientsMethodsStudy population and data collectionThis prospective singlecenter cohort study enrolledfrom March to March all patients with cancer aged years and older who were referred to thegeriatric oncology clinic of Poitiers University HospitalpriorSociodemographic data and cancerrelated information werecollected during the consultationincluding age sexmarital status social environment type of cancer metastasis status and cancerspecific treatment Tumor siteswere classified as follows colorectal breast prostateupper gastrointestinaltract stomach and esophagusanticancertreatmenttoplannedand liver urinary system bladder upper urinary tractand kidney hematologic malignancies and other tumors including ovary uterus lung head and neck skinthyroid and unknown primary The CGA was performed by a senior geriatrician specialized in oncologyand provided data necessary to calculate MPI All eligible patients who had signed the consent form were included in the study The study protocol was validated bythe Poitiers University Hospital ethics committee Poitiers France All the clinical and biological data werecollected and recorded in a cohort databaseœliving with familyMultidimensional prognostic indexThe MPI based on a CGA was calculated after administration of standardized and validated tests exploringeight domains Table [] Living status was categoœinstitutionalized orrized asœalone and functional status was evaluated by Activitiesof Daily Living ADL ranging from total dependenceto independence and Instrumental ADL IADL [] Nutrition was assessed by the Mini NutritionalAssessmentShort Form MNASF questionnaire cognitive status was evaluated by the Short Portable MentalStatus Questionnaire SPMSQ[ ] The ExtonSmith Scale ESS estimated the risk of pressure ulcer[] Comorbidities were evaluated by the CumulativeIllness Rating Scale CIRS which scores the severity of anic systems ranging from absent to mostsevere [] Based on this scale a comorbidity indexCIRSCI records the number of moderate to severean pathologies CIRS scores from to [] Thenumber of medications is classified in three groups œ‰drugs a day œ to drugs or œ‰¥ drugsThe MPI was scored by matching the results of thesetests A value of œ œ or œ was assigned accordingto the conventional cutoff points considering œ as noproblem œ minor problem and œ major problemTable The sum was then divided by to obtain thefinal MPI score which was categorized into groupsthe œMPI1 group final score ‰ defining patientswith low mortality risk at year the œMPI2 group“ moderate risk and the œMPI3 groupgroup higher riskDefinition of outcomesThe primary outcome in the longitudinal analyses was1year mortality Systematic followup was performedafter discharge through clinical visits every months bythe same clinical research assistant When patients werenot present at visit phone calls were made to the general practitioners to assess vital status and to obtain thedate of death if applicable 0cLiuu BMC Geriatrics Page of No problem value ‰¥‰¥‰Table Multidimensional Prognostic Index score assigned to each domain according to the severity of problemAssessment tests rangeADL “IADL “SPMSQ “10aCIRSCI “14bMNASF “ESS “Number of medicationsMinor problem value “““““““Institutionalized‰¥ ‰¥“Living with familySevere problem value ‰‰‰¥‰¥ ‰ “‰¥ Living statusAbbreviations ADL Activities of Daily Living IADL Instrumental Activities of Daily Living SPMSQ Short Portable Mental Status Questionnaire CIRSCI CumulativeIllness Rating Scale Comorbidity Index MNASF Mini Nutritional Assessment Short Form ESS Exton Smith Scalea Number of errorsb Number of pathologiesLiving aloneStatistical analysisDescriptive statistics were reported as mean ± standarddeviation SD or median 25th“75th percentiles forcontinuous variables or absolute number and percentagefor categorical variables The time to event was plottedas KaplanMeiersurvival curves according to MPIgroups and comparison was made using the logranktest The hazard ratio HR of 1year mortality for eachparameter was determined by Cox proportional hazardsregression Two models were used univariate modeland models adjusted for age sex metastatic statustumor sites Interactions between sex tumor site andmetastatic status for the association between MPI and year mortality were evaluated by the addition of interaction terms into the corresponding regression modelThe Akaike™s information criterion AIC was used tocompare globalfit among models with and withoutMPI and the model with the smallest AIC was considered as the best modelGeneralized cstatistics were calculated to assess improvement in 1year mortality risk prediction of MPI inaddition to traditional risk factors age sex metastaticstatus tumor sites [] The CIs for the changes inthe cstatistic were computed based on bootstrapsamples P values were considered statistically significant Statistical analyses were performed with SASversion SAS Institute Cary NCResultsBaseline characteristics of study populationDuring the recruitment period eligible patients aged years and older were included mostly males n with a mean age of ± years Table Themost common tumor sites were prostate skin and breast of patients had a metastaticdisease Anticancer treatment included chemotherapyin patients surgery in and radiotherapy in Patients had comorbid conditionsregarding the CIRSscale and medication and were frequently malnourished Table In this cohort patients were classified in the œMPI1 group patients in œMPI2 and patients in œMPI Except for metastatic status and antineoplastic treatments all variables of interest differed between the threeMPI groups P ‰ MPI and 1year mortalityAmong the patients were lost to followup Mean followup was ± months Overall mortalityat months was in MPI1 in MPI2 and in MPI3 P Fig Since we observed significant statistical interaction between sex and tumor site P we presented resultsfor the multivariate model with the inclusion in themodel of an interaction term We found no significantinteraction between tumor site and metastatic statusP The risk of 1year mortality across MPI groups isshown in Fig All functional scoring but SPMSQ and living statusnumber of daily drugs metastatic status and tumor sitewere significantly associated with mortality Table Compared to colorectal cancer reference categorybreast cancer was associated with significantly lower year mortality and upper gastrointestinal tractliver cancer and other malignancies with significantly higher year mortalityMPI groups were associated with 1year mortality inthe univariate model and remained significantly associated even after adjustment for age sex metastatic statusand tumor site Compared to the MPI1 group patientsof the MPI2 and MPI3 groups had gradual increasedrisk of 1year mortality adjusted hazard ratio [95CI] [“] and [“] respectively P Table 0cLiuu BMC Geriatrics Page of Table Patients™ baseline characteristics and evaluation by multidimensional prognostic index MPI n Sociodemographic characteristicsAgeFemale n Oncological characteristicsMost frequent tumor sitesProstateSkinColorectumBreastHematological malignanciesBladderMetastatic status n Type of antineoplastic treatment aChemotherapySurgeryRadiotherapyTotal cohortN ± MPI1N ± Comprehensive geriatric assessment and multidimensional prognostic index bHormone therapy ADL scoreADL categoryIADL scoreIADL categoryESS scoreESS categoryMNASF scoreMNASF categorySPMSQ scoreSPMSQ categoryCIRS scoreCIRSCI scoreCIRSCI categoryNumber of medicationsNumber of medications categoryLiving status familyinstitutionalone ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± MPI2N ± ± ± ± ± ± ± ± ± MPI3N ± ± ± ± ± ± ± ± ± MPI score ± ± ± ± P Numbers are mean ± SD or n Abbreviations MPI multidimensional prognostic index SD standard deviation ADL activities of daily livings IADL instrumental activities of daily livings ESS ExtonSmith Scale MNASF mini nutritional assessment short form SPSMQ Short Portable Mental Status Questionnaire CIRS Cumulative Illness Rating Scale CIcomorbidity indexa Antineoplastic treatment may combine one or several types of treatmentb Categories are reported as number of patients with nominorsevere problem to calculate MPI scoreDiscriminationWe assessed improvement in risk discrimination for theMPI group compared with the model with traditionalrisk factors age sex metastatic status and tumor siteWe observed a small but significant improvement in year mortality risk prediction difference in Cstatistic P when including the MPI group in themodel Table 0cLiuu BMC Geriatrics Page of Fig KaplanMeier curves of overall mortality in patients according to MPI groups Dotted line MPI dashed line MPI and solid line MPI Logrank test P Discussion and implicationsOur study confirmed the predictive value of the multidimensional prognostic index for 1year mortality in olderpatients with cancer MPI group had a significantlytwo to fivefold higher rate of 1year mortality We alsoshowed that the MPI improved prediction of 1yearmortality going beyond the traditional risk factors reported in the literature []Estimation of patient survival at time of the therapeutic decision is required to assess the balance of benefits and risks of performing or not performing specificoncologic interventions taking cancerspecific mortalityinto consideration Clinicians may need to know if thepatient will die of cancer or with cancer in cases wherecomorbidities or geriatric syndromes are challengingSeveral scales have been created and validated in largeepidemiologic cohorts to estimate overall survival notably at months with the Carey and Walter indexes [ ] These two scores consider dependency comorbidities with cancer and malnutrition Walter and collaborators reported independent associations betweenoneyear mortality in multivariable analysis and risk factors including male gender two medical diagnoses congestive heart failure aOR 95CI “ andcancer aOR “ for localized cancer and aOR “ for metastatic cancerfunctional dependency in any ADL at discharge aOR “for dependencies from to ADLs and aOR “ for dependencies in all ADLs and laboratoryvalues creatinine level mgdL [ μmolL] aOR “ and albumin level ‰ gdL aOR “ from to gdL and aOR “ forvalues below gdL [] Carey confirmed thesefindings and furthered the elaboration of a prognosticindex for mortality in communityliving frail older individuals considering eight independent risk factors ofmortality weighted using Cox regression male sex dependence in toileting malignant neoplasm and renal insufficiency [] None of these tests were specificallydeveloped in cohorts with individuals with cancer andthey may consequently not be informative enough to reflect clinical and functional variability in daily care andto provide personalized corrective interventions Recentevidence reported a positive impact of geriatric interventions and monitoring in survival increase improvementof quality of life and completion of chemotherapy [] The MPI differs from other mortality indexes because it is based on a CGA with each of the eight tests 0cLiuu BMC Geriatrics Table Univariate and multivariate analyses for oneyear mortality model for MPIgroups n VariableADL score pointPvalueAdjusted HR CI“IADL score pointSPMSQ score pointCIRS score pointMNA score pointESS score pointNumber of drugs drugLiving statusliving with familyliving aloneInstitutionalizedAge yearSex male vs femaleMetastatic statusTumor sitesColorectalbreastprostateUpper gastrointestinal tractliverurinary systemhematologic malignanciesother tumorsMultidimensional Prognostic Indexgroup group group HR CI “ “ “ “ “ “ “reference “ “ “ “ “reference “ “ “ “ “ “reference “ “ “““““““ “ “ “reference “ “ “ “ “ “reference “ “Page of P Abbreviations HR hazard ratio CI confidence interval MPI Multidimensional Prognostic IndexMultivariate model adjusted for age sex tumor site metastatic status and MPI groupsassessing one geriatric domain Giantin and collaboratorsconfirmed the good discriminatory power for 12monthmortality in a cohort of cancer patients older than and validated higher mortality prediction compared to astandard CGA [] Use of the MPI in clinical practicemay provide rapid and comprehensive evaluation of patients and help to adapt decisionmaking in oncologyThe MPI has been developed and validated in large cohorts of in and outpatients for many causes to predict notonly mortality but also length of hospital stay P care intensity institutionalization rehospitalization andaccess to homecare services [ ] In an internationalmulticenter cohort of hospitalized older patients patients in group MPI2 OR “ P and the MPI3 group OR “ P were at higher risk of overall mortality compared to thoseof the lower risk group at admission [] This index maybe used as a decisionmaking tree for cancer managementso as to select older patients with lower mortality risk forthe same standard treatment as younger counterpartsthose who could benefit from adapted care or an exclusively supportive strategy in patients with limited life expectancy This classification in three groups is comparableto the geriatric oncology algorithm of Balducci [] ThisTable Predictive performance of MPI during 12month followupBiomarkerclinical modelAkaike criterioncindexclinical model MPIClinical model age sex metastatic status tumor site CI““difference in CstatisticsP value 0cLiuu BMC Geriatrics Page of algorithm defines three groups of patients robust vulnerable and frail according to seven criteria age dependencemeasured by ADL and IADL comorbidities with CIRSCIcognition evaluated with MMSE minimental state examination or delirium depressive mood urinary and fecalincontinence and falls in the last months Risk of deathincreased steadily from the lowest to the highest categorycompared to the fit group the patients with a vulnerableprofile had a twofold mortality risk HR “and a threefold risk in the frail group HR “ P [] More recent classifications were suggested to improve global management for such individualsincluding nutrition data and cognitive assessment[ ]Indeed malnutrition is highly prevalent in geriatriconcology settings [] This geriatric syndrome is a wellknown risk factor for early mortality Our findings confirmed that oneyear mortality is strongly associated withnutritional status and altered MNA in its short formSome questions in this test were selected for the elaboration of the Geriatric8 G8 index to screen for vulnerability in older patients with cancer as recommended bythe International Society for Geriatric Oncology SIOG[ ]The findings ofthis study should be interpretedwith caution Firstits design as an observationalsinglecenter study may limit the extrapolation of ourresults to a more general older population with cancer Recruited patients in this cohort may not be representative as cancer specialists may not refer alltheir patients to the geriatric oncology clinic notablythose screened as œnotvulnerable in geriatric termsas recommended by the SIOG and National Instituteof Cancer in a twostep approach [] Cancer management of these patients may follow standard strategy without geriatric expertise After accounting fortraditional risk factors the magnitude of the improvement in risk prediction by the addition is small butsignificant Moreover our results are consistent withexisting findings in geriatric oncology settingsthisstrategyOur research on the predictive value of MPI foroneyear mortality of older patients with cancershould serve as a foundation for future studies aiming to improve therapeutic strategies for these patients A major part ofinvolvespersonalized geriatric interventions such as specificcare monitoring by nurse and physical rehabilitationIt has shown benefits for elderly cancer patients butso far no study has demonstrated any impact onsurvival [“] MPI appears to be a rapid assessmenttool helping to optimize cancer care guidepatienttailored interventions and predict early mortality These findings should pave the way for prospective interventionaltaking account ofstudiesMPI groups for decisionmaking about cancer treatments and followupConclusionsIn addition to established risk factors MPI improves riskprediction of 1year mortality in older cancer patientsThis practical prognostic tool may help to optimizemanagement of these vulnerable individualsAbbreviationsADL Activities of Daily Living AIC Akaike™s information criterionCGA Comprehensive Geriatric Assessment CI confidence intervalCIRS Cumulative Illness Rating Scale CIRSCI Cumulative Illness Rating Scale comorbidity index ESS ExtonSmith Scale G8 Geriatric8 HR Hazard ratioIADL Instrumental Activities of Daily Living MNASF Mini NutritionalAssessmentShort Form MPI Multidimensional Prognostic IndexSD Standard deviation SIOG International society of geriatric oncologySPMSQ Short Portable Mental Status QuestionnaireAcknowledgmentsAuthors thank Emilie Favard for her assistance in the collection of followupdata and Jeffrey Arsham who edited the English of the manuscriptAuthors™ contributionsEL CH and MP designed the study EL SV and AJ were responsible for theacquisition of data EL and PJS performed the statistical analysis andinterpretation EL PJS and MP wrote the manuscript EL PJS MP TB MLBand AP substantively revised the work All authors EL CH SV TB AJ MLBAP PJS and MP read and approved the final manuscriptFundingThe authors declare no fundingAvailability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateAll eligible patients who had signed the consent form were included in thestudy The study protocol was validated by the Poitiers University Hospitalethics committee Poitiers FranceConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Geriatrics Poitiers University Hospital Poitiers France2Clinical Investigation Centre CIC1402 CHU Poitiers University of PoitiersINSERM Poitiers France 3Department Geriatric Care Orthogeriatrics andRehabilitation Frailty Area EO Galliera Hospital Genova Italy 4Departmentof interdisciplinary Medicine Aldo Moro University of Bari Bari ItalyReceived December Accepted August ReferencesExtermann M Aapro M Bernabei R Cohen HJ Droz JP Lichtman S et alUse of comprehensive geriatric assessment in older cancer patientsrecommendations from the task force on CGA of the International Societyof Geriatric Oncology SIOG Crit Rev Oncol Hematol “Cancer Research UK Worldwide cancer incidence statistics Availablefrom httpwwwcancerresearchukhealthprofessionalcancerstatisticsworld widecancer incidence Accessed Apr Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin “ 0cLiuu BMC Geriatrics Page of Pamoukdjian F Liuu E Caillet P Herbaud S Gisselbrecht M Poisson J Howto optimize Cancer treatment in older patients an overview of availablegeriatric tools Am J Clin Oncol “Kalsi T BabicIllman G Ross PJ Maisey NR Hughes S Fields P The impact ofcomprehensive geriatric assessment interventions on tolerance tochemotherapy in older people Br J Cancer “ Rao AV Hsieh F Feussner JR Cohen HJ Geriatric evaluation andmanagement units in the care of the frail elderly cancer patient J GerontolA Biol Sci Med Sci “ Meyer AM Becker I Siri G Brinkkötter PT Benzing T Pilotto A Polidori MCNew associations of the Multidimensional Prognostic Index Z GerontolGeriatr “ Pilotto A Veronese N Daragjati J CruzJentoft AJ Polidori MC MattaceRasoF Using the multidimensional prognostic index to predict clinicaloutcomes of hospitalized older persons a prospective multicentreinternational study J Gerontol A Biol Sci Med Sci “Ferrat E Paillaud E Caillet P Laurent M Tournigand C Lagrange JL et alPerformance of four frailty classifications in older patients with cancerprospective elderly cancer patients cohort study J Clin Oncol “ Caillet P Liuu E Raynaud Simon A Bonnefoy M Guerin O Berrut GAssociation between cachexia chemotherapy and outcomes in oldercancer patients a systematic review Clin Nutr “ Decoster L Van Puyvelde K Mohile S Wedding U Basso U Colloca G et alScreening tools for multidimensional health problems warranting a geriatricassessment in older cancer patients an update on SIOG recommendationsAnn Oncol “Soubeyran P Bellera C Goyard J Heitz D Curé H Rousselot H et alScreening for vulnerability in older cancer patients the ONCODAGEprospective multicenter cohort study PLoS One 20149e115060 Caillet P CanouiPoitrine F Vouriot J Berle M Reinald N Krypciak S et alComprehensive geriatric assessment in the decisionmaking process inelderly patients with cancer ELCAPA study J Clin Oncol “ Galvão DA Taaffe DR Spry N Joseph D Newton RU Combined resistanceand aerobic exercise program reverses muscle loss in men undergoingandrogen suppression therapy for prostate cancer without bonemetastases a randomized controlled trial J Clin Oncol “ Goodwin JS Satish S Anderson ET Nattinger AB Freeman JL Effect ofnurse case management on the treatment of older women with breastcancer J Am Geriatr Soc “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsBalducci L Extermann M Management of Cancer in the older person apractical approach Oncologist “ Walter LC Brand RJ Counsell SR Palmer RM Landefeld CS Fortinsky RH Development and validation of a prognostic index for 1year mortalityin older adults after hospitalization JAMA “Gouverneur A Salvo F Berdaï D Moore N FourrierRéglat A Noize PInclusion of elderly or frail patients in randomized controlled trials oftargeted therapies for the treatment of metastatic colorectal cancer asystematic review J Geriatr Oncol “Talarico L Chen G Pazdur R Enrollment of elderly patients in clinical trialsfor cancer drug registration a 7year experience by the US Food and DrugAdministration J Clin Oncol “Zeng C Wen W Mans AK Pao W Shu XO Zheng W Disparities by raceage and sex in the improvement of survival for major cancers results from theNational Cancer Institute surveillance epidemiology and end results SEERprogram in the United States to JAMA Oncol “Ellis G Gardner M Tsiachristas A Langhorne P Burke O Harwood RH et alComprehensive geriatric assessment for older adults admitted to hospitalCochrane Database Syst Rev 20179CD006211 Wildiers H Heeren P Puts M Topinkova E JanssenHeijnen ML ExtermannM International Society of Geriatric Oncology consensus on geriatricassessment in older patients with cancer J Clin Oncol “ AvelinoSilva TJ Farfel JM Curiati JA Amaral JR Campora F JacobFilho WComprehensive geriatric assessment predicts mortality and adverseoutcomes in hospitalized older adults BMC Geriatr Angleman SB Santoni G Pilotto A Fratiglioni L Welmer AK MPI_AGEProject Investigators Multidimensional Prognostic Index in Association withFuture Mortality and Number of Hospital Days in a PopulationBasedSample of Older Adults Results of the EU Funded MPI_AGE Project PLoSOne 201510e0133789 Giantin V Falci C De Luca E Valentini E Iasevoli M Siviero P Maggi S et alPerformance of the multidimensional geriatric assessment andmultidimensional prognostic index in predicting negative outcomes inolder adults with cancer Eur J Cancer Care “ httpsdoi101111ecc12585 Pilotto A Ferrucci L Franceschi M D'Ambrosio LP Scarcelli C Cascavilla L Development and validation of a multidimensional prognostic indexfor oneyear mortality from comprehensive geriatric assessment inhospitalized older patients Rejuvenation Res “ Pilotto A Rengo F Marchionni N Sancarlo D Fontana A Panza F et alComparing the prognostic accuracy for allcause mortality of frailtyinstruments a multicentre 1year followup in hospitalized older patientsPLoS One 20127e29090Katz S Downs TD Cash HR Grotz RC Progress in development of the indexof ADL Gerontologist “Lawton MP Brody EM Assessment of older people selfmaintaining andinstrumental activities of daily living Gerontologist “ Pfeiffer E A short portable mental status questionnaire for the assessmentof anic brain deficit in elderly patients J Am Geriatr Soc “ Rubenstein LZ Harker JO Salva A Guigoz Y Vellas B Screening forundernutrition in geriatric practice developing the shortform mininutritional assessment MNASF J Gerontol A Biol Sci Med Sci M366“ Bliss MR McLaren R ExtonSmith AN Mattresses for preventing pressuresores in geriatric patients Mon Bull Minist Health Public Health Lab Serv“Linn BS Linn MW Gurel L Cumulative illness rating scale J Am Geriatr Soc“ Conwell Y Forbes NT Cox C Caine ED Validation of a measure of physicalillness burden at autopsy the cumulative illness rating scale J Am GeriatrSoc “ Pencina MJ D'Agostino RB Overall C as a measure of discrimination insurvival analysis model specific population value and confidence intervalestimation Stat Med “ Giantin V Valentini E Iasevoli M Falci C Siviero P De Luca E Does themultidimensional prognostic index MPI based on a comprehensivegeriatric assessment CGA predict mortality in cancer patients Results of aprospective observational trial J Geriatr Oncol “ Carey EC Covinsky KE Lui LY Eng C Sands LP Walter LC Prediction ofmortality in communityliving frail elderly people with longterm careneeds J Am Geriatr Soc “ Epub Nov 0c"

Thyroid_Cancer

Integrinmediated adhesive properties ofneutrophils are reduced by hyperbaric oxygentherapy in patients with chronic nonhealingwoundMonica BaiulaID1 Roberto Greco2 Lucia Ferrazzano2 Alberto Caligiana1Klarida Hoxha3 Daniele Bandini3 Pasquale LongobardiID3 Santi Spampinato1˜¯Alessandra TolomelliID2˜¯ Department of Pharmacy and Biotechnology Alma Mater Studiorum University of Bologna Bologna Italy Department of Chemistry œGiacomo Ciamician Alma Mater Studiorum University of Bologna BolognaItaly Hyperbaric Centre Ravenna Italy˜¯ These authors contributed equally to this work Current address MediNeos Observational Research Modena Italy alessandratolomelliuniboit AT santispampinatouniboit SSa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSAbstractCitation Baiula M Greco R Ferrazzano L CaligianaA Hoxha K Bandini D Integrinmediated adhesive properties of neutrophils arereduced by hyperbaric oxygen therapy in patientswith chronic nonhealing wound e0237746 101371journalpone0237746Editor Nukhet AykinBurns University of Arkansasfor Medical Sciences College of Pharmacy UNITEDSTATESReceived May Accepted July Published August Copyright Baiula This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscript and its SupportingInformation filesFunding Financial Disclosure This researchstudy was supported by Fondazione delMonte di Bologna e Ravenna wwwfondazionedelmonteit through a grant to ATFdM3980 prot N˚ bis2015 The authorswere also supported by the University of BolognaIn recent years several studies suggested that the ability of hyperbaric oxygen therapyHBOT to promote healing in patients with diabetic ulcers and chronic wounds is due to thereduction of inflammatory cytokines and to a significant decrease in neutrophils recruitmentto the damaged area α4 and 2 integrins are receptors mediating the neutrophil adhesionto the endothelium and the comprehension of the effects of hyperbaric oxygenation on theirexpression and functions in neutrophils could be of great importance for the design of noveltherapeutic protocols focused on antiinflammatory agents In this study the α4 and 2 integrins™ expression and functions have been evaluated in human primary neutrophils obtainedfrom patients with chronic nonhealing wounds and undergoing a prolonged HBOT kPaper minutes The effect of a peptidomimetic α41 integrin antagonist has been also analyzed under these conditions A statistically significant decrease in 2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one monthafter the last treatment while α4 integrin levels remained unchanged However cell adhesion function of both neutrophilic integrins α41 and 2 was significantly reduced and respectively but α41 integrin was still sensitive to antagonist inhibition in the presence of fibronectin suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacyIntroductionHyperbaric oxygen HBO therapy has emerged in the last years as an innovative approachand an effective adjunctive therapy for the treatment of different pathologies The oxygenPLOS ONE 101371journalpone0237746 August PLOS ONE 0cRFO17 RFO18 RFO19 and by Italian Ministryof Education University and Research with aspecific research project PRIN project20157WW5EH and a grant to the Department ofChemistry œGiacomo Ciamician of the Universityof Bologna under the initiative Department ofExcellence L232 del The fundershad no role in study design data collection andanalysis decision to publish or preparation of themanuscriptCompeting interests The authors have declaredthat no competing interests existIntegrins™ role in HBOTpromoted wound healingpressure applied in the chamber is usually from to kiloPascal kPa“ to absoluteatmospheres ATA and the first effect of pressurizing the human body is the increase of partialpressure of gases and the decrease of volume of gasfilled spaces according to Boyle™s law [ ]The additionally available oxygen has the ability to restore oxygenation in areas where hypoxia or hypoperfusion occur and it can help damaged tissue to heal [] Moreover increasedoxygen levels that lead to changes in reactive oxygen species ROS and nitrogen speciesRNS production during HBO therapy HBOT are essential to stimulate specific repair functions of macrophages neutrophils and fibroblasts in the healing process [“] In additionHBOT regulates the inflammatory response reduction of NLRP3 inflammasome proinflammatory cytokines including IL1 IL6 e IL18 TNFα [“]HBOT has been successfully employed to control nonhealing diabetic ulcers and chronicwounds significantly minimizing the number of amputations relative to standard wound carealone in diabetic population []Wound healing is a complex process that involves growth factors components of the extracellular matrix and several cell types Inflammatory cytokines such as tumor necrosis factorαTNFα and interleukin1 IL1 are often present at high levels in the site of inflammationlike in chronic wounds [] The immune system is involved in all the steps of tissue repair[] Inflammatory response evolves in the leukocyteadhesion cascade primarily mediated bytwo major adhesion receptor families selectins and integrins []Neutrophils play a crucial role in wound healing process by sensing their environment andresponding to the extracellular signals by adhesion migration and other effector functions[] After ending their role at the site of inflammation neutrophils undergo apoptosis and areremoved by macrophages this latter event is considered a strong signal for inflammation resolution Although fighting infection neutrophils can also have harmful effects inducing damagein the inflamed tissue and leading to a delay in healing process [] and chronic inflammationNeutrophils express integrins on their surface significantly contributing to the recruitmentphase Among them 2 integrin family members including αL2 and αM2 bind to endothelial intercellular adhesion molecule1 ICAM1 and α41 integrin recognizes vascular celladhesion molecule1 VCAM1 expressed on endothelial cells [] Moreover α41 integrinand 2 integrins are involved in the onset and resolution of inflammatory process mediatingthe adhesion of monocytes lymphocytes and neutrophils to the blood vessels []Several studies carried out on animal models have evidenced that neutrophil recruitment issignificantly reduced by treatment with HBO in case of damage induced by ischemia andreperfusion [“] in other studies HBOT induces the reduction of tissue necrosis [ ]and lipid peroxidation []Moreover it has been shown that HBO treatment inhibits ischemia reperfusioninducedneutrophil adhesion to endothelium by blocking 2 integrin polarization [ ] and may alsoreduce leukocytes recruitment as it impaired adhesion molecule function by Snitrosation[] Conversely the role played by α41 integrin in neutrophilmediated adhesion to endothelium has been poorly investigated but an important role of α41 in 2 integrinindependentmigration of neutrophils across heart endothelium has been demonstrated in vitro suggestinga similar in vivo situation in neutrophil trafficking in reperfused myocardium []HBOT represents an effective therapy for chronic wounds as it reduces inflammation andaccelerates healing [] probably involving integrins In the present study we investigatedwhether HBOT could exert its effects by modulating functions of α41 and 2 integrinsexpressed on neutrophils obtained from patients with chronic nonhealing ulcers In a previous study Thom [] isolated polymorphonuclear leukocytes PMN from young healthyvolunteers exposed to only one session of HBO They observed a reduction in cell adhesionmediated by 2 integrins without any variation of its expression Our aim is to evaluate the rolePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingof integrinmediated adhesion by characterizing the expression of integrin receptors on neutrophils during the HBOT and by analyzing the effect of a peptidomimetic α41 integrin antagonist under these conditions If integrins are a target for both HBOT and synthetic antagonistthat blockade their activation a joint therapy could be hypothesized leading to a faster andstronger decrease of inflammation To this purpose expression and function of α41 and 2integrins were investigated for the first time in human primary neutrophils isolated from theblood of patients with chronic nonhealing ulcer undergoing HBOT or standard wound therapy alone Expression of these integrins was monitored in patients before the beginning ofHBOT exposure and during the therapy using specific antibodies towards α4 integrin or 2integrin family Patient wound area size was measured and proinflammatory cytokine levelswere evaluated both in neutrophils and in plasma Furthermore in vitro cell adhesion assayswere performed in the presence of a peptidomimetic integrin antagonist previously developedby our group [] to investigate if the HBO treatment may influence neutrophil recruitmentand adhesion mediated by α41 integrinMaterials and methodsPatients™ recruitmentThe study protocol was conformed to the ethical guidelines of the Declaration ofHelsinkiThe patients followed exclusively the therapy prescribed by their medical doctor followingthe indications of the Italian public health system No additional treatments were done for thepurposes of the study which did not change any therapeutic protocol nor interfere with thehealing progress The patients of the HBOT group were submitted to the prescribed HBOTand to blood sampling following the standard protocols approved by the Institutional EthicsReview Board of the Comitato Etico della Romagna CEROM Version of June approved by CEROM Ethical Committee Reg Sperimentazioni Prot N45332018I5150 which has full access to the data in order to check conformity of the study to thecurrent regulationThirty patients from Hyperbaric Centre Ravenna Italy were enrolled in the study betweenJuly and January in two different groups control group patients n receivedstandard wound care alone as prescribed by their medical doctor whereas HBOT grouppatients n received HBOT in addition to conventional wound treatment The inclusioncriteria were adults years or older and chronic wounds that fail to demonstrate improvement wound area reduction after a minimum of weeks of standard wound therapyThe exclusion criteria were symptoms of bacterial infection malignancy pregnancy medications that can adversely affect healing including anticonvulsants steroids antibiotics angiogenesis inhibitors and NSAIDs such as drugs known to promote healing including vitaminsthyroid hormone and iron Contraindication for HBOT were claustrophobia uncontrolleddiabetes middle ear problems glaucoma heart failure pacemaker untreated pneumothoraxchronic obstructive pulmonary disease pulmonary emphysema with retention of CO2 prothesis and seizures Patients in the control group were matched by gender age and ethnicityExclusion criteria for control group patients were the same for patients in HBOT groupThe patients allowed donating blood samples and were informed of the aim of the studyand completed the written informed consent process before enrolling in the study At the endof the study all individuals received personal information about the results relative to theirown samplesThe following demographic information was collected for all recruited patients at baselinegender age smokernonsmoker and other medical conditions Photographs of the ulcer werePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingtaken at various times After sharp wound debridement with a sterile scalpel the wound™s surface area was calculated by VISITRAK„¢ Digital System Smith Nephew For each patientthe ulcer was graded and staged by a clinician as described belowSample sizeSample size was based on a power analysis using G�Power [] The power was set at tolimit the risk of committing a type II error to the α level was set at The effect sizewas set at and the number of samples for each group was calculated to be to detect statistically significant differences P value of between groups In this study patientswere selected and allocated to study groups control and HBOT groupHyperbaric oxygen therapy protocolHBOT was conducted at the Hyperbaric Centre in Ravenna Italy The protocol procedureconsisted of HBO exposures in a multiplace hyperbaric chamber daily session five perweek from Monday to Friday HBOT group patients breathed oxygen at kPa per minutes in cycles for minutes separated by minutes medical air breathing intervalsClassification of woundsIn this study ulcers were graded using Falanga Wound Bed Preparation Score Staging copyrighted [] that provides staging of varying degrees based upon descriptions and characteristics of ulcers Table [] Staging of the wounds was done by combining the score of thewound bed appearance with that of the wound exudate Table Clinical assessment ofwound conditions was conducted for HBOT group before HBO treatment T0 immediatelyafter the fifteenth HBOT session T15 and one month after ending HBOT T1M for controlgroup during the first evaluation by a medical doctor T0 and after fifteen days of conventional wound therapy T15Neutrophil isolation from peripheral bloodVenous blood samples were obtained from the antecubital vein of participants in EDTAcontaining vacutainers Neutrophil isolation and further experiments were performed at theDepartment of Pharmacy and Biotechnology University of Bologna Bologna Italy Bloodsamples were obtained for patients in control group during the first evaluation by a clinicianTable Cutaneous ulcers were graded using Falanga wound classification systemWound Bed CharacteristicsWound appearanceGranulation tissueFibrinous tissueEscharABCDWound Exudate ScoreExtent of ControlFullyPartiallyUncontrolledAdapted from []101371journalpone0237746t001“ Any amountExudate AmountNoneminimal““““Dressing requirementNo absorptive dressing required If clinically feasibledressings could stay on for up to a weekModerate amountDressing changes required every “ daysVery exudative woundAbsorptive dressing changes required at least dailyPLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingT0 and after fifteen days of conventional wound therapy T15 while for HBOT grouppatients before T0 and immediately after the fourth T4 the eighth T8 the twelfth T12and the fifteenth T15 HBOT sessions In addition one month after ending HBOT T1Manother blood sample was collected from HBOT group patients For neutrophil isolationblood was carefully layered on top of an equal volume of Lympholyte1poly Cedarlaneand centrifuged at g for min at “ËšC as previously described [] Plasma wascarefully removed and store at ˚C for further analysis Neutrophils were transferred in aclean tube and were resuspended in mL of HBSS Hanks™ Balanced Salt Solution LifeTechnologies Italia without Ca2Mg2 and centrifuged at g for minutes To lyse theresidual red blood cells RBCs mL Red Cell Lysis Buffer Roche were added and the cellswere resuspended vortexing at low speed to avoid neutrophils activation Neutrophils werecentrifuged at g for min resuspended in HBSS without Ca2Mg2 and adjusted todesired concentrationCell viability was determined using Annexin V7AAD assay Guava Nexin Reagent Millipore as previously described [] and was Differential analysis of cells retrieved usingthis procedure showed granulocytes of which were neutrophils Neutrophilswere stored at room temperature and used for functional tests cell adhesion assay and flowcytometry analysis within h of collection An aliquot of the purified neutrophils was immediately stored at ˚C and used for mRNA extractionNeutrophil adhesion assayAdhesion assays on purified neutrophils were performed as previously described [ ]Briefly black 96well plates were coated overnight at ˚C with fibronectin FN or fibrinogenFg both μgmL to study respectively adhesion mediated by α41 and 2 integrins Neutrophils were counted and stained with CellTracker green CMFDA μM min at ˚CLife Technologies Italia Thereafter cells were plated 50000well on coated wells and incubated for min at ˚C After three washes adhered cells were lysed with Triton X100in BSA bovine serum albumin in HBSS min at ˚C and fluorescence was measuredEx485 nmEm535 nm To evaluate the ability of ligand [] named here RG66 to inhibitneutrophils™ adhesion cells were preincubated with various concentrations ˆ’“ˆ’ M ofRG66 or vehicle methanol for min at ˚C before plating cells into coated wells Neutrophil adhesion assays were also carried out in the presence of an antihuman 2 or α4 integrinantibody both μgmL purified mouse antihuman CD18 and purified mouse antihumanCD49d antibody BD Pharmingen Experiments were carried out in triplicate Data analysisand IC50 values were calculated using GraphPad Prism GraphPad Software San DiegoCA USAFlow cytometry analysisPurified neutrophils were suspended in BSA in HBSS at the concentration of cellsmL μLsample and incubated with FITClabeled antiα4 integrin antibody μLsampleFITC Mouse antihuman CD49d BD Pharmingen or FITClabeled anti2 integrin antibody μLsample FITC Mouse antihuman CD18 BD Pharmingen for min at ˚C as previously described [] After two washes with BSA in HBSS cells were resuspended in PBSand analyzed in a Guava EasyCyte Flow Cytometer Millipore and cellssample wereanalyzed Data were normalized with the relative fluorescence for nonspecific binding evaluated by exposing the cells to an isotype control monoclonal antibody FITC mouse IgG BectonDickinson Italia and set to PLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingIn another set of experiments purified neutrophils were suspended in BSA in HBSS atthe concentration of cellsmL μLsample and incubated with the conformational sensitive phycoerythrin PElabeled HUTS21 monoclonal antibody μLsample PE mouseantihuman CD29 antibody BD Pharmingen for min at room temperature Neutrophilswere washed twice with BSA in HBSS resuspended in PBS and analyzed at the flow cytometry cellssample were analyzed Data were normalized to nonspecific binding relativefluorescence evaluated by exposing the cells to an isotype control mAb monoclonal antibodyand set to Quantitative real time PCRTotal RNA was extracted from purified neutrophils with TRI Reagent SigmaAldrich andquantified using a NanoDrop spectrophotometer ThermoFisher Scientific For each sample“ μg of total RNA was treated with RNasefree DNase as previously described [] TheRNA samples were then converted into cDNA using HighCapacity cDNA Reverse Transcription Kits Life Technologies Italia according to the manufacturer™s instructions RealtimePCR was performed using GoTaq1 qPCR Master Mix Promega Corporation Madison WIUSA The protocol consisted of i for L19 and TNFα denaturation at ˚C for minutesfollowed by cycles of ˚C denaturation seconds and ˚C annealingextension minute ii for α4 integrin denaturation at ˚C for minutes followed by cycles of ˚Cdenaturation seconds ˚C annealing seconds and ˚C seconds iii for IL1denaturation at ˚C for minutes followed by cycles of ˚C denaturation seconds˚C annealing seconds and ˚C seconds Notemplate controls and DNA meltingcurve analysis were used as controls to ensure the lack of contaminating DNA in the RNApreparations and to rule out primerdimer formation respectively To amplify integrin andcytokine targets the following primers were used α4 integrin sense primer 50GTCGCATCCCGTGCAACTTTG30 and antisense primer 50GCTGTGCAGCACGACCGAGT30 amplifying a bp fragment TNFα sense primer 50CTTCTCCTTCCTGATCGTGG30 and antisense primer 50TCTCAGCTCCACGCCATT30 amplifying a bp fragment [] IL1sense primer 50CAAGGGCTTCAGGCAGGCCG30 and antisense primer 50TGAGTCCCGGAGCGTGCAGT30 amplifying a bp fragment [] To amplify 2 integrin cDNA primersequences were from PrimerBank [] a sense primer 50TGCGTCCTCTCTCAGGAGTG30and an antisense primer 50GGTCCATGATGTCGTCAGCC30 amplifying a bp fragmentwere usedAs reference control a bp fragment of the L19 ribosomal protein was amplified using asense primer 50CTAGTGTCCTCCGCTGTGG30 and an antisense primer 50AAGGTGTTTTTCCGGCATC30 [] For data analysis relative expression of RTPCR products wasdetermined using the ΔΔCT method [] as previously described [] the threshold cycle Ctvalues were normalized both on the basis of L19 content and on the values derived from T0sample Each sample was tested in triplicate Primers were synthesized by SigmaAldrichCytokine quantification in plasma by ELISACytokine protein levels TNFα and IL1 were determined in plasma of patients usingELISA kits Invitrogen LifeTechnologies Italia Monza Italy according to the manufacturer™sinstructions Briefly 50μLwell of sample were added to a 96well plate together with μL ofbiotin conjugated primary antibody the plate was then incubated for hours at room temperature After washing four times μL of streptavidinHRP solution were added and the platewas incubated for min at room temperature The wells were washed times and afterwards μL of stabilized chromogen were added and incubated for min at room temperaturePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingAfter the addition of μL of stop solution absorbance was read at nm using an EnSpireMultimode Plate Reader PerkinElmer Waltham MA USA The calculated overall intraassay coefficient of variation was for TNFα and for IL1 the interassay coefficient of variation was calculated to be for TNFα and for IL1Synthesis and bioactivity of α41 synthetic ligandThe synthesis of RRG66 has been previously reported [] via a multistep synthesis startingfrom an enantiopure 3RZtertbutyl 3allylamino2ethylidene4methylpentanoate Thedetailed description of the synthetic protocol has been reported in S1 Fig The biological evaluation showed a strong dependence of the bioactivity on the ring stereochemistry could bedetected since S1 turned out to be completely inactive []Data and statistical analysisAll assays were carried out in triplicate for individual sample at each time pointperson and nrefers to the number of individuals Continuous variables are presented as mean ± standarddeviation when normally distributed data were tested using oneway ANOVA followed byNewmanKeuls posttest or using standard Student t test In addition data are presented asmedian and range and analyzed using MannWhitney™s test when non normally distributedCategorical data were analyzed using χ2square test Data analysis and IC50 values referring toadhesion assays in the presence of RG66 compound were fitted using sigmoidal doseresponseequation using GraphPad Prism software Statistical analyses were performed using GraphPadPrism version GraphPad Software Inc La Jolla CA USA P was consideredsignificantResultsDemographic and clinical data patients men and women age ± years presenting a chronic nonhealingwound condition were recruited and volunteered to participate in the study All the patientscompleted the study and no patient was excluded from the data analysis Demographic parameters such as age gender and clinical data are summarized in Table Age distributions demographic and clinical characteristics of patients were compatiblebetween HBOT and control groups P Overall the wounds of the patients enrolled in the study were caused by different etiologies caused by diabetes by venous insufficiency by critical limb ischemia bytrauma and by vasculitisAs regards comorbidities seven of the subjects suffered from diabetes of type1 and oftype2 twelve of them of hypertension six of obesity seven of venous insufficiency eight ofcardiomyopathy and six of hypothyroidism not requiring thyroid hormones Three subjectswere smokers and four previous smokers Common sites of wound were leg patients foot patients great toe patients and other sites patients Before beginning the HBOTall participants passed a standard medical and physical revision at the Hyperbaric Centre inRavenna All the patients both in control and HBOT groups received standard wound careie wounds were cleaned gently minimizing chemical or mechanical trauma at low pressure“ psi with saline solution and daily sterile sharp debridement with scalpel curette or scissors was performed for the necrotic tissue removal with caution to avoid excess tissue damagewhich may delay healing to get a wellbleeding granulating basePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingPTable Demographic and clinical data of patients enrolled in the studyDemographic dataNumber of subjectsAge yearsaFemaleMalebSmokersComorbiditiesbDiabetesHypertensionObesityVenous insufficiencyGlaucomaCardiomyopathyHypothyroidismHBVHCV positiveRheumatoid arthritisControl groupHBOT group “type1 type2 “ extype1 type2 aAge is expressed as median rangeb Gender and comorbidities are expressed as number of individuals101371journalpone0237746t002Integrin expression on neutrophils deriving from patients undergoingHBOTIn order to study the effect of HBOT on neutrophil integrin expression these cells were isolated from blood samples deriving from patients with a chronic nonhealing wound receiving standard wound care alone control group or undergoing HBOT HBOT group Forcontrol group patients blood samples were collected during the first wound evaluation T0and after fifteen days of conventional wound therapy T15 for patients in the HBOT groupblood samples were obtained before T0 and immediately after the fourth T4 the eighthT8 the twelfth T12 and the fifteenth T15 HBOT treatment at the end of three weeks fiveexposuresweek moreover the last blood sample was drawn one month after ending HBOTT1MAs shown in Fig HBOT did not alter α4 integrin expression in primary human neutrophils both at the mRNA and protein levels Fig panels a and b nor induced any significantvariation in α4 integrin expression throughout the duration of HBO treatment No significantchange in α4 integrin expressed on neutrophils was observed in both control and HBOT grouppatientsOn the contrary 2 integrins were significantly reduced by HBOT both at mRNA andprotein levels this decrement was maintained up to the end of HBO treatment and also onemonth after the last HBOT session Fig panels c and d Neutrophils deriving from patientsreceiving conventional wound care alone control group did not show any changes in 2integrins expressionEffects of HBOT on integrinmediated adhesive properties of neutrophilsDuring an inflammatory process neutrophils adhere and transmigrate through bloodvesselwalls α41 and 2 integrins expressed on neutrophil cell membrane are required and stronglymediate rolling and firm adhesion crawling and transmigration steps of adhesion cascade [] as their activation is an essential step of this complex process To understand whetherPLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingFig HBOT does not modify α4 integrin levels panels a and b but reduces 2 expression panels c and d in neutrophilsderiving from patients receiving standard wound care alone control group and patients undergoing HBOT for sessionsHBOT group The decrement of 2 integrins was maintained up to one month after ending HBOT The effects of HBOT onintegrin expression were evaluated both by qPCR mRNA levels panel a c and by flow cytometry measuring integrin expressed oncell surface panel b d Results from qPCR are expressed as mean ± standard deviation of individual samples carried out intriplicate at each time point control group n HBOT group n Data from flow cytometry analysis are expressed as meanfluorescence intensity MFI ± standard deviation of individual samples carried out in triplicate at each time point control groupn HBOT group n MFI values for respective isotype control monoclonal antibody were set to ��� p versus T0both control and HBOT group101371journalpone0237746g001HBOT could influence integrinmediated neutrophil adhesion we performed cell adhesionassays to fibronectin FN or fibrinogen Fg ligands for α41 and 2 integrins respectivelyon neutrophils isolated from patients with chronic nonhealing wound receiving standardwound care alone control group or undergoing HBOT HBOT group Adhesion of neutrophils obtained from patients belonging to both control and HBOT groups was significantlyreduced by the addition of integrin specific antibodies able to block integrin functions Fig demonstrating that neutrophil adhesion to fibronectin or fibrinogen was mainly mediated byα41 or 2 integrin respectively Moreover as shown in Fig exposure to HBO induces a significant reduction of neutrophil adhesion mediated by either 2 or α41 integrins Fig panela and b respectively Interestingly this reduction of neutrophil adhesive properties is retainedthroughout the duration of HBO treatment and up to one month after the last HBOT sessionIn addition these data showed that adhesive properties of α41 integrin expressed on neutrophils were impaired during HBOT although its expression was not modified both at mRNAand protein levels as shown in Fig To better understand the involvement of α41 integrinin neutrophil adhesion we used a conformationspecific antibody that recognizes a specificepitope on integrin 1 subunit exposed only in a defined structural conformation [] Integrins exist in three major conformations a bent or inactive an intermediateactive and an highactivity conformation [] To monitor conformational changes in integrin subunits it is therefore possible to use conformationspecific antibodies [] We employed thePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingFig HBOT reduces significantly integrinmediated neutrophil adhesion to fibrinogen Fg or fibronectin FN Integrinmediated adhesion wasdecreased in neutrophils obtained from patients belonging to HBOT group no changes were observed in control group patients The effects of HBOTon neutrophil adhesion mediated by 2 panel a or α41 panel b integrins were evaluated by adhesion assay to Fg or FN respectively as described inmethod section Adhesion mediated by 2 or α41 integrin is significantly prevented in neutrophils treated with a monoclonal antibody anti2 or antiα4 respectively HBOT exposure significantly reduced anti1 HUTS21 mAb binding to neutrophils namely changing α41 integrin conformationpanel c Mean fluorescence intensity MFI due to the anti1 integrin mAb PE conjugated HUTS21 binding in the presence of fibronectin μgmL was measured Nonspecific binding of an isotype control PE conjugated mAb added to neutrophils produced an MFI of ± that was subtractedfrom all samples Data are expressed as mean ± standard deviation of individual samples carried out in triplicate at each time point control groupn HBOT group n �� p � �� p ���� p versus T0 both control and HBOT group101371journalpone0237746g002PEconjugated HUTS21 mAb to determine whether the reduced adhesive properties of neutrophils mediated by α41 integrin during HBOT are due to a conformational change of 1subunit indicative of its activation status HUTS21 antibody recognizes a ligandinducedbinding site that is hidden in the inactive conformation but it is exposed when the agonistbinds or upon partial integrin activation namely when the integrin is in a high affinity conformation The epitope recognized by HUTS21 antibody is located in the hybrid domain of 1integrin subunit [] PEconjugated anti1 HUTS21 mAb was added to neutrophils in thepresence of fibronectin μgmL and fluorescence was measured by flow cytometry Exposure to HBOT induced a significant reduction of HUTS21 mAb binding to neutrophilsthroughout the duration of the treatment and up to one month after the last HBOT sessionFig panel c meaning th

Thyroid_Cancer

"Tumor microenvironment TME plays an important role in malignant tumors Our study aimed toinvestigate the effect of the TME and related genes in osteosarcoma patientsMethods Gene expression profiles and clinical data of osteosarcoma patients were downloaded from the TARGETdataset ESTIMATE algorithm was used to quantify the immune score Then the association between immune scoreand prognosis was studied Afterward a differential analysis was performed based on the high and lowimmunescores to determine TMErelated genes Additionally Cox analyses were performed to construct two prognosticsignatures for overall survival OS and diseasefree survival DFS respectively Two datasets obtained from the GEOdatabase were used to validate signaturesResults Eightyfive patients were included in our research The survival analysis indicated that patients with higherimmune score have a favorable OS and DFS Moreover genes were determined as TMErelated genes Theunsupervised clustering analysis revealed two clusters were significantly related to immune score and T cells CD4memory fraction In addition two signatures were generated based on three and two TMErelated genesrespectively Both two signatures can significantly divide patients into low and highrisk groups and were validatedin two GEO datasets Afterward the risk score and metastatic status were identified as independent prognosticfactors for both OS and DFS and two nomograms were generated The Cindexes of OS nomogram and DFSnomogram were and respectivelyConclusion TME was associated with the prognosis of osteosarcoma patients Prognostic models based on TMErelated genes can effectively predict OS and DFS of osteosarcoma patientsKeywords Tumor microenvironment Osteosarcoma Prognosis Immune features NomogramBackgroundOsteosarcoma is the most common bone tumor especiallyin children and adolescents [] It was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group [] Currently surgery and Correspondence 407404159qqcom4Wenzhou Medical University Wenzhou ChinaFull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years However due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable[] Previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively [] Thereforeit is necessary to The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cHu BMC Cancer Page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsRecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified For example Chen [] reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients Moreover Huang [] discovered that dysregulated circRNAs serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microRNA In addition lncRNA [] microRNA [] and many clinical data [] were also identified asprognostic biomarkers for osteosarcoma patients However osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humansTherefore it is necessary to find accurate biomarkers forosteosarcomaIn recent years researchers have paid more and moreattention to the role of the tumor microenvironmentTME in malignant tumors The function of TME inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] More importantly Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data ESTIMATE an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in [] Based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [“] Generally the above research indicatedthat TME can serve as the prognostic biomarker in tumorsand many TMErelated genes were determined as the prognostic genes However the role of TME and TMErelatedgenes in osteosarcoma patients remains unclearIn the present study gene expression data and corresponding clinicopathologic data were obtained from TheTherapeutically Applicable Research to Generate EffectiveTreatments TARGET dataset Then the ESTIMATEalgorithm was performed to quantify the immune score ofosteosarcoma and the TMErelated genes were identifiedby the differential expression analysis Subsequently theprognostic value of TME and TMErelated genes weredetermined by a series of bioinformatics methodsMethodsGene expression datasetsLevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom TARGET dataset ocgcancergovprogramstarget accessed on Oct The correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus After data were extracted from the public domainthe ESTIMATE an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in R software version [] Meanwhile the messenger RNAmRNA expressionprofiles and clinical data ofincludingGSE21257 [] and GSE39055 [] were obtained fromthe Gene Expression Omnibus as external validationcohortstwo cohortsSurvival analysis and correlation analysisAfter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score The KaplanMeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival OS and diseasefreesurvival DFS between high and lowscore cohorts Inaddition the association between clinicopathologic dataand TME score was also studied MannWhitney signedrank test was performed to compare the differences ofimmune score between each clinical group All statisticalanalyses in the present study were performed using Rsoftware Except for the special instructions p value twoside was identified as statistically significantin the present studyDEGexpressed geneDifferentially expressed gene analysisDifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group The limma package in Rsoftware was used to perform the differential analysisand genes with log FC and adjusted pvalue qvalue were identified as DEGs []To further understand the function of DEGs identifiedin the present study Gene Ontology GOincludingbiological processes BP molecular functions MF andcellular componentsCC and Kyoto Encyclopedia ofGenes and Genomes KEGG analysis were performedby clusterProfiler package in R software []Evaluation of association with immune cellsTo further investigate the association between DEGs andimmune cells the CIBERSORT package was used toestimate the relative proportions of types of immunecells [] Meanwhile the œConsensusClusterPlus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping DEGs [] Cumulative distribution function CDF and relative change inarea under the CDF curve were used to determine theoptimal number of clusters k Then MannWhitney 0cHu BMC Cancer Page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters []Survival analysis of DEGsBased on the DEGs the univariate COX analysis was performed to determine the prognostic value of immunerelated genes Then the OSrelated genes were validatedin the GSE21257 dataset while the DFSrelated geneswere validated in the GSE39055 dataset Only genes successfully validated were selected for further analysis Afterward based on the validated genes the multivariate COXanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsThe risk score for each patient was calculated based onthe coefficient from the multivariate COX analysis and thecorresponding gene expression Meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score The survival receiver operating characteristic ROC curve was used to show the discrimination of signatures and the KaplanMeier survivalcurve with the logrank test was generated to show thedifferences of OS and DFS between high and lowriskgroups In addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature The KaplanMeier survivalcurve and survival ROC curve were generated to show thepredictive ability of the signature in the validation cohortDevelopment of a nomogram for osteosarcoma patientsNomogram is a tool to visualize the predictive model andconvenient for clinical practice Therefore we attemptedto develop a nomogram based on the TMErelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients Firstlythe univariateCOX analysis was performed to filter prognostic variableswhich will be further included in the multivariate COXanalysis Secondly based on independent prognostic variables two nomograms were established for predicting theOS and DFS respectively The Cindex was used to assessthe discriminatory performance of the nomogram whichrange from to [] A Cindex of means agreement by chance and a Cindex of represents perfectdiscriminatory performance The higher value of the Cindex the better performance of the nomogram is Furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsResultsImmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females The immunescore of the cohort range from ˆ’ to Tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group The survival analysis indicated thatpatients with higher immune score had a favorable OSand DFS Fig 1a and b After adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both OS and DFSFig 1a and b Inaddition the relationship between immune score and clinical features was also investigated However there was nosignificant relationship between immune score and clinicalvariables Supplementary Figure 1A1CDifferential expression analysisAccording to the median of the immune score patients were divided into highscore n and lowFig Association between immune score and prognosis in osteosarcoma patients a KaplanMeier survival analysis of overall survival for patientswith high vs low immune score b KaplanMeier survival analysis of diseasefree survival for patients with high vs low immune score 0cHu BMC Cancer Page of score group n There were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesFig 2a b and Supplementary Table To furtherunderstand the function of DEGs GO analysisand KEGG analysis were performed The top significant results of GO analysis among three types wereillustrated in Fig 2c Interestingly we can find that theresults of GO analysis are mostly associated with immunity which further verify that the immunerelated DEGsare associated with immune features In addition the results of KEGG also confirmed it Such as œPhagosomeœAutoimmune thyroid disease œAntigen processing andpresentation œB cell receptor signaling pathway œIntestinal immune network for IgA production œInflammatorybowel disease œPrimary immunodeficiency œTh1 andTh2 cell differentiation œTh17 cell differentiation œNatural killer cell mediated cytotoxicity and œNFˆ’kappa Bsignaling pathway Fig 2dconsensusunsupervisedEvaluation of DEGs and immune cellsTo further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns Based on the consensus matrix heat map patientswere clearly divided into two clustersFig 3a In additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined Fig 3bc The immune score betweentwo clusters was significantly different Fig 3d In additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot Fig 3e Interestinglywe can see that the T cells CD4 memory activated ofcluster is significantly higher than cluster Fig 5fPrognostic value of TMErelated genesPrevious studies indicated that TMErelated genes canserve as the prognostic biomarker for tumor patientsFig Differentially expressed genes with the immune score in osteosarcoma patients a Heatmap of significantly differentially expressed genesbased on immune score b The volcano figure to show the upregulated and downregulated genes c GO analysis of differentially expressedgenes d KEGG of differentially expressed genes GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes 0cHu BMC Cancer Page of Fig The immune landscape of the tumor microenvironment ac Unsupervised clustering of all samples based on the overlapping DEGs dComparison of immune score between two clusters e The distribution of types of immune cells in osteosarcoma patients f The comparisonof types of immune cells between clusters DEG Differentially expressed geneHence we performed the univariate COX analysis toidentify prognostic DEGs The results showed that and genes were identified as OS and DFSrelatedDEGs respectively Supplementary Table and Afterward five OSrelated genes were successfully validated inthe GSE21257 data set and five DFSrelated genes were successfully validated in the GSE39055 cohort Furthermoremultivariate COX analysis was performed and two prognostic signatures were generated for predicting the OS andDFS respectively The risk score for predicting the OS wasasrisk score FCGR2B0766 GFAP0702 MPP70387 In addition the risk score for predicting theDFS was as follows risk score CYP2S10574 ICAM3 The AUC values of OSrelated signature were follows 0cHu BMC Cancer Page of and in and 3year respectively Fig 4aand the AUC values of DFSrelated signature were and in and 3year respectively Fig 5aMoreover survival curves showed that patients in the highrisk group had worse OS and DFS compared with the lowrisk patients Figs 4b and 5b Heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients Figs 4ceand 5ce Then both signatures were validated in independent cohorts For OS signature the AUC values ofvalidation cohort were and at and3year Fig 4f For DFS signature the AUC values ofvalidation cohort were and at and3year Fig 5f Additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients Figs 4g and 5gHeat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsFigs 4hj and f 5hjDevelopment of a nomogram for osteosarcoma patientsTo generate a nomogram for clinical use the COX analysiswas performed to select the clinical prognostic variables InFig Establishment and validation of the prognostic model for overall survival based on significant DEGs a Receiver operating characteristiccurves of prognostic signature in the training cohort b The survival curve showed the different overall survival status between high and lowriskpatients c The heat map showed the expression of prognostic genes in the training cohort d The risk curve of each sample reordered by riskscore e The scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f Receiver operating characteristiccurves of prognostic signature in validation cohort g The survival curve showed the different overall survival status between high and lowriskpatients h The heat map showed the expression of prognostic genes in the validation cohort i The risk curve of each sample reordered by riskscore j The scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0cHu BMC Cancer Page of Fig Establishment and validation of the prognostic model for diseasefree survival based on significant DEGs a Receiver operatingcharacteristic curves of prognostic signature in the training cohort b The survival curve showed the different diseasefree status between highand lowrisk patients c The heat map showed the expression of prognostic genes in the training cohort d The risk curve of each samplereordered by risk score e The scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f Receiver operatingcharacteristic curves of prognostic signature in validation cohort g The survival curve showed the different diseasefree status between high andlowrisk patients h The heat map showed the expression of prognostic genes in the validation cohort i The risk curve of each sample reorderedby risk score j The scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate COX analysis risk score and metastatic status were identified as both OS and DFSrelated variablesFig 6a and e Afterward risk score and metastatic statuswere determined as both independent OS and DFSrelated variables in the multivariate COX analysis Fig 6band f Based on independent variables two nomogramswere established for predicting the OS and DFS in osteosarcoma patients respectively Fig 6c and g The Cindexvalues were and in OS nomogram and DFSnomogram respectively The results of Cindex mean thatboth two nomograms have good discrimination Meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve Fig 6dand h which indicated a good calibrationDiscussionThe relationship between TME and tumor have beenwidely studied in recent years In the present study ESTIMATE algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from TARGET database We confirmed that theTME is significantly associated with the prognosis ofosteosarcoma patientsInadditionfunctional enrichment analyses of TMErelated genes indicated that immunerelated processesincluding OS and DFS 0cHu BMC Cancer Page of Fig Nomograms based on the tumor microenvironment related genes for osteosarcoma patients a Univariate COX analysis of overall survivalrelated variables b Multivariate COX analysis of overall survivalrelated variables c Nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curveS of overall survival nomogram e Univariate COX analysis of diseasefree survivalrelated variables fMultivariate COX analysis of diseasefree survivalrelated variables g Nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curveS of diseasefree survival nomogramknown to contribute to tumor progression More importantly DEGs based on the TME were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the OS and DFS of osteosarcoma patientsrespectivelyIn recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the TME and the ESTIMATE algorithm is oneof the most important quantitative tools for this researchfield Based on the ESTIMATE algorithm the association between the prognosis and TME has been initially 0cHu BMC Cancer Page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ “] However previousstudies indicated that TME scores serve as a differentrole in different tumors For example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ “] However for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] Therefore we can find great heterogeneity among different tumors from the perspectiveof TME For osteosarcoma patients the present studyindicated that patients with higher immune score had abetter OS and DFS Hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionIn our research TMErelated genes were identified by comparing the highscore and lowscore osteosarcoma patients The functional enrichment includingGO and KEGG analyses showed that TMErelated geneswere mainly involved in the immune features such asregulation of leukocyte activation MHC protein complex MHC protein and complex binding More importantly the unsupervised cluster analysis based on DEGswas performed and all patients were divided into twoclusters Immune score and T cell CD4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between DEGs and immune featuresDue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantThe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients Emilie etal []performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma Recently the prognostic signature based onTMErelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients [] Compared with the study performedby Zhang [] we think that our research have someadvantages Firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts Secondlythe outcome of DFS was not reported in the previousstudy As reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]Hence the DFS nomogram can improve the managementof osteosarcoma patients Finally two nomograms incorporated TMErelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsIn our research five genes were incorporated into thefinal prognostic signatures FCGR2B GFAP and MPP7were identified and validated as OSrelated biomarkerswhile CYP2S1 and ICAM3 were DFSrelated biomarkersThe role of these genes in tumor prognosis had beenwidely reported in previous studies [“] FCGR2Bhas been confirmed as an immunerelated gene previously [] Although the relationship between FCGR2Band prognosis in sarcoma patients had not been reported the prognostic value of FCGR2B had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] In addition NewM etal [] demonstrated that MPP7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomaCYP2S1 described as Cytochrome P450 Family Subfamily S Member was reported significantly associatedwith colorectal cancer In primary colorectal cancerCYP2S1 was present at a significantly higher level ofintensity compared with normal colon [] More importantly the presence of strong CYP2S1 immunoreactivity was associated with poor prognosis [] The roleof ICAM3 in cancer was also widely reported in published studies and the Akt pathway plays an importantrole in the impact of ICAM3 on tumors YG Kim etal[] reported that ICAM3 can induce the proliferationof cancer cells through the PI3KAkt pathway Additionally JK Park etal showed that the ICAM3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing MMP2 andMMP9 via Akt pathway [] showed that the ICAM3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingMMP2 and MMP9 via Akt pathwayAlthough the role of TME and TMErelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated Firstly the treatmentinformation cannot be obtained from the TARGET database which may influence the prognosis of osteosarcomapatients Secondly two nomograms were generated andshowed good performance in our study However externalvalidation by a large cohort is needed Thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear Finally inthe training cohort and DEGs were identified asOS and DFSrelated DEGs respectively However onlyfive OS and five DFSrelated genes were identified in thevalidation cohort The different age structures smaller 0cHu BMC Cancer Page of sample sizes and the platform covering only part of thegenes may contribute to this resultReceived February Accepted July ConclusionIn conclusion TME plays an important role in osteosarcoma patients and related with the progression of thetumor Moreover TMErelated genes can serve as prognostic biomarkers in osteosarcoma patients Howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studySupplementary informationSupplementary information accompanies this paper at doi101186s12885020072162Additional file Additional file Additional file Additional file AbbreviationsTME Tumor microenvironment DEG Differentially expressed genesOS Overall survival DFS Diseasesfree survival ROC Receiver characteristiccurve ESTIMATE Estimation of STromal and Immune cells in MAlignantTumor tissues using Expression data TARGET Therapeutically ApplicableResearch to Generate Effective Treatments GO Gene Ontology BP Biologicalprocesses MF Molecular functions CC Cellular components KEGG KyotoEncyclopedia of Genes and Genomes CDF Cumulative distribution functionAcknowledgementsNoneAuthors™ contributionsC H L Y Sq T C L and Yh W conceived of and designed the study C H R Sand C L performed literature search R S L Y and B C generated the figuresand tables L Y Hl R X Y and Jy L analyzed the data C H wrote themanuscript and Sq T and L Y critically reviewed the manuscript L Ysupervised the research All authors have read and approved the manuscriptFundingWe received no external funding for this studyAvailability of data and materialsThe data of this study are from TARGET and GEO databaseEthics approval and consent to participateThe research didn™t involve animal experiments and human specimens noethics related issuesConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Joint Surgery the Affiliated Hospital of Qingdao UniversityQingdao China 2Department of Medical Oncology the First Hospital ofChina Medical University Shenyang China 3Department of Nursing Sir RunRun Shaw Hospital Affiliated to Zhejiang University Hangzhou China4Wenzhou Medical University Wenzhou ChinaReferencesJaffe N Bruland OS Bielack S Pediatric and adolescent osteosarcoma vol New York Springer Science Business Media Vander RG Osteosarcoma and its variants Orthopedic Clin North Am “Biermann JS Adkins D Benjamin R Brigman B Chow W Conrad EU 3rdFrassica D Frassica FJ Gee S Healey JH Bone cancer J Natl ComprCancer Netw “Simpson S Dunning MD de Brot S GrauRoma L Mongan NP Rutland CSComparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics Acta Vet Scand Chen X Cates JM Du YC Jain A Jung SY Li XN Hicks JM Man TKMislocalized cytoplasmic p27 activates PAK1mediated metastasis and is aprognostic factor in osteosarcoma Mol Oncol “Huang X Yang W Zhang Z Shao Z Dysregulated circRNAs serve as prognosticand diagnostic markers in osteosarcoma by sponging microRNA to regulatethe downstream signaling pathway J Cell Biochem “Liu M Yang P Mao G Deng J Peng G Ning X Yang H Sun H Long noncoding RNA MALAT1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis Int J Surg “Xu K Xiong W Zhao S Wang B MicroRNA106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma Oncol Lett “Zheng W Huang Y Chen H Wang N Xiao W Liang Y Jiang X Su W WenS Nomogram application to predict overall and cancerspecific survival inosteosarcoma Cancer Manag Res Kahlert C Kalluri R Exosomes in tumor microenvironment influence cancerprogression and metastasis J Mol Med “ Binnewies M Roberts EW Kersten K Chan V Fearon DF Merad M CoussensLM Gabrilovich DI OstrandRosenberg S Hedrick CC Understanding thetumor immune microenvironment TIME for effective therapy Nat Med“ Yoshihara K Shahmoradgoli M Martínez E Vegesna R Kim H TorresGarcia WTreviño V Shen H Laird PW Levine DA Inferring tumour purity and stromaland immune cell admixture from expression data Nat Commun Yang S Liu T Nan H Wang Y Chen H Zhang X Zhang Y Shen B Qian PXu S Comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma J Cell Physiol “ Deng Z Wang J Xu B Jin Z Wu G Zeng J Peng M Guo Y Wen Z MiningTCGA database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma Biomed Res Int Zhao K Yang H Kang H Wu A Identification of key genes in thyroid Cancermicroenvironment Med Sci Monit Xu WH Xu Y Wang J Wan FN Wang HK Cao DL Shi GH Qu YYZhang HL Ye DW Prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment AgingAlbany NY Chen B Chen W Jin J Wang X Cao Y He Y Data Mining of PrognosticMicroenvironmentRelated Genes in clear cell renal cell carcinoma a studywith TCGA database Dis Markers Li X Gao Y Xu Z Zhang Z Zheng Y Qi F Identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods Cancer Med “ Pan XB Lu Y Huang JL Long Y Yao DS Prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma Aging Albany NY Wang H Wu X Chen Y Stromalimmune scorebased gene signature aprognosis stratification tool in gastric Cancer F

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evaluation of biochemical and hematological parameters in adults with Down syndromeDavid de Gonzalo‘calvo123 Isabel Barroeta45 Madalina Nicoleta Nan6 Jos Rives6 Diana Garzn45 Mar­a Carmona‘Iragui457 Bessy Benejam457 Laura Videla457 Susana fern¡ndez7 Miren Altuna45 S­lvia Valldeneu45 Rafael Blesa45 Alberto Lle45 Francisco Blanco‘Vaca689 Juan Fortea457 Mireia Tondo6Down syndrome DS is the most common worldwide cause of intellectual disability of genetic origin and the most common chromosomal disorder affecting live‘born infants In addition to intellectual disability individuals with DS have other comorbidities and complex medical conditions The increase in the life expectancy of patients with DS requires expanding the knowledge about their clinical characteristics and related laboratory parameters Several studies exploring laboratory tests in DS patients exist but their focus is limited to specific areas of metabolism Therefore our main goal was to describe the biochemical and hematological findings in a DS cohort and to compare the values to those of a control population A total of DS individuals and control subjects were enrolled DS individuals had a higher frequency of several clinical conditions compared to control individuals and presented with significant differences with respect to the controls in both biochemical and hematological parameters We found age‘ and sex‘related differences in several of the parameters A good understanding of the differences in our cohort might be of aid in the clinical follow‘up of adults with DS especially considering that the lifespan of DS individuals may reach years of age in developed countriesAbbreviationsAD AF ALT AST B12 CKDEPI DS ESR FT4 eGFR GGT HbA1c Alzheimer™s disease Alkaline phosphatase Alanine aminotransferase Aspartate aminotransferase Vitamin B12 Chronic kidney disease epidemiology collaboration Down syndrome Erythrocyte sedimentation rate Free thyroxine Estimated glomerular filtration rate Gammaglutamyl transferase Glycated hemoglobin1Biomedical Research Institute Sant Pau IIB Sant Pau Barcelona Spain 2Institute of Biomedical Research of Barcelona IIBB Spanish National Research Council CSIC Barcelona Spain 3Translational Research in Respiratory Medicine University Hospital Arnau de Vilanova and Santa Maria IRBLleida Lleida Spain 4Sant Pau Memory Unit Department of Neurology Hospital de La Santa Creu i Sant Pau Biomedical Research Institute IIB Sant Pau Universitat Aut²noma de Barcelona Barcelona Spain 5Center of Biomedical Investigation Network for Neurodegenerative Diseases CIBERNED Madrid Spain 6Department of Biochemistry Hospital de La Santa Creu i Sant Pau Biomedical Research Institute IIB Sant Pau CSant Quint­ Barcelona Spain 7Barcelona Down Medical Center Fundaci Catalana de S­ndrome de Down Barcelona Spain 8Center of Biomedical Investigation Network for Diabetes and Metabolic Diseases CIBERDEM Madrid Spain 9Department of Biochemistry and Molecular Biology Universitat Aut²noma de Barcelona Barcelona Spain email mtondosantpaucatScientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cHDLc LDLc MCH MCHC MCV MDRD4 MPV K RDW Na TG TSH Highdensity lipoprotein cholesterol Lowdensity lipoprotein cholesterol Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Mean corpuscular volume Modification of diet in renal disease Mean platelet volume Potassium Red blood cell distribution width Sodium Triglycerides Thyroid stimulating hormoneDown syndrome DS is the most common worldwide cause of intellectual disability of genetic origin and the most common chromosomal disorder affecting liveborn infants with an estimated birth prevalence of per live births1“ Despite the shorter life expectancy when compared to healthy subjects and adults with other causes of intellectual disability4 there has been a progressive increase in the life expectancy of patients with DS in recent decades currently reaching nearly years5 This fact has increased the need to expand the knowledge about the clinical characteristics of DS individuals and the health problems differentiating them from both pediatric and adult populations6 DS is associated with a distinct phenotype involving many body systems In addition to intellectual disability individuals with DS present with a high number of comorbidities and complex medical conditions whose frequencies are modified throughout the lifespan of the individuals7 The increase in life expectancy has led to a higher prevalence of agerelated pathologies including premature Alzheimer™s disease AD8Since optimal medical management is associated with improved quality of life and functioning among persons with DS910 medical professionals including pediatricians and other physicians should closely supervise this population throughout their lifespan and evaluate their laboratory results Previous investigations in DS cohorts have focused on select biochemical parameters such as uric acid and thyroid function biomarkers bone mineral density nutritional zinc status gonadal and endocrine function and glucose and lipid metabolism parameters11“ However no previous work has described a comprehensive panel of biochemical and hematological parameters in a large cohort of DS patientsOur hypothesis is that a thorough analysis of the biochemical and hematological parameters will provide a basis to establish whether commonly observed alterations in DS individuals are intrinsic of the disease or have clinical implications similarly as for the general population Therefore our goals were to describe the biochemical and hematological findings in our DS cohort and to compare the values to those of a control populationMaterial and methodsStudy participants This was a singlecenter descriptive study of adults with DS recruited at Barcelona Down Medical Center Fundaci Catalana S­ndrome de Down and Hospital de la Santa Creu i Sant Pau Barcelona in Catalonia Spain according to a populationbased health plan to screen for neurological comorbidities1718 The Down Medical Center provides medical care specifically for individuals with DS and possesses over medical records more than of the estimated Down syndrome population in Catalonia therefore it reflects the population with DS in our geographic area The period of patient recruitment for this study was February to June In adults with DS ‰¥ a0years a biochemical and hematological analysis was performed as part of their annual health plan visit A total of patients were enrolled in the study Six further patients were ultimately excluded for presenting with conditions unrelated to DS according to their medical records patients with hepatitis C patient with hepatitis B and patient with breast cancer resulting in a final total number of DS individuals included age range “ a0years A total of healthy control participants in the same age range “ a0years were enrolled in the study Volunteers were recruited from the SPIN Sant Pau Initiative on Neurodegeneration cohort santp aumem oryun itcomourresea rchspincohor t or social media SantPauMemory Further details on the clinical protocol of the SPIN cohort can be found elsewhere19Based on current guidelines1720 associated clinical conditions were obtained through a systematic review of the medical records including the following history of arterial hypertension dyslipidemia diabetes mellitus congenital heart disease gastrointestinal pathology dermatological pathology bone pathology hypothyroidism hearing problems otolaryngology pathology ophthalmological pathology psychiatric pathology epilepsy and Alzheimer™s disease Treatment data with a special focus on the treatment of hypothyroidism were also collectedBiochemical and hematological data Analyzed biochemical and hematological parameters were selected according to a defined laboratory blood profile as recommended in the guidelines for management of patients with DS1720Blood collection and processing were performed in accordance with the Standard Operating Procedures for Serum and Plasma Collection from the Early Detection Research Network EDRN Consensus Statement and Standard Operating Procedure Integration Working Group21 Blood samples were collected by venipuncture after an overnight fastWhole blood samples were collected in VACUTAINER tubes and fractionated by centrifugation at a0g for a0min at room temperature to obtain serum Serum was aliquoted into a0mL tubes and the following parameters were measured according to standard commercially available assays adapted to an Architect C4000 Abbott Diagnostics USA using automated procedures thyroid stimulating hormone TSH free thyroxine FT4 Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cAge yearsMalefemaleArterial hypertensionDyslipidemiaDiabetes mellitusCongenital heart diseaseGastrointestinal pathologyDermatological pathologyBone pathologyHypothyroidismTreatment for hypothyroidismHearing problemsOtolaryngology pathologyOphtalmological pathologyPsychiatric pathologyEpilepsyAlzheimer™s disease “ Controln Median P25“P75n “ Down syndromenMedian P25P754n “ pvalue Table Characteristics of the Study Population Data are presented as frequencies percentages for categorical variables Continuous variables are presented as median interquartile range Differences between groups were analyzed using Wilcoxon ranksum test or Fisher™s exact testsodium Na potassium K glucose urea creatinine total bilirubin triglycerides TG total cholesterol aspartate aminotransferase AST alanine aminotransferase ALT alkaline phosphatase AF gammaglutamyl transferase GGT total proteins vitamin B12 and folate The estimated glomerular filtration rate eGFR was calculated according to the MDRD4 Modification of Diet in Renal Disease and CKDEPI Chronic Kidney Disease Epidemiology Collaboration formulasWhole blood samples in EDTAK3 were also obtained for determining blood cell count and indices The tubes were immediately inverted times to mix the anticoagulant additive with blood The blood was processed within a0h of extraction Using the impedance channel of the automated hematology analyzer Sysmex XE2100 Roche Diagnostics Kobe Japan the following parameters were determined red blood cell count RBC white blood cell count WBC platelet count hemoglobin hematocrit mean volume MCV mean corpuscular hemoglobin concentration MCHC mean corpuscular hemoglobin MCH red blood cell distribution width RDW and mean platelet volume MPV The erythrocyte sedimentation rate ESR was calculated with a VES cube Sysmex Analyzer Roche Diagnostics Kobe JapanValues were compared to normal reference ranges used in our laboratory established in a healthy population from our geographical area according to standardized guides22Statistical analysis Descriptive statistics were used to summarize the characteristics of the study population Data are presented as medians [25th percentile P25“75th percentile P75] for continuous variables and as frequencies percentages for categorical variables Data normality was analyzed using the Kolmogorov“Smirnov test Continuous variables were compared between groups using the Wilcoxon ranksum test ANCOVA models adjusted for age and sex were used to compare continuous variables across the study groups Variables were logtransformed to achieve a normal distribution For clarity the original values are shown Categorical variables were compared between groups using Fisher™s exact test Spearman™s rho coefficient was used to assess the correlation between continuous variables The statistical software package R wwwrproje ct was used for statistical analyses A Pvalue was considered statistically significantEthical aspects The study was approved by the Sant Pau Ethics Committee following the standards for medical research in humans recommended by the Declaration of Helsinki and in accordance with Spanish legislation for research in people with intellectual disabilities All participants or their legally authorized representatives gave written informed consent before enrolment in accordance with the guidelines of the local ethics committeeResultsStudy cohort characteristics We enrolled a total of individuals with DS males and females with a median age of “ years and control subjects males and females with a median age of “ years The clinical features of the DS and control populations are listed in Table a0 The frequency of the following clinical conditions was significantly higher in the DS group than in the control group history of congenital heart disease gastrointestinal pathology dermatologiScientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0ccal pathology bone pathology hypothyroidism hearing problems otolaryngology pathology ophthalmological pathology epilepsy and AD No differences were observed in the frequency of diabetes mellitus or psychiatric pathology for either group DS individuals presented with a lower frequency of arterial hypertension and dyslipidemia compared to the control group See Table a0 for further details on the cohort characteristicsBiochemical and hematological parameters in patients with Down syndrome We performed a detailed biochemical and hematological analysis of the DS cohort and compared the profiles obtained with our control population The reference values of the studied parameters the number and percentage of patients out of range and the median P25“P75 of the whole study population are shown in Table a0 Seventythree percent of the studied hematological parameters and of the studied biochemical parameters were significantly different between the DS individuals and the control population The DS individuals presented with higher TSH urea creatinine AST hemoglobin hematocrit MCV ESR MCH and RDW values and lower TG total cholesterol folate eGFR MPV and WBC values These differences remained significant or close to signification after adjusting for confounding factors such as age and sex Statistical differences for RBC and MCHC were observed after adjustment An additional analysis to evaluate the impact of hypothyroidism treatment on TSH was performedNo differences were observed for TSH between both studied groups treated DS individuals “ vs untreated DS individuals “ Pvalue For categorical variables the percentage of DS individuals out of range for some parameters was also statistically significant compared to the control population Parameters with a higher percentage of values out of range in the DS group were TSH urea creatinine total proteins RBC MCV ESR MCH and WBC whereas those with a lower percentage of values out of range were K TG total cholesterol and ASTThe differences in the biochemical and hematological parameters and the number and percentage of patients out of range between DS individuals and the control population according to sex are displayed in Supplemental Tables a0 and For the female DS cohort parameters with significantly higher values were TSH urea creatinine AST hemoglobin hematocrit MCV ESR MCH and RDW whereas those with significantly lower values were TG total cholesterol GGT eGFR RBC MPV and WBC For categorical variables parameters with significantly higher percentages of values out of range were TSH creatinine total proteins MCV ESR MCH and WBC whereas those with a significantly lower percentage of values out of range were total cholesterol and B12 Supplemental Table a0 For the male DS cohort parameters with significantly higher values were TSH hemoglobin hematocrit MCV ESR MCH and RDW whereas those with significantly lower values were TG total cholesterol eGFR MPV and WBC Regarding categorical variables parameters with significantly higher percentages of values out of range were TSH ESR and MCH whereas those with significantly lower percentages of values out of range were K TG total cholesterol and GGT Supplemental Table a0The differences in the biochemical and hematological parameters between males and females as well as the frequency and percentage of patients out of range in the control and DS groups are displayed in Supplemental Table a0 and Table a0 respectively For the control group parameters with significantly higher values in the male subgroup were K creatinine TG ALT hemoglobin hematocrit RBC and MCHC whereas those with significantly lower values were AF eGFR and ESR Among the categorical variables K had a significantly higher percentage of values out of range in the male subgroup and ESR had a significantly lower percentage of values out of range Supplemental Table a0 For the DS cohort parameters with significantly higher values in the male subgroup were creatinine total bilirubin TG ALT GGT hemoglobin hematocrit RBC MCHC and WBC whereas those with significantly lower values were folate MCV ESR RDW platelet count and MPV Regarding categorical variables parameters with significantly higher percentages of values out of range in the male subgroup were total bilirubin B12 RBC and MPV whereas those with significantly lower percentages of values out of range were MCV ESR and MCHC Table a0The correlation between the biochemical and hematological data with age was also explored in both study groups As shown in Table a0 for the control population urea creatinine total cholesterol and AST showed a significant positive correlation with age while eGFR showed a significant negative correlation For the DS population Na urea creatinine TG total cholesterol AST AF MCV ESR MCH and RDW showed a significant positive correlation with age while eGFR ALT B12 hemoglobin hematocrit RBC MCHC and platelet count showed a significant negative correlationDiscussionThe present study evaluated several biochemical and hematological parameters in a large sample of adults with DS Several studies exploring laboratory tests in DS patients exist but their focus is limited to specific areas of metabolism11“ DS is among the most complex genetic conditions compatible with life characterized by accelerated aging and affecting gene expression beyond chromosome The sheer number of affected genes and epigenetic changes suggests that numerous pathways of human metabolism are altered and subsequently might be reflected in laboratory test parameters Here we performed a comprehensive approach by analyzing parameters related to different physiological mechanisms We found significant differences with respect to nontrisomic controls in both biochemical and hematological parameters even after adjusting for potential confounding factors Furthermore we found age and sexrelated differences in several of the parameters The fact that women with DS experience menopause earlier than healthy women24 may explain some of these sexrelated differencesClinically and as previously described48925 our DS cohort presented with a higher incidence of congenital heart disease gastrointestinal pathology dermatological pathology bone pathology hypothyroidism otolaryngology pathology ophthalmological pathology epilepsy and AD than the control population Arterial Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cVariableBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT UL““““ ‰¤ a0years “ a0years “Females Males Females Males Females Males Females “Males “Females Males “““Total proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC — 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count — 109LMPV fLWBC — 109LFemales “Males “Females “Males “Females “Males “““““““““nn OOR “ “ “ “ “ “ “ “ “ “n OOR “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ControlDown syndromeReference valuesMedian P25“P75nMedian P25“P75pvalue categoricalpvalue continuouspvalue continuous adjusted Table Biochemical and hematological parameters in the control group and the cohort of patients with Down Syndrome Differences between groups were analyzed using Wilcoxon Ranksum test ANCOVA models adjusted for age and sex or the Fisher™s exact test OOR out of range NA not applicablehypertension and dyslipidemia were less prevalent whereas no difference was observed regarding the diabetes mellitus incidence as discussed belowWith respect to laboratory studies the hematological profile was largely altered in DS individuals when compared to the control population Of note significant differences were found for almost all the hematological parameters when comparing males and females suggesting the need to consider sex when evaluating the hematological profile in a DS individual It is well known that trisomy impacts hematopoietic cell biology through multiple and complex pathways In adults the metabolic and redox derangements observed in the RBCs from individuals with DS have been previously linked to alterations in cell survival and size in particular macrocytosis26 Different studies have also proposed that the additional copy of chromosome has a profound impact on fetal hematopoiesis which ultimately impacts the function and number of hematopoietic lineages27“ Additionally between and of newborn infants with DS develop transient myeloproliferative disorder32“ Although the disease usually resolves without treatment in the first few months of life it is estimated that “ of individuals with transient myeloproliferative disorder will go on to develop subsequent leukemia3536 Finally the fact that folate concentrations are significantly lower in DS individuals matches the observed hematological alterations Taken together these impaired hematological parameters suggest the existence of abnormalities Scientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cFemalenVariableBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT ULTotal proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC — 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count — 109LMPV fLWBC — 109L n OOR Median P25“P75n OOR Median P25“P75pvalue categoricalpvalue continuousMalen “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ Table Differences between sex in the Down syndrome group Differences between groups were analyzed using Wilcoxon Ranksum test or the Fisher™s exact test OOR out of range NA not applicablein hematopoiesis and provide information on how an extra copy of chromosome may lead to phenotypic consequencesConcerning the biochemical profile our results support the findings from previous independent studies We showed that of our DS individuals presented with values out of range for TSH level Of those out of were treated for hypothyroidism Impaired TSH and FT4 levels have been largely described in DS populations37 Moreover subclinical hypothyroidism in children with DS is an abundantly common occurrence with a prevalence of approximately and has been attributed to the dysregulation of the hypothalamicpituitarythyroid axis37 Regarding urea metabolism of our DS individuals presented with a high urea concentration which may be due to impaired renal function among other causes Indeed and as previously reported39 almost of our DS individuals also presented with impaired creatinine values Serum creatinine is the most reliable parameter for detecting kidney damage due to its high diagnostic specificity From its concentration and based on formulas in which age sex and weight are taken into account it is possible to estimate the glomerular filtration rate eGFR Our DS cohort also presented with a lower eGFR which is in agreement with a previous study exploring renal disease in DS individuals40 Despite the significantly altered parameters related to renal function our DS individuals presented with a very low frequency of arterial hypertensionConcerning the lipid profile we found significantly lower total cholesterol and TG concentrations in DS individuals compared to the control population It would have been interesting to study the fractioned forms of cholesterol together with their apolipoprotein concentrations however because the current study was not designed to answer questions regarding lipid metabolism lowdensity lipoprotein cholesterol LDLc and highdensity lipoprotein cholesterol HDLc were not measured Several works measuring circulating total cholesterol Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cControlnSpearman™s rhoDown syndromenSpearman™s rhopvaluepvalueBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT ULTotal Proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC — 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count — 109LMPV fLWBC — 109Lˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ Table Correlations between biochemical and hematological parameters and age NA not applicableLDLc HDLc and TG concentrations in the DS population exist However they report contradictory results and prevent firm conclusions from being drawn Some studies have reported an unfavorable41“ or favorable lipid profile46 However most of the studies reported no change in serum TC LDLc or HDLc in individuals with DS compared to a control group or to population norms414547“ In our study these lower total cholesterol and TG concentrations may have translated into a significantly lower prevalence of hyperlipidemia in DS individuals It has been described that DS individuals may be protected against atherosclerosis4752“ leading to a low incidence of cardiovascular events53 However a work carried out with individuals with DS found that they were at high risk of cerebrovascular events but a lower risk of coronary events in males55 Therefore risk of major cerebrovascular events in people with DS should not be ruled out Concerning diabetes mellitus a similar incidence of type diabetes mellitus50 and a higher incidence of type diabetes mellitus has been described for individuals with DS56 We found no difference in type diabetes mellitus frequency among our DS and control populations as previously described in a different study16 In regard to arterial hypertension prevalence our results are in line with numerous studies that have described a lower incidence of this condition in DS individuals50515758 Despite these observations cholesterol fractioned forms and glycated hemoglobin HbA1c concentrations were not measured making it difficult to draw conclusions regarding dyslipidemia and diabetes mellitus in our cohort Yet an increased degree of hypolipidemia should not be ruled out Overall future studies elucidating the mechanisms behind the low cholesterol and TG concentrations and lower prevalence of arterial hypertension observed in our DS cohort should be performedIt is important to emphasize that our main goal was to help determining if the observed biochemical and hematological alterations have direct clinical implications for DS individuals While the altered biochemical and hematological profiles may be developmental features ie a consequence of the specific genetic characteristics of individuals with DS or the result of accelerated aging it should be recalled that they may also be reflecting comorbidities or the use of medication From a clinical standpoint to elucidate if the observed differences are consequence of concomitant conditions or features of the syndrome itself could be of help in the management of DS individuals Unfortunately due to the design of our study these questions remain unanswered Future Scientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cstudies focusing on specific areas of metabolism of DS individuals with different comorbidities could shed some light on this matterOur study has several strengths We collected relevant clinical biochemical and hematological data in a large DS cohort and performed a systematic analysis The fact that our controls were chosen from a healthy background broadens the actual differences and strengthens the present results Ultimately according to the wide inclusion criteria and the broad range of represented ages we believe that the results from our study may help clinicians when interpreting laboratory analyses in DS individuals Some limitations should also be taken into account The control and DS populations were not strictly age and sex matched and the control group had a reduced number of males when compared to females Nonetheless both populations were within the same age range and additional analysis including adjustment for age and sex were performed Furthermore despite our large cohort of DS individuals the number was still not sufficient to perform statistical analysis stratification according to the observed clinical conditions Moreover and as stated previously some of the observed biochemical andor hematological alterations may have been a consequence of the use of drugs for the treatment of other comorbidities Finally our defined clinical biochemical and hematological profiles were somehow general and unable to cover all the possible comorbidities present in DS individualsIn conclusion adults with DS show a specific profile of biochemical and hematological parameters A good understanding of the differences in our cohort with those in the general population might aid in the clinical followup of adults with DS especially considering that the life span of DS individuals can now reach a0years of age in developed countriesReceived March Accepted July References Parker S E et al Updated national birth prevalence estimates for selected birth defects in the United States “ Birth Canfield M A et al National estimates and raceethnicspecific variation of selected birth defects in the United States “ Defects Res A Clin Mol Teratol “ Birth Defects Res A Clin Mol Teratol “ Besser L M Shin M Kucik J E Correa A Prevalence of down syndrome among children and adolescents in metropolitan Atlanta Birth Defects Res A Clin Mol Teratol “ Yang Q Rasmussen S A Friedman J M Mortality associated with Down™s syndrome in the USA from to A populationbased study Lancet “ Bittles A H Glasson E J Clinical social and ethical implications of changing life expectancy in Down syndrome Dev Med Child Neurol “ Morris J K Alberman E Trends in Down™s syndrome live births and antenatal diagnoses in England and Wales from to Analysis of data from the National Down Syndrome Cytogenetic Register BMJ b3794 Startin C M et al Health comorbidities and cognitive abilities across the lifespan in Down syndrome J Neurodev Disord “ Hithersay R et al Association of dementia with mortality among adults with Down syndrome older than years JAMA Neurol Bull M J Health supervision for children with Down syndrome Pediatrics “ Roizen N J Patterson D Down™s syndrome Lancet “ Hawli Y Nasrallah M ElHajj Fuleihan G Endocrine and musculoskeletal abnormalities in patients with Down syndrome Nat Rev Endocrinol “ Sakadamis A Angelopoulou N Matziari C Papameletiou V Souftas V Bone mass gonadal function and biochemical assessment in young men with trisomy Eur J Obstet Gynecol Reprod Biol “ Niegawa T et al Evaluation of uric acid levels thyroid function and anthropometric parameters in Japanese children with Down syndrome J Clin Biochem Nutr “ Costa R et al Bone mineral density distribution curves in Spanish adults with Down syndrome J Clin

Thyroid_Cancer

Oral squamous cell carcinoma OSCC is a common kind of squamous cell carcinoma of the head and neck which is a threat to public health Long noncoding RNAs lncRNAs are associated with the development of various diseases including cancers LncRNA titin antisense RNA TTN‘AS1 is known as a crucial regulatory factor in several cancers Nevertheless the specific functions of TTN‘AS1 in OSCC remains obscureMethods The expression of TTN‘AS1 in OSCC samples or cells was analyzed through qRT‘PCR Colony formation assay EdU assay flow cytometry assay TUNEL assay and wound healing assay were conducted to estimate the func‘tions of TTN‘AS1 in OSCC cells RIP and luciferase reporter assays were utilized to detect the interaction between TTN‘AS1 and miR‘‘3p as well as between miR‘‘3p and NFAT5Results TTN‘AS1 expression was stronger in OSCC cells Knockdown of TTN‘AS1 effectively restrained cell prolifera‘tion and migration but had inductive role in apoptosis Moreover TTN‘AS1 could function as the miR‘‘3p sponge in OSCC and miR‘‘3p exerted the inhibitory functions on OSCC cell growth In addition NFAT5 was proven as the target of miR‘‘3p Rescue assay indicated that overexpressing NFAT5 could reverse the inhibitory function of TTN‘AS1 depletion on cell growthConclusion lncRNA TTN‘AS1 contributed to the progression of OSCC via miR‘‘3pNFAT5 axisKeywords TTN‘AS1 miR‘‘3p NFAT5 Oral squamous cell carcinomaBackgroundOral squamous cell carcinoma OSCC is one of the commonest squamous cell carcinomas occurs in the head and neck It ranks sixth in occurrence and had a high mortality rate [ ] According to many years of investigation and research the pathogenesis of OSCC is related to the internal factors such as drinking and smoking but its specific pathogenesis is still unclear [ ] Although the surgery for OSCC is effective the situation for the overall survival of OSCC patients is still unfavorable [ ] Thus Correspondence fusuwei2009163comDepartment of Stomatology Henan Provincial People™s Hospital People™s Hospital of Zhengzhou University No7 Weiwu Road Zhengzhou Henan Chinaindepth study of the potential molecular mechanisms of OSCC is of great significance for developing new therapeutic strategiesLong noncoding RNAs lncRNAs are classified as the subgroup member of noncoding RNAs family with over nucleotides in length which are not able to encode proteins [ ] Recently lncRNAs are confirmed to involve in different cell progression such as cell proliferation and cell apoptosis Moreover the crucial functions of lncRNAs in the occurrence and development of assorted cancers have also been reported through a flow of researches [ ] Different kind of lncRNAs exerted different functions in cancers For example PVT1 accelerated esophageal carcinoma cell migration and invasion via sponging miR145 and regulating FSCN1 [] The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cFu a0et a0al Cancer Cell Int Page of SARCC alters he androgen receptormiRNA1433p signals thereby suppresses the progression of renal cell carcinoma [] And GAPLINC facilitated gastric cancer cell growth through serving as a sponge of miR378 to regulate MAPK1 [] Titin antisense RNA TTNAS1 is a novel lncRNA that takes part in the regulation of cancer development in accordance with existing researches For illustration TTNAS1 with high expression in lung adenocarcinoma cells can expedite cellular functions of lung adenocarcinoma through serving as a sponge of miR1425p to regulate CDK5 [] Nevertheless its specific function of TTNAS1 in OSCC remains unclearHere we selected TTNAS1 as the object of our research and investigated the regulatory mechanisms and functions in OSCCMethodsTissues samplesPaired tissues adjacent normal and tumor were collected from patients with OSCC who were diagnosed at Henan Provincial People™s Hospital Patients participated in this study didn™t receive any kind of therapy before surgery All patients enrolled in this study had signed informed consent This study received the approval of the Ethics Committee of Henan Provincial People™s Hospital Samples were stored at ˆ’ a0 °C until useCell linesHuman normal squamous epithelial cell line NOK obtained from Shanghai Honsun Biological Technology CoLtd Shanghai China human tongue squamous carcinoma cell lines including SCC4 SCC9 CAL27 procured from ATCC Manassas VA USA and BICR cell obtained from European Collection of Authenticated Cell Cultures ECACC UK were used in current study NOK cell was cultured in DMEM Gibco Rockville MD USA with antibiotics and FBS Gibco CAL27 cell was cultured in DMEM containing FBS SCC4 cell was cultured in DMEM F12 Medium containing a0ngml hydrocortisone and FBS SCC9 cell was cultured in DMEM F12 Medium containing a0mM a0lglutamine a0gL sodium bicarbonate a0mM HEPES a0 mM sodium pyruvate supplemented with a0 ngml hydrocortisone and FBS BICR16 cell was cultured in DMEM with 500ugml G418 and FBS Cell culture was conducted under a condition with CO2 and a0°CTotal RNA extraction and a0qRT‘PCRTRIzol Reagent Invitrogen Carlsbad CA was responsible for total RNA extraction from samples or cells Afterwards RNA samples were converted into cDNA Japan PowerUp„¢ SYBR® Green Master Mix Life Techby employing Reverse Transcriptase Kit Takara Shiga nologies Grand Island NY USA was utilized for PCR analysis [] After amplification ˆ’ΔΔCt method was applied to quantify PCR products U6 snRNA or GAPDH was used as the internal control for lncRNA mRNA or miRNA All primers used in this experiments were provided in Additional file a0 Table a0 S1 Each samples were assayed for more than triplicateTransfectionsThe shRNAs designed for TTNAS1 or NFAT5 and nonspecific shRNAs as well as pcDNA31NFAT5 and empty vector theses transfection plasmids were procured from GenePharma Shanghai China In addition the miR4113p mimicsinhibitor and NC mimicsinhibitor were procured from Genechem Shanghai China SCC4 and SCC9 cells were collected for a0h of plasmid transfections by use of Lipofectamine Invitrogen Sequence for all plasmids used in current study were listed in Additional file a0 Table a0 S1 Each samples were assayed for more than triplicateCCK‘ assayAs previously described [] CCK8 Kit Beyotime Shanghai China was applied to detect cell viability under manufacturer™s protocols Cells cellswell were planted in 96well plates After and a0h the CCK8 reagents were added into each well Cell viability was detected using a microplate reader to measure the absorbance at the wave length of a0nm Each samples were assayed for more than triplicateColony formation assayAfter indicated transfections SCC4 and SCC9 cells were planted into 6well plates with cells in each well Following 14day of cell culture the resulting colonies were fixed using PFA for a0min stained using crystal violet solution for a0min and finally counted manually [] Each samples were assayed for more than triplicateEdU assayfor cell proliferation detection by use of BeyoClick„¢ EdU assay was undertaken in cells of SCC4 and SCC9 EdU Cell Proliferation Kit Beyotime Shanghai China with Alexa Fluor [] The DAPI staining solution was acquired from Beyotime for detecting cell nucleus After washing in PBS cells were studied using inverted microscope Olympus Tokyo Japan Each samples were assayed for more than triplicate 0cFu a0et a0al Cancer Cell Int Page of Flow cytometryCell apoptosis of transfected SCC4 and SCC9 cells was assayed employing the flow cytometer BD Biosciences Franklin Lakes NJ in the presence of Annexin VPI double staining kit Invitrogen Cell samples were collected from 6well plates via centrifugation then stained in Binding Buffer and assayed with flow cytometry [] Each samples were assayed for more than triplicateTUNEL assayThe transfected cell samples of SCC4 and SCC9 were washed employing PBS and fixed using PFS for TUNEL assay [] in the presence of TUNEL assay reagent Merck KGaA Darmstadt Germany Following addition of DAPI staining solution cell samples were analyzed using optical microscopy Olympus Each samples were assayed for more than triplicateWound healingThe transfected cell samples of SCC4 and SCC9 were seeded in 6well plates and cultivated until confluence [] Then the artificial wounds were created with a0μL of pipette tip At and a0h after incubation in serumfree medium the distance of wound healing were imaged under microscope Olympus Each samples were assayed for more than triplicateSubcellular fractionationThe TTNAS1 content in cytoplasmic and nuclear fracPARIS„¢ Kit Invitrogen as requested by provider Cell tions of SCC4 and SCC9 cells was studied by use of samples were lysed with cell fractionation buffer and cell disruption buffer then centrifuged for separating cell cytoplasm and cell nucleus [] For quantification GAPDH and U6 served as the cytoplasmic indicator and nuclear indicator respectively Each samples were assayed for more than triplicateFISHThe subcellular location of TTNAS1 in SCC4 and SCC9 cells was also studied with FISH assay using the deigned specifically TTNAS1probe Ribobio Guangzhou China After fixation the digested cells were airdried and cultured with probes in the hybridization buffer then treated in DAPI staining buffer [] Olympus fluorescence microscope was used for imaging Each samples were assayed for more than triplicateApplying the Magna RIP„¢ RNABinding Protein Immunoprecipitation Kit [] RIP assay was conducted RNA immunoprecipitation RIPfor RNA interaction in SCC4 and SCC9 cells as guided by provider Millipore Bedford MA RIP lysis buffer Thermo Fisher Scientific Waltham MA USA was applied to obtain the lysates Lysis was incubated with the magnetic beads Invitrogen Carlsbad CA USA conjugated with antiAgo2 antibody or antiIgG antibody at a0 °C overnight Complex was washed and purified according to the protocol of RIP kit used in this experiment The enrichment of RNAs were examined via RTqPCR Each samples were assayed for more than triplicateLuciferase reporter assayTTNAS1 fragment covering wildtype or mutant miR4113p binding sites were employed to construct TTNAS1WT or TTNAS1Mut vectors by use of the pmirGLO dualluciferase vectors Promega Madison WI SCC4 and SCC9 cells were cotransfected with miR4113p mimics or NC mimics and TTNAS1WT or TTNAS1Mut vectors for a0h followed by analysis of dualluciferase reporter assay system Promega [] Renilla luciferase activity was used as the internal control Each samples were assayed for more than triplicateWestern blotCells were lysed via RIPA buffer BCA Protein Assay kit Pierce Biotechnology Rockford IL was used to assess the concentration of protein Separation of equal amount of proteins was conducted via SDSPAGE BioRad Laboratories Hercules CA followed by the transformation to PVDF membranes Millipore Bedford MA The membranes were blocked with skim milk and incubated with primary and secondary antibodies All antibodies were obtained from Abcam Cambridge MA USA Protein bands were detected using a ECL detection kit Pierce Biotechnology Rockford IL Each samples were assayed for more than triplicateAnimal studySix 4weekold BALBc nude mice Shanghai Laboratory Animal Center was subjected to animal study in line with the ethical standards and guidelines of Henan Provincial People™s Hospital SCC6 cells × stably transfected with shNC or shTTNAS11 were injected into the right dorsal flanks of six mice Tumor sizes and volume were monitored by a caliper every a0days Four weeks later the mice were killed followed with the resection of tumors for measuring tumor weightStatistical analysesData of three or more independent assays were exhibited as the mean ± SD In addition Student™s ttest or onewaytwoway ANOVA followed by Tukey post hoc test 0cFu a0et a0al Cancer Cell Int Page of use of GraphPad Prism ® GraphPad Software Inc La was employed for comparing the group difference by Jolla CA USA Experimental data were collected when p ResultsKnockdown of a0TTN‘AS1 restrains the a0proliferation and a0migration of a0OSCC cellsAt first the relative higher level of TTNAS1 was observed in OSCC samples rather than adjacent normal ones Additional file a0 Fig S1A Next we detected the expression of TTNAS1 in OSCC cells through qRTPCR analysis We discovered that TTNAS1 expression was extremely high in OSCC cells in comparison of normal human squamous epithelial cell NOK cell Fig a01a At the same time we also found that TTNAS1 expression in SCC4 and SCC9 cells was highest Thus we knocked down TTNAS1 expression in SCC4 and SCC9 cells and identified that the TTNAS1 expression was exactly declined Fig a0 1b Following functional experiments were implemented to test the influence of inhibiting TTNAS1 on cells proliferation apoptosis and migration CCK8 assay unveiled that TTNAS1 depletion had significantly suppressive effect on cell viability Additional file a0 Fig S1B The number of colonies and EdU positive cells were reduced after silencing TTNAS1 indicating that cell proliferation could be restrained by TTNAS1 depletion Fig a01c d Then it was found by flow cytometry and TUNEL experiments that apoptosis was accelerated when decreased the level of TTNAS1 Fig a01e f Finally wound healing assay revealed that the migrated capability of SCC4 and SCC9 cells was hampered by silencing TTNAS1 Fig a0 1g In a word knockdown of TTNAS1 restrained cell proliferation and migration of OSCCTTN‘AS1 acts as a0miR‘‘3p sponge in a0OSCCThen we tested the distribution of TTNAS1 in SCC4 and SCC9 cells The results indicated that TTNAS1 tended to be located in the cytoplasm of SCC4 and SCC9 cells Fig a0 2a b indicating the potential posttranscriptional regulatory role of TTNAS1 in OSCC A flow of evidence suggested that lncRNA could serve as a ceRNA to regulate mRNAs through sponging miRNAs at posttranscriptional level [ ] Then we utilized starBase website to predict the possible miRNA which could have the binding site of TTNAS1 and one potential miRNA miR4113p was found out Fig a02c Then qRTPCR analysis was implemented to test the expression of miR4113p in OSCC samples and cells And the results indicated that miR4113p expression was lower in OSCC tissues and cells Additional file a0 Fig S1C and Fig a0 2d The lowest level of miR4113p was detected in SCC4 and SCC9 cells After that we discovered the binding site of miR4113p and TTNAS1 from starBase website Fig a02e and conducted Ago2RIP assay to evaluate the binding possibility of them We discovered that miR4113p and TTNAS1 were markedly enriched in antiAgo2 group Fig a02f and Additional file a0 Fig S1D which indicated that they were coexisted in RISC Following we overexpressed miR4113p and conducted the luciferase reporter assay We discovered that miR4113p overexpression caused a notable reduction on the luciferase activity of TTNAS1WT while the luciferase activity of TTNAS1Mut displayed no visible change Fig a02g h indicating that TTNAS1 could bind to miR4113p Overall TTNAS1 sponges miR4113p in OSCCUpregulation of a0miR‘‘3p represses OSCC cell growth and a0migrationIn order to search the role of miR4113p in OSCC functional experiments were implemented Firstly colony formation and EdU assays indicated that overexpressing miR4113p suppressed the proliferation of SCC4 and SCC9 cells Fig a0 3a b Moreover apoptosis of SCC4 and SCC9 cells was accelerated by miR4113p mimics through flow cytometry analysis and TUNEL assays Fig a03c d As illustrated in Fig a03e overexpression of miR4113p visibly reduced cell migration Taken together overexpression of miR4113p suppressed growth and migration in OSCCNFAT5 is a0the a0downstream target of a0miR‘‘3p in a0OSCCFor the sake of further verifying ceRNA hypothesis we searched the targets of miR4113p Combining the searching results from miRmap microT and PicTar databases candidate target genes were found under the condition Program number programs Fig a0 4a Then qRTPCR assay was applied to detect the influence of miR4113p overexpression and TTNAS1 inhibition on the levels of these mRNAs The results displayed a significant downregulation of mRNAs TLL2 MGAT4A RAB21 and NFAT5 when miR4113p was overexpressed and TTNAS1 was knocked down while other mRNAs were almost unchanged Fig a0 4b Then we tested the expressions of TLL2 MGAT4A RAB21 and NFAT5 in OSCC cells through qRTPCR for further detection We discovered that only NFAT5 displayed a high expression in OSCC cells Fig a04c High level of NFAT5 was further determined in OSCC tissues compared to adjacent normal ones Additional file a0 Fig S2A Thus we selected NFAT5 to conduct the further experiments Following we discovered the binding site of NFAT5 and miR4113p from starBase Fig a04d And RIP assays were implemented to evaluate the relationship of TTNAS1 NFAT5 and miR4113p The results 0cFu a0et a0al Cancer Cell Int Page of Fig Knockdown of TTN‘AS1 restrains the proliferation and migration of OSCC cells a The expression of TTN‘AS1 was tested through qRT‘PCR in OSCC cells b The interference efficiency of TTN‘AS1 was detected by qRT‘PCR in SCC‘ and SCC‘ cells c d Cell proliferation ability was measured by colony formation and EdU experiments when TTN‘AS1 was inhibited e f Cell apoptosis was evaluated through flow cytometry and TUNEL experiments after silencing TTN‘AS1 g Wound healing assays were utilized to estimate cell migration when TTN‘AS1 was subjected to knockdown P P 0cFu a0et a0al Cancer Cell Int Page of Fig TTN‘AS1 acts as the miR‘‘3p sponge in OSCC a b The cellular location of TTN‘AS1 was identified in SCC‘ and SCC‘ through Subcellular fractionation and FISH c StarBsae website was utilized to predict the possible miRNAs that could bind with TTN‘AS1 d MiR‘‘3p expression was detected through qRT‘PCR in OSCC cells e The binding site of TTN‘AS1 in miR‘‘3p f RIP assay was utilized to evaluate the relationship between miR‘‘3p and TTN‘AS1 g The efficiency of miR‘‘3p overexpression was tested through qRT‘PCR h Luciferase reporter assays were conducted to verify the correlation of miR‘‘3p and TTN‘AS1 P P showed that TTNAS1 NFAT5 and miR4113p were enriched in Ago2 indicating that TTNAS1miR4113pNFAT5 axis combined with RISC Fig a04e and Additional file a0 Fig S2B Then miR4113p was silenced and the interference efficiency was detected We could observe that miR4113p expression exactly declined after inhibition Fig a04f Following we detected the expression of NFAT5 when TTNAS1 and miR4113p were inhibited through qRTPCR Results indicated that NFAT5 expression could be hampered by TTNAS1 depletion but then recovered by miR4113p inhibition Fig a0 4g and Additional file a0 Fig S2C It demonstrated that NFAT5 and TTNAS1 were positively associated while NFAT5 and miR4113p were negatively correlated Then we investigated the function of NFAT5 in OSCC cells Firstly we knocked down the expression of NFAT5 in SCC4 and SCC9 cells and tested the knockdown efficiency Fig a04h and Additional file a0 Fig S2D NFAT5 expression could be hampered effectively after knockdown Then colony formation and EdU assays were carried out and the 0cFu a0et a0al Cancer Cell Int Page of Fig Upregulation of miR‘‘3p represses cell proliferation and migration in OSCC a b Cell proliferation was estimated through colony formation and EdU experiments when miR‘‘3p was overexpressed c d Flow cytometry and TUNEL experiments were implemented to measure cell apoptosis after overexpressing miR‘‘3p e Wound healing assays were adopted to test cell migration ability when miR‘‘3p was subjected to upregulation P See figure on next pageFig NFAT5 is a target gene of miR‘‘3p in OSCC a mRNAs which had the binding site with miR‘‘3p were predicted by starBase b The qRT‘PCR analysis was utilized to screen out the mRNAs which could be inhibited by NFAT5 depletion and miR‘‘3p overexpression c The expressions of TLL2 MGAT4A RAB21 and NFAT5 in SCC‘ and SCC‘ cells through qRT‘PCR d The binding site of NFAT5 and miR‘‘3p e RIP assay was adopted to test the relationship between TTN‘AS1 miR‘‘3p and NFAT5 f The interference efficiency of miR‘‘3p was tested by qRT‘PCR analysis g The expression of NFAT5 was detected when NFAT5 and miR‘‘3p was silenced h The interference efficiency of NFAT5 was tested by qRT‘PCR analysis i j Cell proliferation was evaluated through colony formation and EdU experiments when NFAT5 was knocked down k l Cell apoptosis was measured through flow cytometry and TUNEL experiments after inhibiting NFAT5 m Wound healing assays were carried out for estimating cell migration after NFAT5 was subjected to inhibition P 0cFu a0et a0al Cancer Cell Int Page of result indicated that silencing NFAT5 repressed the proliferation of SCC4 and SCC9 cells Fig a0 4i j Moreover cell apoptosis capability was expedited by NFAT5 depletion in flow cytometry and TUNEL assays Fig a04k l Finally wound healing assays indicated that silencing NFAT5 could hamper cell migration capability Fig a04m 0cFu a0et a0al Cancer Cell Int Page of Collectively NFAT5 was a target gene of miR4113p in OSCC and it accelerated the progression of OSCCTTN‘AS1 promotes OSCC progression via a0miR‘‘3pNFAT5 axisFor the sake of proving whether TTNAS1 could accelerate OSCC progression via miR4113pNFAT5 axis rescue assays were implemented Ahead of rescue assays qRTPCR was adopted to test the overexpression efficiency of NFAT5 in SCC4 and SCC9 cells The results displayed that NFAT5 expression was visibly increased after transfecting with pcDNA31NFAT5 Fig a05a Next we detected the mRNA and protein levels of NFAT5 in SCC4 and SCC9 cells after transfection It was uncovered that NFAT5 levels decreased by TTNAS1 depletion were rescued by the inhibition of miR4113p or the upregulation of NFAT5 Additional file a0 Fig S2E Then colony formation and EdU rescue assays were conducted we discovered that cell proliferation was hampered by TTNAS1 depletion but then it was recovered by NFAT5 overexpression or miR4113p inhibition Fig a0 5b c Through flow cytometry and TUNEL assays we found that knockdown of miR4113p or upregulation NFAT5 could reverse the cell apoptosis ability which was accelerated by TTNAS1 depletion Fig a05d e In the end it was indicated through wound healing assay that the inhibited cell migration caused by knockdown of TTNAS1 was restored by NFAT5 overexpression or miR4113p inhibition Fig a05f Thus we confirmed that TTNAS1 promoted OSCC cell growth and migration by miR4113pNFAT5 axisTTN‘AS1 promoted OSCC cell growth in a0vivoIn vivo study was conducted to support above in a0 vitro findings We observed that tumor size volume and weight in shNC group were all smaller than those in shTTNAS11 group Fig a06a“c Importantly IHC staining indicated that silencing of TTNAS1 caused a reduction in the positivity of Ki67 and PCNA Fig a06d All these experiments unveiled that TTNAS1 promotes OSCC progression via miR4113pNFAT5 axisDiscussionOral squamous cell carcinoma OSCC is a common squamous cell carcinoma of the head and neck It has a relatively high incidence worldwide As the regulatory functions of lncRNA in assorted cancers are constantly being explored lots of lncRNAs have also been confirmed to play a crucial role in promoting the development of OSCC For example PLAC2 could promote cell growth through activating wntβcatenin pathway in OSCC [] CEBPAAS1 was considered to correlate with the bad prognosis and it also could facilitate tumorigenesis through CEBPABcl2 in OSCC [] Moreover P4713 was reported to contribute to the malignant phenotypes of OSCC through activating the JAKSTAT3 pathway [] In our research we investigated the functions of TTNAS1 in OSCC TTNAS1 was a novel lncRNA and it served as the oncogene in lung adenocarcinoma [] In this study TTNAS1 was discovered to be highly expressed in OSCC cells And TTNAS1 depletion impaired cell proliferation and migration but it accelerated cell apoptosis in OSCC Overall TTNAS1 exerted the carcinogenic effect in OSCCMiRNAs are small RNAs with “ nucleotides in length without ability of coding protein [] In recent years an increasing number of evidences discovered that lncRNA could function as a crucial element of competing endogenous RNA ceRNA network by sponging miRNA to regulate mRNA so as to take part in the regulation of cancer progression [ ] For example lncRNA ATB functioned as a ceRNA to expedite YAP1 through sponging miR5905p in malignant melanoma [] PAGBC acted as a sponge of miR133b and miR and accelerated gallbladder tumorigenesis [] AFAP1AS1 could act as a ceRNA of miR4235p to expedite nasopharyngeal carcinoma progression [] In our research we utilized bioinformatics tools to find the possible miRNA which could bind to TTNAS1 After screening miR4113p was selected With the conduction of RIP and luciferase experiments we proved that TTNAS1 could act as ceRNA to sponge miR4113p in OSCC MiR4113p was verified as the tumor suppressor gene in ovarian cancer and it could restrain cell proliferation migration and invasion of ovarian cancer [] Thus we investigated the functions of miR4113p in OSCC As we expected miR4113p could repress cell proliferation and migration but accelerate cell apoptosis in OSCC In short our research confirmed that TTNAS1 sponged miR4113p and overexpressing miR4113p could repress the progression of OSCCNFAT5 is a mRNA and it has been reported to be associated with several cancers For example NFAT5 was proved to conduce to the glycolytic phenotype rewiring and pancreatic cancer progression through transcription of PGK1 [] Moreover NFAT5 cpuld also promote glioblastoma celldriven angiogenesis through EGFL7 which was mediated via SBF2AS1 and miR3383p [] In our research we discovered that NFAT5 was highly expressed in OSCC cells And based on the mechanism experiments we also proved that NFAT5 was the target of miR4113p and overexpressing it could accelerate the progression of OSCC Rescue experiment indicated that upregulation of NFAT5 could offset TTNAS1 knockdownmediated functions on the progression of OSCC 0cFu a0et a0al Cancer Cell Int Page of Fig TTN‘AS1 promotes OSCC progression via miR‘‘3pNFAT5 axis a The qRT‘PCR analysis was utilized to examine the overexpression efficiency of NFAT5 in SCC‘ and SCC‘ cells b c Cell proliferation capability in SCC‘ and SCC‘ cells was measured by colony formation and EdU assay in different groups d e Cell apoptosis was tested through flow cytometry and TUNEL assays in different groups f Wound healing assays were implemented to detect the cell migration ability in different groups P 0cFu a0et a0al Cancer Cell Int Page of Fig TTN‘AS1 promoted OSCC cell growth in vivo a Tumors removed from the mice injected with sh‘NC‘transfected cells or sh‘TTN‘AS11‘transfected cells b c Volume and weight in different groups were measured d IHC staining of tumor tissues collected from different groups with anti‘Ki‘ and anti‘PCNA P proving the functions of TTNAS1miR4113pNFAT5 axis in OSCCtransfected with sh‘TTN‘AS11 was examined by qRT‘PCR and western blot analyses after co‘transfection with miR‘‘3p inhibitor or pcDNA31NFAT5 P ConclusionTaken together TTNAS1 could contribute to the progression of OSCC via miR4113pNFAT5 axis which may provide the new idea for the exploration of OSCC treatmentsSupplementary informationSupplementary information accompanies this paper at https doi101186s1293 ‘‘ ‘Additional file a0 Sequence for all plasmids used in current studyAdditional file a0 Figure S1 A TTN‘AS1 expression in adjacent normal and tumor tissues was examined by qRT‘PCR analysis B CCK‘ assay was applied to analyze the viability of SCC‘ and SCC‘ cells transfected with sh‘NC sh‘TTN‘AS11 or sh‘TTN‘AS12 C The level of miR‘‘3p was assessed in pairs of OSCC tissues and adjacent normal tissues D Agarose gel electrophoresis for the Ago2‘RIP assay in Fig 2F P Additional file a0 Figure S2 A NFAT5 expression in paired tissues obtained from OSCC patients B Agarose gel electrophoresis for the Ago2‘RIP assay in Fig 4E C Protein level of NFAT5 in cells transfected with sh‘NC sh‘TTN‘AS11 or co‘transfected with sh‘TTN‘AS11 and miR‘‘3p inhibitor D Protein level of NFAT5 in cells transfected with sh‘NC sh‘NFAT51 and sh‘NFAT52 E mRNA and protein level of NFAT5 in cells AbbreviationsOSCC Oral squamous cell carcinoma TTN‘AS1 Titin antisense RNA lncRNAs Long non‘coding RNAs ceRNAs Competing endogenous RNAs miRNAs microRNAs mRNA Messenger RNA ATCC American type culture collection DMEM Dulbecco™s modified Eagle™s medium FBS Fetal bovine serum RIPA Radioimmunoprecipitation assay SDS‘PAGE Sulphate‘polyacrylamide gel electrophoresis PVDF Polyvinylidene fluoride RT‘qPCR RNA extraction and quantitative real‘time polymerase chain reaction HRP Horseradish peroxidase FISH Fluorescence in situ hybridization WT Wild‘type Mut Mutant SD Stand‘ard deviation ANOVA Analysis of varianceAcknowledgementsWe appreciate all the people involved in this studyAuthors™ contributionSF project administration study design and review experiments YZ SL and ZS methods investigation data JZ and QH preparation draft manuscript All authors read and approved the final manuscriptFundingNoneAvailability of data and materialsNot applicable 0cFu a0et a0al Cancer Cell Int Page of Ethics approval and consent to participateAll patients enrolled in this study had signed informed consent This study received the approval of the Ethics Committee of Henan Provincial People™s HospitalConsent for publicationAuthors confirmed that this work can be published The content of this manu‘script is original and it has not yet been accepted or published elsewhereCompeting interestsNo competing interest existReceived February Accepted June References Krishna Rao SV Mejia G Roberts‘Thomson K Logan R Epidemiology of oral cancer in Asia in the past decade“an update ‘ Asian Pac J Cancer Prev APJCP “Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin “ Warnakulasuriya S Global epidemiology of oral and oropharyngeal cancer Oral Oncol ““Sacco AG Cohen EE Current treatment options for recurrent or metastatic head and neck squa

Thyroid_Cancer

"Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAURKA a cell cycleregulated kinase is associated with malignant transformation and progression in many cancer types Weanalyzed the expression change of AURKA in pancancer and its eï¬ect on the prognosis of cancer patients using the TCGAdataset We revealed that AURKA was extensively elevated and predicted a poor prognosis in most of the detected cancer typeswith an exception in colon cancer AURKA was elevated in colon cancer but the upregulation of AURKA indicated betteroutcomes of colon cancer patients Then we revealed that undermethylation of the AURKA gene and several transcriptionfactors contributed to the upregulation of AURKA in colon cancer Moreover we demonstrated that AURKA overexpressionpromoted the death of colon cancer cells induced by Oxaliplatin whereas knockdown of AURKA significantly weakened thechemosensitivity of colon cancer cells to Oxaliplatin Mechanistically AURKA inhibited DNA damage response by suppressingthe expression of various DNA damage repair genes in a TP53dependent manner which can partly explain that ARUKA isassociated with a beneficial outcome of colon cancer This study provided a possibility to use AURKA as a biomarker to predictthe chemosensitivity of colon cancer to platinum in the clinic IntroductionAurora Kinase A AURKA is a cell cycleregulated kinaseinvolved in centrosome maturation mitotic entry bipolarspindle assembly and chromosome separation [] The elevated expression of AURKA is frequently reported in manycancer types [] AURKA alone or combined with otherfactors can trigger cell malignant transformation [ ] andpromote the malignant phenotype of cancer cells [ ]AURKA shows oncogenic activity by regulating multipleoncogenic and tumorsuppressive proteins [] Of theseproteinstumor suppressor TP53 has been intensivelystudied Phosphorylation of TP53 at Ser215 and Ser315 byAURKA results in TP53 degradation by MDM2mediatedubiquitination and abrogation of TP53 DNA bindingtransactivation activity respectively [ ] In turn TP53downregulation increases the expression of ARUKA atboth transcriptional and posttranslationallevels [ ]Negative feedback regulation between AURKA and TP53greatly promotes carcinogenesis and progressionMaintaining genome stability by transactivating theDNA damage response DDR genes is the critical mediatorof TP53dependent tumor suppression [ ] thus TP53deficiency causes the loss of various DDR mechanisms and 0cBioMed Research Internationalthereby facilitates genome instability and cancer development [] Meanwhile platinuminduced DNA damage cantrigger the DDR which is a major contributor to chemoresistance to platinum [] In view of the association betweenAURKA TP53 and DDR the upregulated AURKA in cancer might promote the cancer progression but meanwhileenhance the chemosensitivity of DNA damageinducingdrugs in the clinicIn this study we analyzed the expression state and regulation mechanism of AURKA in colon cancer We also testedthe eï¬ect of dysregulated AURKA on chemosensitivity to theplatinum drug and explored the underlying molecular mechanism in colon cancer These results provided a novel insightinto the function of AURKA in cancer Material and Methods Dataset and Processing The data of AURKA mRNAexpression in types of cancers and matched normal tissueswere downloaded from The Cancer Genome Atlas TCGAdatabase including Bladder Urothelial Carcinoma BLCABreast Invasive Carcinoma BRCA Cervical Squamous CellCarcinoma and Endocervical Adenocarcinoma CESCColon Adenocarcinoma COAD Head and Neck SquamousCell Carcinoma HNSC Kidney Renal Clear Cell Carcinoma KIRC Kidney Renal Papillary Cell CarcinomaKIRP Liver Hepatocellular Carcinoma LIHC Lung Adenocarcinoma LUAD Lung Squamous Cell CarcinomaLUSC Pancreatic Adenocarcinoma PAAD Prostate Adenocarcinoma PRAD Rectum Adenocarcinoma READSarcoma SARC Skin Cutaneous Melanoma SKCM Stomach Adenocarcinoma STAD Thyroid Carcinoma THCAand Uterine Corpus Endometrial Carcinoma UCEC Thecorrelation between the AURKA level and the overall survivalOS of cancer patients was also analyzed through the GEPIAhttpgepiacancerpkucnindexhtml The mRNA expression data are shown in Supplementary We compared theexpression level of AURKA mRNA by calculating the meanvalue and standard deviation The eï¬ect of AURKA copynumber variant CNV on AURKA expression level was alsoanalyzed based on the Colon Adenocarcinoma COAD datafrom the TCGA database The eï¬ect of methylation on theexpression of AURKA was assessed using the MEXPRESS datamexpressbe The transcription factor TF targeting ARUKA was screened based on the ChipSeq data in theUCSC databank httpgenomeucsceduENCODE Thetargeted regulatory capacity of TP53 on DDR genes wasassessed using the Cistrome Data Browser httpcistromedb Meanwhile the correlation between AURKAand TF genes was analyzed in colon cancer through theGEPIA httpgepiacancerpkucnindexhtml Cell Culture Two colon cancer cell lines SW1116 andHCT116 and 293TN cell line were used in this study Missense mutation presents in TP53 in SW1116 whereasHCT116 has a wildtype TP53 according to the Cancer CellLine Encyclopedia CCLEportalsbroadinstituteccleabout Additionally all the DDR genes involvedin our study are the wildtype but for BRCA2 which has aframeshift in HCT116 They were cultured using Dulbecco™sModified Eagle Medium DMEM HyClone Logan UTUSA with fetal bovine serum FBSInvitrogenCarlsbad CA USA μgml streptomycin and IUml°penicillin at C in a humidified atmosphere containing CO2 Cells used to detect phosphorylated TP53 were treatedwith a specific proteasome inhibitor MG132 Sigma StLouis MO USA μM for six hours The Construction of Stable Cell Lines Overexpression orknockdown of AURKA was achieved by using lentivirus ps to infect colon cancer cells described as before [] Inbrief the ORFs of AURKA cloned by PCR and synthesizedshRNA against AURKA were inserted into PlvxPuro andSHC201 vectors respectively The scramble sequences wereinserted into these vectors to be used as control Thesevectors were transfected into 293TN cells with the packingvectors System Bioscience Mountain View CA USA toget pseudo lentiviral ps After being filtered andconcentrated by PEG precipitation System Biosciencelentiviral ps were added to the culture medium toinfect colon cancer cells for h After routine culture for h the stable cells were selected and purified by puromycin μgml MTT Assay Colon cancer cells were seeded in 96wellplates at a density of cells per well and incubated overnight The culture medium was replaced with fresh culturemedium containing a diï¬erent concentration of Oxaliplatin and μgml with replicates each After h of incubation μl MTT gl was added to each wellfor h in the incubator The supernatant was removed and μl DMSO was added to each well After being vibratedfor min the plate was read on a microplate reader at nm to calculate the cell viability rate All assays werereplicated three times The result was analyzed using the cellviability percentage the total number of viable cells at eachdrug concentration relative to the number of viable cellstreated with the solvent control Western Blot Total proteins were extracted from coloncancer cells using the RIPA buï¬er Beyotime Institute ofBiotechnology Shanghai China μg protein was separated in SDSPAGE gel by electrophoresis and transferredonto PVDF membrane The blots were blocked by BSA°at C overnight The membrane was incubated with primaryantibodies AURKA rabbit polyclonal antibody ProteinTech Wuhan China No 102971AP diluted at TP53 rabbit polyclonal antibody Proteintech WuhanChina No104421AP diluted at phosphoTP53Ser315 mouse monoclonal antibody Santa Cruz Biotechnology Nosc135772 MDM2 rabbit polyclonal antibodyProteintech Wuhan China No 190581AP and GAPDHmouse monoclonal antibody ProteinTech Wuhan ChinaNo 600041Ig diluted at After washingthemembranes were incubated with peroxidaseconjugatedsecondary antibody Santa Cruz Biotechnology for h at°C The ECL system Thermo Scientific Rockford IL 0cBioMed Research InternationalUSA was used to visualize the blots All assays were replicated three times RealTime PCR Total RNA was extracted from coloncancer cells using TRIzol Invitrogen Carlsbad CA USAEasyScript® Reverse Transcriptase TransGen Biotech CoBeijing China was used to reverse RNA into cDNA Thelevel of DDR gene ATR XLF XRCC1 RPA1 BRCA2 andRAD51 was quantified using the SYBR Green PCR mixBioresearcher Beijing China through CFX96TM RealTime System BioRad The reaction mixture underwent° cycles consisting of denaturation for s at C and°annealing and prolongation for s each at C GAPDHwas used as the endogenous controls All assays were replicated three times The primers used for PCR are shown inSupplementary Statistics Analysis The expression of AURKA in a diï¬erent type of tumors and the diï¬erential expression of genesbetween two groups were analyzed by a twosided Student™sttest Survival analyses were conducted with the KaplanMeier method using the logrank test and the median valueseparation model based on the AURKA expression is presented The hazard ratio was calculated based on the CoxPH model The correlation between methylation statusand AURKA expression was analyzed using the Pearsoncorrelation and Wilcoxon ranksum test Pearson™s correlation and Z test were used to analyze the correlationbetween AURKA and TFs The eï¬ect of CNV on AURKAexpression was assessed by the KruskalWallis test MTTresults were analyzed using variance analysis ˆ—p ˆ—ˆ—p ˆ—ˆ—ˆ— p Results AURKA Was Upregulated and Predicted a BeneficialOutcome in Colon Cancer To explore the eï¬ect of AURKAon cancer progression and prognosis we firstly employedthe TCGA dataset to analyze the mRNA expression ofAURKA in types of tumors Compared with the matchednormal tissues AURKA was significantly upregulated incancer tissues in out of cancer types Figure 1a Nextwe assessed the correlation between the AURKA level andoverall survival OS in cancer types using the GEPIAWe showed that the AURKA level was adversely correlatedwith OS in of cancers including LUAD KIRP PAADSKCM and LIHC However a high level of AURKA wasassociated with a longer OS in COAD Figure 1b Theseresults suggested that AURKA overexpression might playan important role during the carcinogenesis and progressionof cancer however the elevated expression of AURKA predicted a beneficial outcome only in colorectal cancer DNA Undermethylation and Several TranscriptionFactors Might Contribute to the Elevated Expression ofAURKA in Colon Cancer To explore the mechanism bywhich AURKA was upregulated in colon cancer we firstlyanalyzed the eï¬ect of methylation status on AURKA expression By using the MEXPRESS there were methylationsites in the AURKA gene identified Of them methylationsites were significantly adverse correlated with the level ofAURKA Figure 2a Meanwhile we screened the potentialTFs activating AURKA expression based on the ChipSeqdata using the UCSC database and found that a total of TFs potentially regulate AURKA transcription Of themthe expression of TFs was positively correlated with thelevel of AURKA in colon cancer tissues according to theGEPIA correlation analysis Moreover of them have beenidentified to be overexpressed in colon cancer tissues compared with the matched normal tissues through the GEPIAexpression analysis Figure 2b The top four TFs highlycorrelated with AURKA r p were E2F1MYBL2 MYC and BRCA1 The expression and correlationwith AURKA of these four TFs are shown in Figures 2cand 2d We also analyzed the eï¬ect of AURKA CNV onthe expression level of AURKA The result indicated thatthe expression level of AURKA in the AURKA CNV gaingroup was much higher than that in the AURKA CNV neutral group in COAD whereas there was no diï¬erencebetween the AURKA CNV loss and CNV neutral groupFigure 2e But the incidence of CNV gain was lower incolon cancer patients These results indicated that undermethylation the elevated TFs and gene amplification mightcontribute to the elevated expression of AURKA in coloncancer AURKA Increased the Chemosensitivity of Colon CancerCells to Oxaliplatin We found that upregulated AURKAwas associated with the improved prognosis of colon cancerpatients thus we speculated that if AURKA increases chemosensitivity of platinum by increasing the genomic instability in colon cancer We firstly constructed the stable cell lineswith AURKA overexpression or knockdown Figure 3aand then assessed the eï¬ect of AURKA on the chemosensitivity of colon cancer cells The resultindicated thatAURKA overexpression promoted the death of HCT116and SW1116 colon cancer cells induced by Oxaliplatinwhereas knockdown of AURKA significantly weakened theresponse of colon cancer cells to Oxaliplatin Figures 3band 3c These results showed that AURKA may improvethe prognosis of colon cancer patients by increasing the chemosensitivity of colon cancer cells to the DNAdamagingdrug AURKA Downregulated the Expression of DDR Genes byInhibiting TP53 Previous research showed that AURKAinhibits the expression of TP53 which mediates the expression of DDR genes at the transcriptional level We detectedthe eï¬ect of AURKA on TP53 expression by immunoblotin colon cancer cells The result indicated that TP53 wasdownregulated when AURKA was overexpressed whereasupregulated when AURKA was knocked down in coloncancer cells Figures 4a and 4b Next we screened a setof DDR genes that play an important role in DNA damageinduced by chemotherapeutics Meanwhile most of themfunction after the activation of TP53 [] Using theCistrome Data Browser we assessed the transcriptionalregulatory potential of TP53 on these genes and found someof them had higher scores in two sets of data with high 0c AKRUA fo level evitaler ecid2ŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽBioMed Research InternationalŽŽŽŽŽŽŽŽŽCESCCOADHNSCKIRCKIRPLIHCLUADLUSCPAADPRADREADSARCSKCMSTADTHCAUCECTNBLCABRCATumorNormalCOAD Overall survivalLogrank p0034HRhigh06pHR0036nhigh135nlow135lavivrus tnecrePaLUAD Overall survivalLogrank p0047HRhigh13pHR0049nhigh238nlow239lavivrus tnecrePKIRP Overall survivalLogrank p0007HRhigh23pHR00088nhigh141nlow141MonthsMonthsMonthsLow AURKA TPMHigh AURKA TPMPAAD Overall survivalLogrank p00059HRhigh18pHR00068nhigh89nlow89Low AURKA TPMHigh AURKA TPMSKCM Overall survivalLogrank p0014HRhigh14pHR0014nhigh229nlow229lavivrus tnecrePlavivrus tnecrePLow AURKA TPMHigh AURKA TPMLIHC Overall survivalLogrank p000022HRhigh19pHR000028nhigh181nlow181lavivrus tnecrePlavivrus tnecrePMonthsLow AURKA TPMHigh AURKA TPMMonthsMonths Low AURKA TPMHigh AURKA TPMbLow AURKA TPMHigh AURKA TPMFigure AURKA was upregulated in colon cancer and predicted a benefit outcome a Compared with the matched normal tissuesAURKA was significantly upregulated in cancer tissues in out of cancer types b AURKA expression level was adversely correlatedwith OS in of cancers but positively correlated with OS in COADquality control Supplementary We then applied realtime PCR to verify the expression of six representative genesATR XLF XRCC1 RPA1 BRCA2 and RAD51 The resultsindicated that the six DDR genes were downregulated incolon cancer cells with AURKA overexpression but upregulated when knocking down AURKA in colon cancer cellsFigures 4c and 4d which implied that AURKAincreased the chemosensitivity of colon cancer cells to DNAdamageinducing drugs by inducing the degradation ofTP53 and then decreasing the expression of DDR genes 0cBioMed Research InternationalGpG islandAURKA chr2054967393chr2054944445 bpchr2054966401 chr2054967165 chr2054967495 chr2054967671 chr2054967718 r r r r r p p p p p TFE2F1MYBL2MYCBRCA1CBX3rapTFZNF217ELK1EZH2ZBTB33SMARCC1MYBLŽbrMYCŽpTFARID3ATCF3YY1FOXM1TEAD4rpBRCA1ŽE2F1ŽCOADnumT275 numN349COADnumT275 numN349COADnumT275 numN349COADnumT275 numN349cFigure Continued 0cBioMed Research International MPTFEgol MPTCYMgolp value r p value r MPTLBYMgollog2AURKA TPMlog2AURKA TPMp value r log2AURKA TPMŽŽp value r MPTACRBgollog2AURKA TPMdnoisserpxe AKRUACNV loss CNV neutral CNV gainCNV_TypeCNV_TypeCNV LossCNV NeutralCNV gaineFigure Undermethylation upregulation of TFs and gene amplification potentially contributed to the elevated expression of AURKAa Five methylation sites in AURKA DNA were significantly adversely correlated with the level of AURKA b Based on the public data analysisa total of TFs potentially regulated AURKA transcription The expression of TFs was positively correlated with the level of AURKAMoreover of them have been identified to be overexpressed in colon cancer tissues compared with the matched normal tissues c d Theexpression of the top four TFs highly correlated with AURKA was higher in colon cancer tissues compared with normal tissues e Theexpression level of AURKA in the AURKA CNV gain group was significantly higher than that in the AURKA CNV neutral group in COAD 0cBioMed Research InternationalCtrol AURKACtrolCtrol AURKACtrolshAURKAshAURKAAURKAGAPDH ytilibaiv lleC ytilibaiv lleCAURKAGAPDHSW1116aHCT116p00009Drug concentration 𝜇gmlControlAURKAIC50 𝜇gml 𝜇gml ytilibaiv lleCSW1116p00427Drug concentration 𝜇gmlControlAURKAIC50 𝜇gml 𝜇gmlb ytilibaiv lleCcHCT116HCT116p00029Drug concentration 𝜇gmlControlshAURKA1037shAURKA1184IC50 𝜇gml 𝜇gml 𝜇gmlSW1116p00043Drug concentration 𝜇gmlControlshAURKA1037shAURKA1124IC50 𝜇gml 𝜇gml 𝜇gmlFigure AURKA increased the chemosensitivity of colon cancer cells to Oxaliplatin a AURKA was upregulated or knocked down in twocancer cell lines b c AURKA overexpression promoted the death of HCT116 and SW1116 colon cancer cells induced by Oxaliplatinwhereas knockdown of AURKA significantly weakened the response of colon cancer cells to Oxaliplatin DiscussionIn this study we evaluated the expression of AURKA in types of tumor tissues and matched normal tissues Theresult indicated that AURKA was upregulated in most testedcancer types compared with their normal tissues OS analysisshowed that higher AURKA was correlated with a worse outcome of most of the cancer types whereas it only indicated afavorable outcome in colon cancer The prognostic role ofAURKA has ever been assessed in colorectal cancer patientsby a research team in [] Despite the lack of statisticalsignificance they still put forward that AURKA may have a 0cBioMed Research InternationalCtrolAURKACtrolCtrolAURKA CtrolshAURKAshAURKAAURKAMDM2TP53GAPDHpTP53GAPDHAURKAMDM2TP53GAPDHpTP53GAPDHGMGMANRm fo level evitaler ecid2ANRm fo level evitaler ecid2SW1116aŽŽŽŽŽŽŽATRBRCA2 RPA1 XRCC1 RAD51 NHEJ1SW1116 CtrolSW1116 AURKAŽŽŽŽŽŽATRBRCA2 RPA1 XRCC1 RAD51 NHEJ1HCT116 CtrolHCT116 AURKAANRm fo level evitaler ecid2cANRm fo level evitaler ecid2dGMGMHCT116bŽŽŽŽŽŽŽŽŽŽŽATRRPA1BRCA2SW1116 CtrolSW1116 shAURKA1024SW1116 shAURKA1037ŽŽŽŽXRCC1RAD51NHEJ1ŽŽŽŽŽŽŽ ŽŽŽŽŽŽŽATRRPA1BRCA2HCT116 CtrolHCT116 shAURKA1024HCT116 shAURKA1037XRCC1RAD51NHEJ1Figure AURKA downregulated the expression of DDR genes by inhibiting TP53 a b Overexpression of AURKA promoted thephosphorylation of TP53 and decreased the level of total TP53 whereas knockdown of AURKA reduced the phosphorylation of TP53 andincreased the level of total TP53 in colon cancer cells by immunoblot AURKA had no eï¬ect on the expression of MDM2 c d Sixrepresentative DDR genes were downregulated in colon cancer cells with AURKA overexpression but upregulated when knocking downAURKA in colon cancer cells by realtime PCRpositive eï¬ect on survival and emphasized the necessity tostudy the eï¬ect of AURKA on response to treatment []Further study showed that undermethylation and upregulation of TFs potentially contribute to the elevated expressionof AURKA in colon cancer at least partly Some studiesindicated that gene amplification is another contributor tothe elevated AURKA [ ] We also identified that geneamplification in colon cancer patients can result in AURKA 0cBioMed Research Internationalupregulation however its incidence rate was very low incolon cancer patients Finally we demonstrated that AURKAmight improve the prognosis of colon cancer patients byincreasing the chemosensitivity of colon cancer to Oxaliplatin via inhibiting the DDR Our results uncovered thedoubleedged sword eï¬ects of AURKA by inhibiting TP53in colon cancerThe genomic instability has been recognized as a hallmark of cancer and it is associated with carcinogenesis andprogression of cancer [ ] AURKA functions as an oncogene during the development of multiple malignant tumorsby inducing centrosome amplification and genomic instability [ ] In colon cancer the overexpressed AURKA is thecontributor to chromosomal instability [ ] MoreoverAURKA has been revealed to impair the function of DNAdamage repair through inhibiting the expression of DDRgenes such as RAD51 and BRCA12 [“] In additionto the DDR genes involved in Homologous RecombinationRepair HRR TP53 showed the transcriptional regulatorypotential on Mismatch Repair MMR genes according tothe binding scores from the ChipSeq data Supplementary The inhibitory eï¬ect of AURKA on TP53 which has beendemonstrated to transcriptionally activate many DDR genesenlarges the potential of AURKA facilitating DNA damage[] Some studies also indicate that TP53 is essential for chemoresistance rendered by AURKA [ ] In order to verifythe function of TP53 during this process we respectivelyassessed the correlation between AURKA level and OS inpatients with wildtype or mutant TP53 The results indicated that the patients with the higher AURKA had a longerOS time in TP53 wildtype groups although only a marginalsignificance was achieved due to the reduced number ofsamples But no diï¬erence was found in TP53 mutant groupsSupplementary The current research supports that AURKA is involved incolon carcinogenesis through promoting genomic instabilitybut the increased AURKA provides a good chance forenhancing the sensitivity of chemotherapy based on DNAdamageinducing drugs The eï¬ect of AURKA on chemosensitivity has been studied in diï¬erent cancer types Up to nowthey all concluded that AURKA impaired the chemosensitivity which is the exact opposite of our finding Forexample it was reported that inhibiting AURKA enhancesthe chemosensitivity of cancer cells to the taxane and paclitaxel [ ] cisplatin [] doxorubicin [ ] and5fluorouracil 5Fu [] In particular platinum chemosensitivity is inhibited by AURKA in various cancers includingovarian cancer [] hepatocellularcarcinoma [] medulloblastoma [] acute myeloid leukemia []as well as headand neck cancer [] Our finding that AURKA increasedthe platinum chemosensitivity in colon cancer was diï¬erentfrom the previous studies in other cancer types which coincided with our finding that higher AURKA indicated betterprognosis only in colon cancer but not in other cancersThough cancer stem cell is a small subpopulation of cancercells AURKA silencing sensitized the response of colorectalcancer stem cell CRCSC to Oxaliplatin by upregulatingantiapoptotic factors [] which is diï¬erent from our findings in colon cancer cells The diï¬erence might be associatedwith heterogeneity induced by tumor microenvironment andgenomic instability [] AURKAmediated TP53 inhibitionmight result in diï¬erent consequence in diï¬erent geneticcontexts However this hypothesis might be determined byfurther experiments ConclusionAURKA was upregulated in various cancer types but onlypositively correlated with the prognosis of colon cancerpatients The mechanism might be that AURKA improvesthe chemosensitivity of colon cancer cells to Oxaliplatin byinhibiting the expression of TP53regulated DDR genes andthen facilitating DNA damage This study provides a possibility to use AURKA as a biomarker to predict the chemosensitivity of colon cancer to platinum in the clinicData AvailabilityAll the data used to support the findings of this study areincluded within the Conflicts of InterestThe authors declare that there is no conflict of interestregarding the publication of this paperAuthors™ ContributionsBaocong Shan Ran Zhao Jian Zhou and Minghui Zhangcontributed equally to this workAcknowledgmentsThis work was partly supported by the National NaturalScience Foundation of China to Xiaobo Li Natural Science Foundation of Heilongjiang Province H2018009to Tianzhen Wang H2018010 to Yiqi Wu the FundamentalResearch Funds for the Provincial Universities to Ran Zhao to Yuanyuan Zhu and KYYWF0289 to Weiwei Yang and Heilongjiang Postdoctoral Fund LBHZ17136 to Weiwei YangSupplementary MaterialsSupplementary Figure The correlation of AURKA leveland OS in patients with wildtype TP53 or mutant TP53 AThe patients with the higher AURKA had a longer OS timein TP53 wildtype groups although only a marginal significance was achieved due to the reduced number of samplesB No diï¬erence was found in TP53 mutant groups C Mostof the mutations in TP53 were missense variant followed bystop gained and frameshift variant Supplementary Table We compared the expression level of AURKA mRNA by calculating the mean value and standard deviation The eï¬ect ofAURKA copy number variant CNV on AURKA expressionlevel was also analyzed based on the Colon adenocarcinomaCOAD data from the TCGA database SupplementaryTable The primers for PCRSupplementary Table Theassessment of transcriptional regulatory potential of p53 on 0cBioMed Research InternationalDDR genes based on ChipSeq data from cell lines Supplementary Table The assessment of transcriptional regulatory potential of p53 on Mismatch Repair MMR genesbased on ChipSeq data from celllines SupplementarymaterialsReferences[] B Goldenson and J D Crispino œThe aurora kinases in cellcycle and leukemia Oncogene vol no pp “[] A S Nikonova I Astsaturov I G Serebriiskii R L DunbrackJr and E A Golemis œAurora A kinase AURKA in normaland pathological cell division Cellular and Molecular Life Sciences vol no pp “ [] H Zhou J Kuang L Zhong œTumour amplified kinase_STK15_ _BTAK_ induces centrosome amplification aneuploidy and transformation Nature Genetics vol no pp “ [] A H SillarsHardebol B Carvalho M Tijssen œTPX2and AURKA promote 20q amplicondriven colorectal adenoma to carcinoma progression Gut vol no pp “ [] G Vader and S M A Lens œThe Aurora kinase family in celldivision and cancer Biochimica et Biophysica Acta BBA Reviews on Cancer vol no pp “ [] M Yan C Wang B He œAuroraA Kinase a potentoncogene and target for cancer therapy Medicinal ResearchReviews vol no pp “ [] H Katayama and S Sen œAurora kinase inhibitors as anticancer molecules Biochimica et Biophysica Acta BBA GeneRegulatory Mechanisms vol no pp “[] Q Liu S Kaneko L Yang œAuroraA abrogation of p53DNA binding and transactivation activity by phosphorylationof serine  Journal of Biological Chemistry vol no pp “ [] H Katayama K Sasai H Kawai œPhosphorylation byaurora kinase A induces Mdm2mediated destabilization andinhibition of p53 Nature Genetics vol no pp “[] C C Wu T Y Yang C T R Yu œp53 negatively regulates Aurora A via both transcriptional and posttranslationalregulation Cell Cycle vol no pp “ [] S S Chen P C Chang Y W Cheng F M Tang and Y S LinœSuppression of the STK15 oncogenic activity requires atransactivationindependent p53 function The EMBO journal vol no pp “ [] A Janic L J Valente M J Wakefield œDNA repairprocesses are critical mediators of p53dependent tumor suppression Nature Medicine vol no pp “ [] A B Williams and B Schumacher œp53 in the DNAdamagerepair process Cold Spring Harbor Perspectives in Medicinevol no [] L Galluzzi L Senovilla I Vitale œMolecular mechanismsof cisplatin resistance Oncogene vol no pp “ [] X Li J Zhang L Gao œMiR181 mediates cell diï¬erentiation by interrupting the Lin28 and let7 feedback circuitCell Death Diï¬erentiation vol no pp “ [] N Hosoya and K Miyagawa œTargeting DNA damageresponse in cancer therapy Cancer science vol no pp “ [] S Goktas M Yildirim D Suren œPrognostic role ofAuroraA expression in metastatic colorectal cancer patientsJournal of BUON official journal of the Balkan Union ofOncology vol no pp “ [] Synnöve Staï¬ J Isola M Jumppanen and M TannerœAuroraA gene is frequently amplified in basallike breastcancer Oncology Reports vol no pp “ [] S Yamamoto M YamamotoIbusuki Y YamamotoS Fujiwara and H Iwase œA comprehensive analysis ofAurora A transcript levels are the most reliable in associationwith proliferation and prognosis in breast cancer BMC cancer vol no [] A Janssen and R H Medema œGenetic instability tipping thebalance Oncogene vol no pp “ [] W M Grady œGenomic instability and colon cancer CancerMetastasis Reviews vol no pp “ [] X Wang Y X Zhou W Qiao œOverexpression of aurorakinase A in mouse mammary epithelium induces geneticinstability preceding mammary tumor formation Oncogenevol no pp “ [] N Nishida T Nagasaka K Kashiwagi C R Boland andA Goel œHigh copy amplification of the AuroraA gene isassociated with chromosomal instability phenotype in humancolorectal cancers Cancer Biology Therapy vol no pp “ [] Y Baba K Nosho K Shima œAuroraa expression isindependently associated with chromosomalinstability incolorectal cancer Neoplasia vol no pp “[] T Sourisseau D Maniotis A McCarthy œAuroraAexpressing tumour cells are deficient for homologydirectedDNA double strandbreak repair and sensitive to PARP inhibition EMBO Molecular Medicine vol no pp “ [] S Sankaran D E Crone R E Palazzo and J D ParvinœAuroraA kinase regulates breast cancer associated gene inhibition of centrosomedependent microtubule nucleationCancer Research vol no pp “ [] G Yang B Chang F Yang œAurora kinase A promotesovarian tumorigenesis through dysregulation of the cell cycleand suppression of BRCA2 Clinical Cancer Researchvol no pp “ [] H Yang L He P Kruk S V Nicosia and J Q ChengœAuroraA induces cell survival and chemoresistance by activation of Akt through a p53dependent manner in ovariancancer cells International journal of cancer vol no pp “ [] P Cammareri A Scopelliti M Todaro œAuroraA isessential for the tumorigenic capacity and chemoresistance ofcolorectal cancer stem cells Cancer Research vol no pp “ [] T Hata T Furukawa M Sunamura œRNA interferencetargeting aurora kinase a suppresses tumor growth andenhances the taxane chemosensitivity in human pancreaticcancer cells Cancer Research vol no pp “[] Y Lin F M Richards B F Krippendorï¬ œPaclitaxel andCYC3 an aurora kinase A inhibitor synergise in pancreatic 0cBioMed Research Internationalcancer cells but not bone marrow precursor cells British Journal of Cancer vol no pp “ [] M A Miller C Palaniswamy D Sharma and V Y ReddyœInappropriate shock from a subcutaneousimplantablecardioverterdefibrillator due to transcutaneous electricalnerve stimulation Heart Rhythm vol no pp [] Q Zhu X Yu Z W Zhou C Zhou X W Chen and S FZhou œInhibition of Aurora A Kinase by alisertib inducesautophagy and cell cycle arrest and increases chemosensitivityin human hepatocellular carcinoma HepG2 cells CurrentCancer Drug Targets vol no pp “ [] J Wang K Nikhil K Viccaro L Chang J White andK Shah œPhosphorylationdependent regulation of ALDH1A1by Aurora kinase A insights on their synergistic relationship inpancreatic cancer BMC Biology vol no p [] Y Shionome W H Lin H Y Shiao H P Hsieh J T A Hsuand T Ouchi œA novel auroraA inhibitor BPR1K0609S1sensitizes colorectal tumor cells to 5fluorofracil 5FU treatment International Journal of Biological Sciences vol no pp “ [] M Li H Li F Liu œCharacterization of ovarian clear cellcarcinoma using target drugbased molecular biomarkersimplications for personalized cancer therapy Journal ofOvarian Research v

Thyroid_Cancer

Research letterthe COVID19An analysis of SARSCoV2 cellentry genes identifies the intestineand colorectal cancer assusceptible tissuesEditorSARSCoV2 is the causative agentforpandemicCOVID19 has necessitated rapidchangesin surgical practice andanisation through both the initialpeak and ongoing recovery period1SARSCoV2 infects cells by interacting with the host cellsurfaceprotein ACE2 and utilises TMPRSS2in viralspike protein priming tofacilitate cell entry Fig 1a2 WhilstCOVID19 is predominantly a respiratory disease approximately of patients have concurrent gastroinsymptoms3 SARSCoV2testinalRNA and live virus have been identified in stool from COVID19 patientsand SARSCoV2 readilyinfectsintestinal anoids46 Despite thesecircumstantial data gastrointestinaltransmission has not yet been formally confirmed Cancers commonlyexpress different genes from the tissueof origin and it is largely unexploredtumours can be infectedwhetherwith SARSCoV2 We soughttoexplore the expression of ACE2 andTMPRSS2 in large publicly available normaltissue and pancancerexpression data sets to understandwhether levels of these genes identifysusceptible tissuesAnalysis ofthe normaltissueGenotype Tissue Expression projectGTEx dataset showed high ACE2expression in the testis small intestinekidney heart thyroid and adipose tissue Fig S1a supporting informationTMPRSS2 levels were highest in theprostateintestinepancreas lung salivary gland kidneythyroid and liver Fig S1b supportinginformation Whilst initial analysissuggested only kidney and thyroidcoexpressed high levels of ACE2 andTMPRSS2 closer inspection of smallstomachsmall BJS Society LtdPublished by John Wiley Sons Ltdinformationintestinal and colonic samples revealedheterogeneity with a subpopulationalso coexpressing highlevels that was confirmed by Kmeansclustering Fig 1bc Fig S1cd supportingIn colonictissue high levels of both genes werefound in younger patients and greaterTMPRSS2 expression in femalesalthough neither factor defined thesubpopulation observed Fig S2ajsupporting information Biopsylocation and tissuecompartmentfrom colon samples were explored andhigher levels of both genes were foundin the mucosa and proximal colonalthough these factors also failed tofully define the high coexpressingsubpopulation Fig S2kmsupporting information These normalexpression data appear to identify aproportion of individuals who in thegastrointestinal tract express high levels of both genes known to be involvedin cell entry of SARSCoV2The Cancer Genome Atlas programTCGA is the largest pantumourcollection of genomicand transcriptomic sequencing data Havingidentified the heterogeneous geneexpression within the normal gastrointestinaltract we interrogatedTCGA and GTEx gene expressiondata to identify relative expressionof ACE2 and TMPRSS2 in tumourscompared to their tissue of originGenerally there was no correlationbetween high expression of ACE2and TPMRSS2 in normal tissue andhigh tumour expression Fig S3adsupporting information Howeverwe identified colorectal cancer asunique amongst human malignancies by coexpressing higher levels ofboth ACE2 and TMPRSS2 relativeto normal Fig 1de We exploredTCGA data to identify if molecularsubgroups of colorectal cancers specific mutations or other commonlycollectedcouldvariablesclinicaldefine tumours with varying expression of ACE2 and TMPRSS2 Therewas a subtle yet significanttrendfor higher ACE2 expression withyounger age but no association withsex Fig S4a supporting information Possession of a BRAF mutationwas found to predict lower levels oftumour ACE2 however TMPRSS2expression was unchanged Fig S4bcsupporting information StrikinglyACE2 levels were very low in tumourswith microsatellite instability MSIFig 1f Cumulatively these data identify aproportion of healthy individuals assusceptible to putative SARSCoV2intestinal infection and that patientswith colorectal cancer may be at evengreater risk of infection Further clinical studies are urgently required toexplore this mode of transmission ofCOVID19Conflicts of Interest and Sourcesof FundingThere are no conflicts of interestsScientistSJAB is supported by an AdvancedFellowshipCliniciangrantfrom Cancer Research UKC14094A27178 and core fundingfrom Wellcome and MRC to theWellcomeMRC Cambridge StemCell InstituteMahnaz DarvishDamavandi1 JamesLaycock2 Christopher Ward1 MilouS van Driel1 Mae A Goldgraben3and Simon JA Buczacki121WellcomeMRC Cambridge Stem CellInstitute University of CambridgePuddicombe Way Cambridge UK2Cambridge Colorectal Unit CambridgeUniversity Hospitals NHS Trust HillsRoad Cambridge UKand 3Department of Medical GeneticsUniversity of Cambridge Addenbrooke™sTreatment Centre Cambridge UKSCICambridgeSiBucz 101002bjs11911BJS 0cFig ACE2 and TMPRSS2 are expressed heterogeneously in the normal intestine and colorectal cancerResearch letterabdfcea Schematic demonstrating the mode of entry of SARSCoV2 into cells via interactions with the cell surface proteins ACE2 and TMPRSS2 b Scatterplot of the expression of ACE2 and TMPRSS2 across all colon GTEx samples Yellow circle highlights high coexpressing samples c Scatter plot of theexpression of ACE2 and TMPRSS2 across all small intestine GTEx samples Yellow circle highlights high coexpressing samples d Box and whisker plotof expression of ACE2 between normal large intestine Grey TCGA and GTEx and colon and rectal cancer Red TCGA samples Median IQROneway ANOVA P TPM Transcripts per million e Box and whisker plot of expression of TMPRSS2 between normal large intestineGrey TCGA and GTEx and colon and rectal cancer Red TCGA samples Median IQR Oneway ANOVA P TPM Transcripts permillion f Box and scatter plot of ACE2 expression levels from TCGA COADREAD data sets between tumour subtypes CIN chromosomal instabilityMSI microsatellite instability GS genome stable and POLE DNA polymerase epsilon Median IQR KruskalWallis p BJS Society LtdPublished by John Wiley Sons LtdwwwbjscoukBJS 0cResearch letter S¸reide K Hallet J Matthews JBSchnitzbauer AA Line PD Lai PBS Immediate and longterm impactof the COVID19 pandemic ondelivery of surgical services Br J Surg 101002bjs [Epub ahead of print] Hoffmann M KleineWeber HSchroeder S Kruger N Herrler TErichsen S SARSCoV2 CellEntry Depends on ACE2 andTMPRSS2 and Is Blocked by aClinically Proven Protease InhibitorCell “280e8 Yang L Tu L Implications ofgastrointestinal manifestations ofCOVID19 Lancet Gastroenterol Hepatol “ Wu Y Guo C Tang L Hong ZZhou J Dong X Prolongedpresence of SARSCoV2 viral RNA infaecal samples Lancet GastroenterolHepatol “ Wang W Xu Y Gao R Lu R Han KWu G Detection of SARSCoV2in Different Types of ClinicalSpecimens Jama “ Lamers MM Beumer J van der Vaart JKnoops K Puschhof J Breugem TI SARSCoV2 productivelyinfects human gut enterocytes Science 101126scienceabc1669 [Epub ahead of print]Supporting informationAdditional supporting informationcan be found online in theSupporting Information section atthe end of the BJS Society LtdPublished by John Wiley Sons LtdwwwbjscoukBJS 0c'

Thyroid_Cancer

"variability around prevalence estimates of multimorbidity due to poorconsensus regarding its definition and measurement Medicationbased measures of morbidity may be valuableresources in the primarycare setting where access to medical data can be limited We compare the agreementbetween patient selfreported and medicationbased morbidity and examine potential patientlevel predictors ofdiscordance between these two measures of morbidity in an older ‰¥ years communitybased populationMethods A retrospective cohort study was performed using national pharmacy claims data linked to The IrishLongituDinal study on Ageing TILDA Morbidity was measured by patient selfreport TILDA and two medicationbased measures the RxRisk years and RxRiskV ‰¥ years which classify drug claims into chronic diseaseclasses The kappa statistic measured agreement between selfreported and medicationbased morbidity at theindividual patientlevel Multivariate logistic regression was used to examine patientlevel characteristics associatedwith discordance between measures of morbidityResults Two thousand nine hundred twentyfive patients were included years N and ‰¥ years N Hypertension and high cholesterol were the most prevalent selfreported morbidities inboth age cohorts Agreement was good or very good κ “ for diabetes osteoporosis and glaucoma andmoderate for high cholesterol asthma Parkinson™s and angina κ “ All other conditions had fair or pooragreement Age gender marital status education poordelayed recall depression and polypharmacy weresignificantly associated with discordance between morbidity measuresConclusions Most conditions achieved only moderate or fair agreement between selfreported and medicationbased morbidity In order to improve the accuracy in prevalence estimates of multimorbidity multiple measures ofmultimorbidity may be necessary Future research should update the current RxRisk algorithms inline with currenttreatment guidelines and reassess the feasibility of using these indices alone or in combination with othermethods to yield more accurate estimates of multimorbidityKeywords Agreement Selfreport Rxrisk RxriskV Morbidity Polypharmacy Older people Correspondence caitrionacahirrcsiie Clionadh Mannion and John Hughes are joint first authors2Division of Population Health Sciences Royal College of Surgeons in IrelandDublin IrelandFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMannion BMC Geriatrics Page of Key pointsKey findings and implications Agreement between patient selfreported morbidityand medicationbased measures of morbidity RxRisk and RxRiskV was mainly moderate or fairDiabetes was the only condition for which the levelof agreement was found to be very good The results of our study indicate that neithermeasure of morbidity is completely reliable and wesuggest that researchers may require multiplemeasures selfreport and medicationbased measures of morbidity to fully capture accurate prevalence estimates of multimorbidity Our study identified several limitations of thecurrent versions of the RxRisk indices which require updating if medicationbased measures ofmorbidity are to be used to assess the epidemiologyof chronic conditions and multimorbiditytheofIndeedattentionBackgroundMultimorbidity is commonly defined as the presence oftwo or more chronic medical conditions and its prevalence has been shown to increase with age [] As theworld™s older population continues to grow multimorbidity has become an important public health issue caphealthcareturingresearchersprofessionals as well as policy makersforhealthcare systems to effectively adapt and manage thedelivery of healthcare to our growing older populationan accurate description of the epidemiology of chronicconditions is required However to date studies in theliterature reveal wide disparities in prevalence estimatesof multimorbidity ranging from to [ ] Thislarge variability is thought to be due to the lack of standards defining multimorbidity and validated methods forhow it should be measured [] A recent systematic review reported definitions of multimorbidity involving differenttheappropriateness of different measures of multimorbidityis also variable depending on both the outcome of interest as well as the type of data that is available []In additioncriteria[]Measures of multimorbidity include diagnosisbasedmeasures eg Charlson Index based on hospital diagnosis codes ICD codes [] medicationbased measureseg RxRisk and RxRiskV for those aged ‰¥ yearsbased on pharmacy data [] and patient selfreportDiagnosisbased measures of multimorbidity are themost common measures and are generally based on hospital or physician records [] Medicationbased measures of multimorbidity include the RxRisk and RxRiskV “ two algorithms which determine an individual™scurrent comorbidities based on their dispensed medication The RxRisk indexes only include morbidities forwhich a medicine could be prescribed and include categories of morbidities based on the World Health anisation WHO Anatomical Therapeutic Classification ATC system [“] The RxRisk and RxRiskVhave good reliability and criterion validity against ICD9diagnoses and have been shown to predict costs of caremortality and health care utilisation [] Previous studies have reported medicationbased measures of morbidity such as the Medicines Disease Burden Index MDBIand RxRiskV to be useful in epidemiological studieswhen adjusting for comorbidity [] However there arefew studies describing the use of these indices to directlymeasure chronic conditions Patient selfreport is also avalid method of identifying disease categories A study ofolder patients with multimorbidity reported good agreement between patient selfreport and general practitioner GP report for a wide range of diseases []A number of studies have compared the differentmeasures of multimorbidity with differing results [ ] A study of older primary care patients inIreland found that medicationbased measures ofmultimorbidity such as RxRiskV performed betterthan diagnosisbased measures of multimorbidity inpredicting emergency and ambulatory care sensitiveACS admissions [] Studies comparing patientselfreport and diagnosisbased measures of multimorbidity have reported a stronger association between selfreport measures of multimorbidity andqualitythandiagnosisbased measures [ ] However no previous research has compared selfreported morbidityin the primary care or community setting with theRxRisk measures of morbidity Comparison betweenselfreported morbidity data and pharmacy records isimportant in order to understand the relative meritsof each measure of morbidity and the potential formisclassification particularly in the community setting where access to medical or clinical data can belimitedfunctionaloutcomesandlifeofStudies have also indicated that agreement betweenselfreport measures and other measures of morbiditymight be influenced by patient recall bias [] Patientrecall has been reported to be influenced by age maritalstatus and education [] There is also some evidencethat cognition and memory influence patient recall []The impact of these factors needs to be explored furtherwhen assessing and comparing measures of morbidityThe aim of this study was to compare the agreementbetween patient selfreported morbidity and medicationbased morbidity RxRisk and RxRiskV and examine potential patientlevel predictors of discordance between theincludingdemographic cognitive and mental health factors in anolder community based populationtwo measures of morbidity 0cMannion BMC Geriatrics Page of MethodsThe STrengthening the Reporting of ObservationalStudies in Epidemiology STROBE guidelines were usedin the reporting of this study []Study populationThis was a retrospective cohort study using data froma national pharmacy claims database the Health Service ExecutivePrimary Care Reimbursement ServiceHSEPCRS General Medical Services GMS schemelinked to the first wave of The Irish LongituDinalstudy on Ageing TILDA TILDA is a nationally representative sample of community dwelling individualsaged ‰¥ years in Ireland The sampling framework isbased on the Irish Geodirectory a comprehensive anduptodate listing and mapping ofresidential addresses in Ireland compiled by the Ordinance SurveyOffice and participants aged ‰¥ years were randomlyselected using the RANSAM sampling procedureThis meant that each residential address in Irelandhad an equal probability of selection and thus ensured that the TILDA sample was representative ofthe Irish population aged ‰¥ years The first wave ofdata collection began in October through toFebruary N participants aged ‰¥ yearswhere participants completed a computeraided personal interview CAPI and a health assessment measuring their health economic and social circumstancesFurther information on TILDA™s study design andsampling framework is described in detail elsewhere[]The HSEPCRS GMS scheme is the largest pharmacy claims dataset in Ireland covering more than of the general Irish population [] It is meanstested and provides free health servicesincludingmedications to eligible persons in Ireland Qualification for the GMS scheme is on the basis of incomerelated meanstesting Automaticforthose aged ‰¥ years occurred between July andDecembercurrent study period meanstesting was introducedbut with a higher income threshold than the generalpopulation As of of men and ofwomen in the general population aged ‰¥ yearswere eligible [] The HSEPCRS GMS pharmacyclaims data were available for consenting TILDAparticipants aged ‰¥ years with GMS eligibility N entitlementhoweversinceJanuaryWithin the HSEPCRSGMS pharmacy claims dataprescriptions are coded using the WHO ATC classification system and prescriber information defineddaily doses strength quantity method and unit ofadministration of each drug dispensed are all available Pharmacy claims data was extracted for yearprior to each participant™s TILDA interview GMSpatientstypically receive their medications on amonthly basis []ifthey had any ofSelfreported morbidityAs part of the TILDA interview participants wereasked to reportthe followingdoctordiagnosed chronic diseases high blood pressure or hypertension high cholesterol angina congestive heart failure heart attack diabetes stroke orministroke abnormal heart rhythm arthritis osteoporosis cancer Parkinson™s disease emotional nervous or psychiatric problems alcohol or substanceabuse dementia serious memory impairment stomach ulcers glaucoma incontinence or chronic painParticipants were also asked to selfreport urinaryincontinence in the past months as well as painmoderate or severe and if they were taking medication for pain management If participants reportedthat they had arthritisthey were asked to clarifythe type of arthritis eg osteoarthritis rheumatoidarthritis some other kind of arthritis Similarlyifparticipants reported emotional nervous or psychiatric problems they were asked to clarify from a listof conditions eg anxiety depression emotionalproblems psychosis manic depressionfillsthatclassify prescription drugMedicationbased measures of morbidity “ Rxrisk andRxriskVThe RxRisk and RxRiskV indices were applied tothe HSEPCRS pharmacy claims data The RxRiskindex was applied to the population aged yearswhile the RxRiskV was applied to the populationaged ‰¥ years The RxRisk and RxRiskV are algorithmsintochronic disease classes for older populations basedon the WHO ATC classification system [“]Within the RxRiskV cardiac disease is separatedinto a number of categories anticoagulation antiplatelet agents arrhythmias congestive heart failureCHFhypertension hypertensionischaemic heartdisease IHDangina and ischaemic heart diseaseIHDhypertension [] For a medication to be eligible as a measure of morbidity per RxRisk and RxRiskV chronic disease classes a patient was required to have been dispensed two or more consecutive prescriptions of the medication in question eg˜donepezil™ was required to be dispensed on ‰¥ consecutive prescriptions to link this medication withthe RxRiskV condition ˜dementia™ This definitionhas previously been used by other pharmacoepidemiological studies [] 0cMannion BMC Geriatrics Page of Comparison of selfreported morbidity with Rxrisk andRxriskVEach selfreported condition in TILDA was matched tothe equivalent RxRisk and RxRiskV condition at theindividual patient level for those aged years and ‰¥ years respectively This was performed by consensusbetween two pharmacists FM CM For some selfreported conditions the ATC classes of medicationsspecific to these conditions “ eg antiwere notthrombotic agents B01AC04 “ B01AC30 were matchedto the selfreported condition of a heart attack and alsoto stroke There were four selfreported TILDA conditions which could not be matched to an RxRisk or RxRiskV condition but the prevalence was low Appendix in Tables and The RxRisk and RxRiskV alsoreported conditions which patients had not been askedabout during their TILDA interview Appendix in Tables and Patientlevel characteristics associated with discordancebetween the two measures of morbidityPatient characteristics were assessed to determine discordance patient recall bias between selfreported morbidity TILDA and the RxRisk years and RxRiskV ‰¥ years medicationbased measures of morbidity These characteristics were age gender maritalstatus education poor delayed recall depression andpolypharmacy Marital status was subcategorised intomarried never married separated or divorced Educationwas categorised into primarynone secondary or thirdhigher level education Delayed recall based on participants being presented with words during the interview and being later asked to recall as many as possiblewas defined as poor where or fewer words wererecalled Depression was defined as scoring or greateron the Centre for Epidemiologic Studies DepressionScale CESD [] Polypharmacy was defined as reporting regular use of five or more prescription medications[]Statistical methodsAgreement between selfreported morbidity TILDAand the RxRisk and RxRiskV measures of morbiditypharmacy claims was assessed using Cohen™s Kappastatistic as neither source was considered to be a goldstandard for reporting morbidity Interpretation of thevalue of Kappa was as follows poor fair “ moderate “ good “ and verygood “ []Multivariate logistic regression was used to examinethe association between the patientlevel characteristicsand discordance between the two measures of morbidityAdjusted odds ratios OR and confidence intervalsCIare presented Discordance was defined asparticipants reporting to have the condition in the absence of any dispensed medication for the condition perRxRisk years or per RxRiskV ‰¥ years andparticipants reporting to not have the condition butmedication was found to be dispensed for the conditionper RxRisk years or RxRiskV ‰¥ years Allsignificance tests were twotailed Statistical significancewas set at P after adjustment for a false discoveryrate of [] Analyses were performed using Stata SEVersion statistical package StataCorp College Station TXResultsStudy populationIn total patients were included in this cohortstudy patients were aged years and were aged ‰¥ years Characteristics ofthe study participants are presented in Table On average patients aged years had SD conditionsper the RxRisk and patients aged ‰¥ years had SD conditions per the RxRiskV The proportion ofpatients with thirdhigher level education was relatively years N low across both age ‰¥ years N Poor delayed recall years N ‰¥ years N years N ‰¥ years N were significantlymore prevalent in the older cohort compared to theyounger cohort p polypharmacygroupsandAgreement between selfreported morbidity andmedicationbased measures of morbidity Rxrisk and RxriskVTables and present a comparison between the number and percentage of patients™ selfreported morbiditiescompared to the RxRisk Table aged years andRxRiskV Table aged ‰¥ years measures of morbidity High blood pressure or hypertension yearsN ‰¥ years N and highcholesterol years N ‰¥ years N were the most prevalent selfreportedmorbidities in both age cohorts in the TILDA datasetHigh cholesterol was also found to be highly prevalentin the RxRisk N and RxRiskV N measures of morbidity Other prevalentRxRisk and RxRiskV conditions included arthritisRxRisk N stomach ulcers RxRiskN RxRiskV N strokeRxRiskV N heart attack RxRiskVN and other heart trouble RxRiskVN There was very good agreement between the selfreported TILDA measure of diabetes and the RxRiskand RxRiskV measures κ There was also good 0cMannion BMC Geriatrics Page of Table Characteristics of study participants by age years and ‰¥ years years N “Age‰¥ years N “GenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimarynoneSecondaryThirdHigher LevelPoor delayed recall YesDepression YesPolypharmacy Yes Data presented as N or mean CI unless otherwise statedagreement between selfreported measures of osteoporosis κ and glaucoma κ and the RxRiskV measure of these morbidities in the older cohort Despite the high prevalence of high cholesterolin both measures of morbidity there was only moderate agreement κ RxRisk κ RxRiskVbetween the two measures There was moderateagreement also for asthma κ RxRisk Parkinson™s κ RxRiskV and angina κ RxRisk V Agreement was fair for selfreported highblood pressure or hypertension RxRisk and RxRiskV heart attack RxRisk stroke RxRisk abnormalheart rhythm RxRiskV cancer RxRisk depression RxRisk and RxRiskV and pain RxRiskVand RxRisk measures of these conditions κ “ All other conditions had poor agreement κ “ including arthritis RxRisk chronic lungdisease and incontinence RxRiskV and emotionalnervous psychiatric problems anxiety and stomach ulcers RxRisk and RxRiskV Tables Patientlevel characteristics associated with discordancebetween the two measures of morbidityAge gender marital status education poor delayedrecall depression and polypharmacy were all associated with discordance between the two measures ofmorbidity Table Females were five times morelikely to have discordance in reporting osteoporosisOR Confidence Intervals CI P Females were also more likely to have discordance in reporting anxiety OR CI emotional problems OR CI and depression OR CI as well as use of pain medication OR CI and incontinence OR CI They were less likely to have discordance in reporting stroke and high cholesterol TablePatients who were never married were less likely tohave discordance in reporting a heart attack OR CI and stroke OR CI Patients with third level educationwere lesslikely to have discordance in reportinghypertension OR CI comparedto those with primary level education Table Patients with poor delayed recall and depression weremore likely to have discordance in reporting anxietyand depression In general discordance was higher inpatients with polypharmacy Table found thatagreement between patientDiscussionWithin a population based study of ageing in Irelandweselfreported morbidity and medicationbased measures ofmorbidity RxRisk and RxRiskV was generally notgood with most conditions achieving only moderateor fair agreement There was ˜very good™ agreementκ between selfreported diabetes and pharmacy dispensing records across both age cohortsThis was the only morbidity common to both age cohorts for which the level of agreement was found tobe ˜very good™ Many research studies confirm this 0cGlaucomaHigh CholesterolAsthmaHigh blood pressure orHypertensionCancer or a malignant tumourDepressionStroke cerebral vascular diseaseParkinsonHeart attack including myocardialinfarction or coronary thrombosisManic depressionEmotional nervous or psychiatricproblem such as depression oranxietyCirrhosis or serious liver damageStomach ulcersArthritis including osteoarthritis orrheumatismN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX Hyperlipidaemia C10AA01C10BX17 Asthma R03AAR03AL R03BAR03BX R03CAR03CC R03DAR03DX Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02C09BA09 C09DA01C09DA07 C02AB01C02AC05 C02DB02C02KX01 Malignancies L01AA01L01XX31 Depression N06AA01N06AG02 N06AXAntiplatelet therapy B01AC04B01AC30Parkinson™s disease N04AA01N04BX02Antiplatelet therapy B01AC04B01AC30Bipolar disorder N05AN01 Anxiety N05BA01N05BA12Anxiety N05BA01N05BA12Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11 GORD Peptic ulcer A02B A02BB A02BC Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01L04AB01L04AB05 L04AD01 L04AX03Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Anxiety Mannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRisk algorithm yearsTILDAStandardErrorSelfreported morbidityDiabetes or high blood sugarRxRisk Pharmacy ClaimsMedicationbased Morbidity ATCKappaκNAny other heart troubleRheumatoid arthritis only Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01Ministroke or TIAL04AB01L04AB05 L04AD01 L04AX03Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06ATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient coprescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm []same level of agreement for diabetes [ ] Thiswas expected given that previous research has demonstrated the reliability of reporting to be better inmorbidities for which there are clear diagnostic criteria eg diabetes [] Furthermore with many educational resources promoting selfmanagement of thiscondition patients with diabetes are more likely toplay an active role in managing their condition egregular selfmonitoring of blood glucose levels dietarymanagement recognising and dealing with symptomssuch as hypo and hyperglycaemia andor medication taking and are therefore more likely to selfreport accurately []There was ˜good™ agreement between both measures ofmorbidity for osteoporosis and for glaucoma in the olderage group A MultiCare cohort study of primary carepatients in Germany found only moderate agreement between patientreported and GPreported osteoporosis[] A retrospective cohort study of older patients in asecondarycare setting in Canada also found moderateagreement for glaucoma between physician and patientreports [] Similar to diabetes patients are required toplay an active role in the management of osteoporosiswhile glaucoma is very often a comorbidity of diabetes[]There was ˜moderate™ agreement between the measures of morbidity for asthma in the younger age cohort years Similar results have been reported for agreement between selfreported asthma and medical recorddata in older hospitalised patients [] There was also˜moderate™ agreement for high cholesterol in both agecohorts and for angina and Parkinson™s disease in the 0cMannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRiskV algorithm ‰¥ yearsTILDASelfreported morbidityDiabetes or high blood sugarRxRiskV Pharmacy claimsMedicationbased Morbidity ATC KappaκNStandardErrorN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX OsteoporosisPaget™s disease M05BA01M05BB09 M05BX03Pain taking pain medication Pain Opioids N02AA01N02AX02 GlaucomaOsteoporosisParkinsonAnginaHigh CholesterolManic depressionHigh blood pressure orHypertensionG03XC01 A12AX92Parkinson™s disease N04AA01N04BX02 Angina C01DA02C01DA14 C01DX16 C01EB17C01EB18 Hyperlipidaemia C10AA01C10BX17 Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02Bipolar disorder N05AN01C09BA09 C09DA01C09DA09 C02AB01C02AC05 C02DB02C02KX01PainAbnormal Heart RhythmDepressionDementiaChronic lung disease such aschronic bronchitis or emphysemaCancer or a malignant tumourEmotional nervous or psychiatricproblem such as depression oranxietyPain Inflammation M01AB01 M01AH06 Pain Opioids N02AA01N02AX02Pain Inflammation M01AB01 M01AH06 Arrhythmia C01AA05 C01BA01C01BD01 C01BD07 Depression N06AA01N06AG02 N06AX Dementia N06DA02 N06DA01Chronic airways disease R03AC02R03DC03 Malignancies L01AA01L01XX31 Anxiety N05BA01 N05BA12Congestive heart failureCirrhosis or serious liver damageHeart attack including myocardialinfarction or coronary thrombosis Chronic heart failure C03CA01C03CC01 C09AA01C09AA10C09CA01 C09CA03 C09CA06C09CA07Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11Antiplatelet therapy B01AC04B01AC30AnxietyStomach ulcersAlcohol or substance abuseAnxiety N05BA01N05BA12 GORD Peptic ulcer A02BA A02BCAny other heart trouble Stroke cerebral vascular diseaseMinistroke or TIA Alcohol dependence N07BB01 N07BB04Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Antiplatelet therapy B01AC04B01AC30Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06 B01AB10Incontinence Neurogenic Bladder Urinary Incontinence V07ANPsychotic illness N05AA01 N05AX17PsychosisATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient prescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm [] 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskVAge yearsGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecall YesDepression YesPolypharmacyYesAgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyHypertension HeartAttack “ “StrokeTIAHigh Cholesterol “ “ “HeartTrouble “Cancer “EmotionalProblems “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Depressiononly “ “ “ “ “ “Stomachulcers “ “ “ “ “ “Asthma “ “ “ “ “ “Arthritisgeneral “ “ “ “ “ “RheumatoidArthritis only “ “ “ “ “ “ “ “ “Angina “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Congestive HeartFailure “Abnormal HeartRhythm “ “ “ “ ““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ ““““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ ““ “ “ “““““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Anxiety “ “ “ “ “ “ “ “ “ “LungDisease “ “ “ “ “ “ “ “ “ “ 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskV ContinuedHypertension HeartAttackOsteoporosis “Psychosisonly “StrokeTIAHigh CholesterolHeartTroubleCancerEmotionalProblemsAnxietyIncontinence PainPain meds “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “AgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyExcluded diabetes Parkinson™s disease manic depression cirrhosis glaucoma alcohol or substance abuse and dementia as number of patients misreporting wassmall N p older age cohort Other studies have reported loweragreement for high cholesterol and higher agreement forangina and Parkinson™s diseases [ ] Discordancehere may be explained by patients managing their cholesterol using nonpharmacological means eg lifestylemodifications[]Interestingly the prevalence of selfreported angina inTILDA was higher than the prevalence reported by RxRiskV This may reflect poor patient adherence if prescribed medications were not dispensedincluding cardioprotective dietThere was only ˜fair™ agreement between both measures of morbidity for hypertension despite hypertensionbeing the most prevalentselfreported morbidityacross both age cohorts Higher agreement betweenselfreported antihypertensive drug use and pharmacyrecords has been reported in a populationbasedstudy and a cohort study of older people in theNetherlands [ ] The discordance observed hereis likely attributable to the omission of a major group[]increasingantihypertensivesofcalciumchannelblockersCCBs in the current version of the RxRisk and RxRiskV algorithms [ ] This is significant giventhat CCBs are recommended as firstline therapy inpatients aged years [] Equally since hypertension is considered to be a condition without symptomsthis may influence patient adherence toantihypertensive medications and their proclivity tofill a prescription for these medications There wasalso ˜fair™ agreement for pain in the older age groupwith agreementsomewhat when selfreported pain specified ˜taking pain medication™ Theprevalence of selfreported pain was higher than themedicationbased RxRiskV prevalenceand thismay be due to patients managing their pain throughnonpharmacological or lifestyle interventions such asphysiotherapy and cognitive behavioural therapy []In both age cohorts there was œpoor to fair agreement between selfreporting of emotional problems 0cMannion BMC Geriatrics Page of poorfoundagreementeg depression anxiety and medicationbased measures These findings are consistent with previous research whichbetweenphysician diagnosis and patient selfreports of anxiety and depression [] This low level of agreementmay be due to a potential stigmatisation bias as only of patients regularly dispensed antidepressants selfreported as having depression in theolder age cohort [ ] Equallyit may be thatcertain antidepressants eg amitriptyline are beingused for other indications such as neuropathic pain[ ] There was also ˜poor™ agreement in bothage cohorts for stomach ulcers and for incontinenceand chronic airways disease COPD in the older cohort Like depression poor agreement here may bedue to gastrointestinal medications being used by patients for other indications such as preventative orsymptomatic reasons [] The poor agreementforchronic airways disease may reflect the nonspecificquestion used in TILDA to measure this selfreportedmorbidity as there is evidence in the literature thatquestionnaire design is an important determinant ofpatient recall In a US study the prevalence of selfreported COPD was found to increase when more explicit questions were asked about emphysema chronicbronchitis and COPD in combination [] The pooragreement between the two measures for incontinenceis most likely reflective of the current version of theRxRiskV which compares selfreported urinary incontinence with dispensed ˜diapers and pads supplies™ []agepoordelayedincreasingA number of factors were associated with discordance between the two measures of morbidity particularlyrecalldepression and polypharmacy A study determiningthe agreement between selfreported and diagnosisbased multimorbidity in older community dwellingwomen reported similar findings where agreementwas found to decrease with decreasing cognition andeducation increasing age and fo

Thyroid_Cancer

Neurogenesis From Neural CrestCells Molecular Mechanisms in theFormation of Cranial Nerves andGangliaKarla MndezMaldonado12  Guillermo A VegaLpez34  Manuel J Aybar34 andIv¡n Velasco15 Instituto de Fisiolog­a Celular “ Neurociencias Universidad Nacional Autnoma de Mxico Ciudad de Mxico Mexico Departamento de Fisiolog­a y Farmacolog­a Facultad de Medicina Veterinaria y Zootecnia Universidad Nacional Autnomade Mxico Ciudad de Mxico Mexico Instituto Superior de Investigaciones Biolgicas INSIBIO CONICETUNT SanMiguel de Tucum¡n Argentina Instituto de Biolog­a œDr Francisco D Barbieri Facultad de Bioqu­mica Qu­mica yFarmacia Universidad Nacional de Tucum¡n San Miguel de Tucum¡n Argentina Laboratorio de Reprogramacin CelularInstituto Nacional de Neurolog­a y Neurocirug­a œManuel Velasco Su¡rez Ciudad de Mxico MexicoThe neural crest NC is a transient multipotent cell population that originates in thedorsal neural tube Cells of the NC are highly migratory as they travel considerabledistances through the body to reach their final sites Derivatives ofthe NC areneurons and glia of the peripheral nervous system PNS and the enteric nervoussystem as well as nonneural cells Different signaling pathways triggered by BoneMorphogenetic Proteins BMPs Fibroblast Growth Factors FGFs Wnt proteins Notchligands retinoic acid RA and Receptor Tyrosine Kinases RTKs participate in theprocesses of induction specification cell migration and neural differentiation of the NCA specific set of signaling pathways and transcription factors are initially expressed inthe neural plate border and then in the NC cell precursors to the formation of cranialnerves The molecular mechanisms of control during embryonic development havebeen gradually elucidated pointing to an important role of transcriptional regulatorswhen neural differentiation occurs However some of these proteins have an importantparticipation in malformations of the cranial portion and their mutation results in aberrantneurogenesis This review aims to give an overview of the role of cell signaling and of thefunction of transcription factors involved in the specification of ganglia precursors andneurogenesis to form the NCderived cranial nerves during anogenesisKeywords cranial nerve peripheral nervous system hindbrain cell signaling transcriptional regulatory networktrigeminal nerve facial nerve vagus nerveAbbreviations BMP bone morphogenetic proteins CN cranial nerve CNS central nervous system DRG dorsal rootganglia FP floor plate hESCs human embryonic stem cells Msx Muscle segmentrelated homeobox NC neural crestNCCs neural crest cells NT neural tube PA pharyngeal arches Pax Paired box PNS peripheral nervous system rrhombomere RA retinoic acid RTK receptor tyrosine kinase Shh Sonic hedgehog Sox Srybox VSMC vascular smoothmuscle cells Wnt Wingless and Int1 WRPW TrpArgProTrp Zic Zinc finger protein of cerebellumEdited byMichael PiperThe University of QueenslandAustraliaReviewed byRoberto MayorUniversity College LondonUnited KingdomRebecca McLennanStowers Institute for MedicalResearch United StatesCorrespondenceManuel J AybarmanuelaybarfbqfunteduarIv¡n Velascoivelascoifcunammx These authors have contributedequally to this workSpecialty sectionThis was submitted toStem Cell Researcha section of the journalFrontiers in Cell and DevelopmentalBiologyReceived April Accepted June Published August CitationMndezMaldonado KVegaLpez GA Aybar MJ andVelasco I Neurogenesis FromNeural Crest Cells MolecularMechanisms in the Formationof Cranial Nerves and GangliaFront Cell Dev Biol 103389fcell202000635Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alINTRODUCTIONDuring the embryonic development of vertebrates one of themain events after the gastrulation process is neurulation whichallows the formation of the neural tube NT The neuralectoderm generates not only the central nervous system CNSbut also another set of cells between the NT and the nonneural ectoderm located in the most dorsal part of the NT calledthe neural crest NC Hall SimµesCosta This versatile and plastic cell population was first described byWilhelm His years ago Hall The NC is one of themost important features that separate vertebrates from otherchordate anisms It arises at the posterior and lateral bordersof the neural and nonneural ectoderm the neural plate borderFigure Cerrizuela the embryo NCCs are divided into cranialNC cells NCCs are multipotent and give rise to several celltypes depending on the site of origin along the anteroposterioraxis oftrunkincluding cardiac vagal and sacral A Minouxand Rijli SimµesCosta and Bronner VegaLopez Cranial nerves CN transmit sensory and motorinformation between the brain and tissues of the head andcervical region The CN are formed from the contribution oftwo specialized embryonic cell populations cranial NC andectodermal placodesOrigin of the Neural CrestNCCs which are multipotent delaminate from their origin andmigrate throughout the body to diï¬erentiate into several celltypes including cells of the peripheral nervous system PNSmelanocytes cranial cartilage and bone neuroendocrine cellsand several other phenotypes B In humans at least cell types have been defined as NC derivatives Vickaryousand Hall Proper NC migration relies on environmentalcues such as EphEphrins Smith Semaphorin3F Gammill Versican Szab thechemokine Stromal cellderived factor Theveneau or Robo2 Shiau The migration patternsof NCCs have been clearly described for model anisms likebirds frogs and mice In all vertebrates cranial NCCs emergefrom the forebrain midbrain and hindbrain regions Coulyand Le Douarin Serbedzija Depending ontheir axial origin cranial NCCs will either migrate through thefacial mesenchyme and into the frontonasal process or willpopulate the branchial arches Noden Lumsden Serbedzija The sensory module of the PNSin the cranial region is composed of an array of paired gangliaadjacent to the hindbrain that transduce the perception of touchpain temperature position and special sensory informationfrom the periphery to the CNS Cranial NCCs migrate to formsensory ganglia such as the trigeminal V the facial VII theglossopharyngeal IX the vagus X CN and also to form themotor ganglia for the oculomotor III and accesory XI CNTable and Figures acomplex and multistep processinitially directed by cell signaling molecules including BoneMorphogenetic Proteins BMPs Wnts Wingless and Int1NC formation isNeural CrestDerived Cranial NervesFibroblast Growth Factor FGF and retinoic acid RA Thesesignals reveal the tissue interactions into the ectodermal cellpopulations the neural plate the nonneural ectoderm and theunderlying mesoderm in a highly coordinated manner VegaLopez It has been proposed that NC specificationoccurs during gastrulation as a consequence of the action oftwo successive gradients of secreted signals A combinationof intermediate levels of activity of BMP and Wnt signalingacting on the ectoderm to induce and specify NC precursorsat the neural plate border and a subsequent requirement ofboth signals is needed for maintenance of specification duringneurulation Aybar and Mayor Steventon Inchick embryos it was shown that NCCs are specified as earlyas the blastula stage Prasad It was demonstratedthat during gastrulation Pax7 expression is restricted to cellslocated in a region in the medial epiblast which are NCfated andcontribute to the neural folds and later to migrating NCCs Basch The inhibition of Pax7 function in chicks inhibitedthe expression of key NC markers such as Snai2 OMIM Sox9 Srybox OMIM Sox10 OMIM andHNK1 beta13glucuronyltransferase like OMIM Basch and BronnerFraser This evidence suggests thatthe neural plateprospective ectoderm interaction at the neuralplate border might not be a requisite for NC specification orinduction and that neural plate border formation and NCinduction might be separable eventsThe variousresearch works carried outto study theorigin of NCCs have identified genes anized into a generegulatory network that participate in and control the inductionspecification and diï¬erentiation of NC SimµesCosta An example of this are the transcription factors involvedin induction such as FoxD3 Forkhead Box D3 OMIM Snai2 and Sox9 SaukaSpengler and BronnerFraser Garc­aCastro and coworkers identified a novel preneuralborder state characterized by early Wntβcatenin signalingtargets that displayed diï¬erent responses to BMP and FGFsignaling from the neural border genes in human cells Leung These preborder genes Gbx2 Gastrulation brainhomeobox OMIM SP5 OMIM Zic3 OMIM and Zeb2 OMIM had their induction andpeak of expression before the classical neural plate borderspecifier genes such Msx12 Muscle segmentrelated homeobox OMIM Pax37 OMIM and Zic1 OMIM Such specifier genes together withsignaling molecules direct the expression of NCspecific geneslike AP2 OMIM FoxD3 Snai2 Sox9 and Sox10Specifiers regulate NC eï¬ector genes involved in migrationSox9 Sox10 Cad7 and diï¬erentiation [Col1a Collagen typeI alpha OMIM Ngn1 Neurogenin OMIM MitfMicrophthalmiaassociated transcription factor OMIM] in human NC development Betters The NC population migrates to diï¬erent regions of the mouseembryo from the NT after the epithelialmesenchymal transitionmaintaining its multipotential character until completingdiï¬erentiation in its final destination Baggiolini To study the ontogeny of the NC diï¬erent model anismsboth in vivo and in vitro have been used Several proteinsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Neural crest origin regions in human and mouse embryos and some of its cranial derivatives A The topleft part of the scheme shows the origin of theneural crest cells green that migrate through the embryo On the topright side the level of axial origin see axial color key of different regions of the neural crest isrepresented in developing mouse or equivalent human embryos the migration of neural crest is represented in green inside the embryos and the direction ofmigration is marked with black arrows Depending on their axial level of origin and migratory pathways neural crest cells adopt different fates and contribute tovarious tissues and ans B The main cranial derivatives labeled in green are shown Abbreviations d days E mouse embryonic stage NCCs Neural CrestCells s somite St human stage VSMC vascular smooth muscle cellsincluding transcription factors as well as epigenetic modifiersthat take part in the specification and diï¬erentiation of the NChave been described The study of transcription factors and ofthe signaling pathways in which they participate is importantto understand the diï¬erentiation programs and how thesemultipotent cells are committed to a specific destination On theother hand transcriptome analysis during the development of theNC from specification to migration Meulemans and BronnerFraser and a more recent study covering the migrationto the diï¬erentiation of the NC show the importance of theinteraction between the diï¬erent transcription factors and thesignaling pathways at every stage of NC development SimµesCosta However these authors acknowledge thatit is difficult to have a complete global map since only a fewtranscriptional regulators have been characterized and little isknown about the function of the products of the eï¬ector genesacting on NC migration Betancur SimµesCosta andBronner VegaLopez NC and cranial placodes are thought to appear togetherduring the evolution of vertebrates to give rise to specific sensorystructures of the head Northcutt and Gans Northcutt The components of the sensory nervous system of the headare derived from the NC and from an embryonic cell populationdeveloping in close proximity the cranial sensory placodes theolfactory lens otic trigeminal epibranchial and paratympanicplacodes A series of events induce develop and anizethese cell precursors which through reciprocalinteractionswith NCCs build the functional sensory system in vertebratesSteventon Singh and Groves Migrating NCCsarrive first at the site of ganglia development ie the trigeminalFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cFrontiersinCellandDeveopmentallliBoogywwwfrontiersniAugustlVoumeAlrticeTABLE Contributions of neural crest cells and placodes to ganglia and cranial nervesCranial nerveGanglion and typeOrigin of neuronsReferencesCNI “ Olfactory Ensheating gliaof Olfactory nervesCNIII “ Oculomotor mCiliary visceral efferentCNV “ Trigeminal mixTrigeminal general afferentCNVII “ Facial mixSuperior general and special afferentCNVIII “ Vestibulocochlear sCNIX “ Glossopharyngeal mixCNX “ Vagus mix Superiorlaryngeal branch and recurrentlaryngeal branchCNXI “ Accessory mInferior geniculate general and special afferentSphenopalatine visceral efferentSubmandibular visceral efferentAcoustic cochlear special afferent and Vestibularspecial afferentSuperior general and special afferentInferior petrosal general and special afferentOtic visceral efferentSuperior general afferentInferior nodose general and special afferentVagal parasympathetic visceral efferentNo ganglionTelencephalonolfactory placode NCCs at forebrainNCCs at forebrainmidbrain junction caudal diencephalonand the anterior mesencephalonNCCs at forebrainmidbrain junction from r2 into 1st PAtrigeminal placodeHindbrain NCCs from r4 into 2nd PA 1st epibranchialplacode1st epibranchial placode geniculateHindbrain NCCs 2nd PAHindbrain NCCs 2nd PAOtic placode and hindbrain from r4 NCCsHindbrain NCCs from r6 into 3rd PA2nd epibranchial placode petrosalHindbrain NCCs from r6 into 3rd PAHindbrain NCCs from r7r8 to 4th and 6th PAHindbrain NCCs 4th and 6th PA 3rd nodose and 4thepibranchial placodesHindbrain NCCs 4th and 6th PAHindbrain from r7r8 to PA NCCs 4th PABoyd Muller O™Rahilly andM¼ller Barraud Noden Couly Wahl Lee d™AmicoMartel and Noden Forbes and Welt D™amicoMartel and Noden D™amicoMartel and Noden Lumsden Barlow and Northcutt Begbie andGraham Barlow Krimm Sandell Narayanan and Narayanan D™amicoMarteland Noden O™Rahilly and M¼ller Barlowand Northcutt Narayanan and Narayanan D™amicoMarteland Noden Muller and O™Rahilly O™Rahilly and M¼ller Abbreviations CN Cranial Nerve m purely motor nerve mix mixed nerve sensory and motor NC neural crest PA pharyngeal branchial arch r rhombomere s purely sensory nerve There is no known ganglionof the accessory nerve The cranial part of the accessory nerve sends occasional branches to the superior ganglion of the vagus nerveMndezMadonadoletalNeuralCrestDerivedCranailNerves\x0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Contribution of neural crest cells to the formation of cranial nerves I III V VII VIII IX X and XI These selected cranial nerves are formed by thecontribution of cranial placodes and neural crest cells indicated in green Neural crestderived Schwann cells produce peripheral myelination of cranial nerves III“XIIThe sensory nerves are the olfactory I the optic II and the vestibulocochlear VIII The motor nerves are the oculomotor III the trochlear IV the abducens VIand the accessory XI The remaining are mixed nervesganglion but the diï¬erentiation of these cells is delayed untilthe migration and diï¬erentiation of the corresponding placodalcells in chicks Covell and Noden Placodal specificationand development as well as its contribution to the assembly ofplacodal derivatives is a complex and wideranging topic thatis beyond the scope of this review We will focus on discussingthe main signaling pathways and relevant transcription factorsinvolved in the specification of cranial NCCs precursors theirdiï¬erentiation to form CNs and ganglia that are exclusively NCderived and the alterations caused by the mutations of certaingenes that are important for the neurogenesis of NC derivativesSIGNALING PATHWAYS INVOLVED INCRANIAL NEURAL CRESTDEVELOPMENTareseveralsignaling pathwaysThereand transcriptionfactors that are known to regulate NC and CN formationduring development We discuss some important pathwaysinvolved in cranial NCCs induction and specification in closerelationship with the cranial ganglia and nerves derived from theNC Figure BMPsBone morphogenetic proteins are proteins that control severalimportant steps in the formation and diï¬erentiation of the CNSof vertebrates These proteins act in diï¬erent regions of theCNS to regulate fate proliferation and diï¬erentiation Aftergastrulation the presence of BMPs and the activation of thissignaling pathway are essential for the diï¬erentiation of thenonneural ectoderm whereas the inhibition of this pathway isrequired for the proper formation of the neural plate It has beenproposed that the later activation of BMPs receptors participatesin the induction of the NC through a very fine regulation wherethe presence of BMPs at a specific time will give rise to the NCin mouse and human Embryonic Stem Cells ESCs Figure 3BMizuseki Leung Seminal studies in Xenopus have shown that there is an activitygradient of BMPs controlled by their antagonists and that anintermediate level is needed to induce the formation of the NCLaBonne and BronnerFraser Marchant Barth Tribulo Thus the BMP antagonistsChordin OMIM and Noggin OMIM areexpressed in a spatiotemporal manner thatinfluences theformation of the NC In mouse at embryonic day E Nogginis expressed in the neural folds and in the dorsal region afterthe closure of the NT The expression of Chordin is low atthe level of the neural plate and in the paraxial mesodermThese antagonists participate in the induction of NC as wellas in delamination but also protect from apoptosis induced byBMP during migration and diï¬erentiation of NCCs Importantlyit was observed that the decrease in the expression of theseBMP antagonists alters the PNS derived from the NC andcraniofacial skeletal elements Noggin knockout mice presentedall cranial nerves but the vagus X and glossopharyngeal IX aredisanized and fused Doubleknockout mice of Noggin andChordin lack CN and only a structure similar to the trigeminalganglion V is present Anderson In the chickembryo the activity of BMP signaling during the formation ofNC precursors is modulated by CKIPSmurf factors throughthe regulation of Smad degradation resulting in intermediateFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Gene regulatory network involved in neural crest contribution to the formation of cranial nerves The cranial ganglia and cranial nerves are formed inprecise positions along the dorsoventral and anteroposterior axes of the midbrainhindbrain region A The drawing represents a human embryo at stage days somites equivalent to mouse day E9510 somites and chick stage h somites The cell signaling pathways that providedevelopmental cues to neural crest precursors are colorcoded when these factors diffuse the target regions are indicated with arrows with the same color In panelB an idealized scheme of the hindbrain shows the cell signaling gradients and the genes that establish the dorsoventral pattern C The illustration of the human days stage and chick stage hindbrain rendered flat to eliminate cerebral flexures The levels of origin of the neural crest cells NCCs and placodeswhich contribute to the formation on cranial nerves are indicated on the left NCCs from the corresponding rhombomeres also populate other embryo structures in asegmental fashion and generate different craniofacial derivatives The positions of the cranial ganglia and the otic vesicles are indicated on the right side thecontribution of NCCs is indicated in green The segmental nested expression of HOX genes is colorcoded On the right signaling pathways and the expression oftranscription factors involved in cranial nerve CN formation are indicated Adapted from Lumsden and Keynes Noden Yamamoto and Schwarting BallyCuif and Wassef Takahashi and Osumi and M¼ller and O™Rahilly Abbreviations CN cranial nerve FP floor plate Mmesencephalon NCCs neural crest cells OV otic vesicle r rhombomere PA pharyngeal archeslevels of BMP activity required for proper NC formationPiacentino and Bronner In contrast placode progenitorshave diï¬erential BMP signaling requirements as they can bespecified under low or no BMP signaling Thiery A study of human ESCs hESC showed that if BMPs areblocked with Noggin for h on days or of thediï¬erentiation protocol there is a dramatic decrease in theinduction of human NCCs However if the inhibition is madeon day the inhibition is partial so the participation of BMPsat the beginning of the induction of the NC is very importantwhile the inhibition of this pathway promoted the expressionof neural genes such as SOX1 OMIM HES5 OMIMFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial Nerves and SOX2 OMIM Leung Thisprotocol produced sensory peripheral neurons and it will beof interest to investigate if such neurons can contribute to thesensory CN after grafting them in experimental animals aswell as the eï¬ect of modulating BMPs on peripheral neurondiï¬erentiation Interestingly BMP antagonism upregulates theseneural stem cell markers but several reports indicated that Sox1Hes5 and Sox2 are involved in the suppression of neuronaldiï¬erentiation by maintaining neural stem and progenitorcells in an undiï¬erentiated state in mammalian cells Kan BaniYaghoub The generation ofneurons from stem cells depends on the decrease of Sox13expression caused by proneural proteins However if Sox13target genes were repressed independently of proneural activityneural progenitor cells diï¬erentiated prematurely and someneuronal features emerged These results demonstrate a dualrole of proneural proteins in the acquisition of a definitiveneuronal fate and indicate that the proneural proteindirectedrepression of Sox13 expression is a required and irreversiblestep in the commitment to neuronal diï¬erentiation in severalspecies including mammals Guillemot Farah Bylund BMP4 OMIM and Smad proteins have beeninvolved in an interesting mechanism called retrograde signalingin trigeminal ganglia from rats Ji and Jaï¬rey Thismechanism elicits a specific transcriptionalresponse thatcontributes to the specification of diï¬erent subpopulations ofsensory neurons in the trigeminal ganglia CN V As axonsfrom the neurons oftrigeminal ganglia grow and extendinto their three main peripheral axonal branches ophthalmicmaxillary and mandibular that innervate the correspondingregions of the face they encounter BMP4 which results in aretrograde signal that leads to transport back transcription factorsSMAD1 and from axons to the somata where nuclearaccumulation of the phosphorylated and transcriptionally activeSmad forms contributes to neuronal specification and gangliapatterning Nohe Ji and Jaï¬rey BDNF BrainDerived Neurotrophic Factor OMIM signaling wasalso found to regulate axonal levels of SMAD1 and inconcert with BMP4 for patterning of the trigeminal gangliaJi and Jaï¬rey Hippo PathwayGenetic studies have demonstrated that Hippo signaling is crucialin an size regulation controlling cell number by modulatingcell proliferation and apoptosis processes Huang Hippo is a criticalfactor for proliferation and epithelialmesenchymal transition during embryonic development andcancer In the neural tube of the mouse chicken and frogYAP YesAssociated Protein OMIM is expressed inthe ventricular zone progenitor cells and colocalizes with theneural progenitor cell marker Sox2 Milewski Cao It has been observed that the ectopic expressionof one ofthis pathwayTAZ Transcriptional Coactivator With PDZBinding MotifOMIM in mammalian cells stimulates cell proliferationthe transcriptional regulators ofreduces the inhibition by contact and promotes the epithelialmesenchymal transition Lei A relationship between this signaling pathway and the classicalNC genes such as interaction with Pax3 is through TAZ andthe phosphoprotein YAP65 These proteins participate as coactivators of Pax3 It has been suggested using transgenicmice that Tead2 TEA Domain Family Member OMIM is an endogenous activator of Pax3 in NCCs Milewski Through expression assays Pax3 and Yap65were colocalized in the nucleus of NC progenitors in thedorsal region offorSchwann cell proliferation and diï¬erentiation in a stagedependent manner Nuclear TAZYAP complexes activate cellcycle regulators to promote Schwann cell proliferation whiledirecting diï¬erentiation regulators in cooperation with Sox10 formyelination in rodents Deng the NT HippoTAZYAP are criticalNeurofibromatosis Nf2 OMIM is a tumorsuppressor that inhibits YAP during dorsal root ganglia DRGdevelopment Merlin encoded by the NF2 tumorsuppressivegene was identified through genetic studies in mouse embryosand proved to be an important upstream regulator of theHippoYap pathway Neurofibromatosis is an inherited diseasecharacterized by the development of bilateral Schwann celltumors originated from CN VIII Mouse with specific Schwanncellinactivated Nf2 alleles developed schwannomas and SChyperplasia McClatchey Giovannini Merlin has also been shown to act as a suppressor ofmouse neural progenitor proliferation by inhibiting TAZYAPpathway activity Lavado The mechanism bywhich Merlin regulates YAP activity might involve p21 Proteinactivated kinase PAK1 OMIM activation whichinduces phosphorylation of Merlin thus abrogating its scaï¬oldfunction for YAP and LATS12 OMIM andthereby attenuates YAP phosphorylation by LATS12 in mousecells Sabra it has been suggested that nuclearexport signals of Merlin mediate YAP nuclear export in epithelialmammalian cells Furukawa Hindley and coworkers investigated the role of HippoYAPsignaling in NC development and neural diï¬erentiation Theyshowed thatthe activity of YAP promotes an early NCphenotype accompanied by premature migratory behavior andthat HippoYAP interacts with RA signaling in hESCs Hindley A recent study demonstrates that YAP is necessaryfor the migration of a premigratory pool of NCCs sincethey incorporated YAP signaling into a BMPWntdependentmolecular network responsible for the migration of trunklevelNC in avians Kumar Notch SignalingNotch is a family of conserved receptors whose activation isinduced by specific ligands Delta1 OMIM Delta OMIM Delta4 OMIM Jagged1 OMIM and Jagged2 OMIM through interactionwith four possible receptors Notch14 Perdigoto and Bardin Once the Notch receptors are activated through thecellcell interaction proteolytic cuts are carried out resultingin the release ofthe Notch Intracellular Domain NICDFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesMumm NICD translocate to the nucleus andforms a transcriptional complex together with the DNA bindingprotein CBF1 C promoter binding factor OMIM This complex recognizes the specific sequence CTGTGGGAAin itstarget genesfor example Hes1 OMIM Kageyama Notch1 receptor is present during development oftherhomboencephalon at E95 in mice showing strong expressionwithin the hindbrainincluding the trigeminal geniculatepetrosum and nodose placodes which give rise to CN VVII IX and X respectively and is also expressed in the oticand olfactory vesicle Reaume A study wherehuman induced pluripotent stem cells were induced toward NCdiï¬erentiation showed that when Notch signaling is blockedusing a Îsecretase inhibitor DAPT or shRNA for JAGGED the genes specifying NC [DLX5 Distalless homeobox OMIM PAX3 SNAI2 SOX10 and TWIST1 OMIM] are downregulated However the ectopic expression ofNICD1 increased its expression demonstrating that Notch alsoparticipates significantly in NC induction Noisa Mead and Yutzey evaluated the function of Notch signaling inmurine NCderived cell lineages in vivo They demonstratedthat cellautonomous Notch has an essential role in properNCCs migration proliferation and diï¬erentiation with criticalimplications in craniofacial cardiac and neurogenic developmentMead and Yutzey Sonic HedgehogSonic Hedgehog Shh signaling is involved in the correctdevelopment of NC and therefore in the generation of its cellularderivatives Figure 3B Shh is a member of the family ofthe secreted Hedgehog proteins Sonic Shh OMIM Indian Ihh OMIM and Desert Hedgehog Dhh OMIM Shh regulation during NC diï¬erentiation is crucialduring head and face morphogenesis Mutant mice and humanslacking Shh present holoprosencephaly and cyclopia due to thelack of separation of the forebrain lobes Chiang It is suggested that Shh inhibition maintains Pax3 expressionso the lack of Shhmediated regulation for Pax3 inductionpromotes the constitutive induction of NC generating theaforementioned phenotypes A subset of Fox genes regulated byShh signaling is important during lip morphogenesis in miceEither Shh addition or Foxf2 OMIM overexpressionwas shown to be sufficient to induce cranial NCCs proliferationEverson On the other hand enhanced Shh signaling in mousemediated by lossoffunction Ptch1WigWig of the Shh receptorPatched1 Ptch1 OMIM suppressed canonical Wntsignaling in the CN region This critically aï¬ected the survivaland migration of cranial NCCs and the development ofplacodes as well as the integration between NC and placodesKurosaka Ptch1WigWig mutants exhibited severelydisanized trigeminal CNV and facial nerves CNVII thatdid not develop properly and failed to project to their appropriatetargettissues Kurosaka High levels of Shhsignaling have been correlated with Moebius Syndrome whichis characterized by cranial nerve defects including trigeminalabducens CNVI and facial alterations concurrent with othercraniofacial defects Verzijl VegaLopez NCCs migration is particularly sensitive to Shh levels since inmice lacking Shh these cells continue their migration beyondthe normal position and fuse medially condensing into a singlemidline ganglion Fedtsova Mutation in the mouseHedgehog acyltransferase Hhat OMIM gene producedhypoplasia and aberrant fusion of cranial ganglia CN V VII IXand X and aï¬ected NC and placode gene markers expressionsuggesting that a regionalized action of the Hedgehog signalingis required for proper cranial ganglia and nerve developmentand patterning Dennis In vitro analyses showedthat Shh increased the number of cranial NC progenitors fromquail embryos yielding neural and mesenchymal lineages Shhcan decrease the neuralrestricted precursors without aï¬ectingsurvival or proliferation These data also suggestthemesenchymalneural precursor was able to yield both the PNSand superficial skeleton Calloni thatReceptor Tyrosine Kinase RTK FamilyHumans have known RTKs which fall into subfamiliesA few years ago a systematic work summarized the contributionof the mouse model to the understanding of the role of a subsetof RTKs in regulating the activity of NCCs in developmentFantauzzo and Soriano With respect to its downstreamsignaling RTKs induce the activation of various pathwaysincluding PLCÎ PI3K MAPK JNK Shc Erk and the JAKSTATpathways In this section we discuss insights pointing tomechanisms of action of some RTK families in relation tothe development of the cranial NC that have emerged fromrecent evidenceEph ReceptorsEphrin ligands and Eph erythropoietinproducing humanhepatocellular carcinoma receptors comprise an increasinglywell studied family of signaling molecules Ephrins bind totwo families of transmembrane tyrosine kinase receptors EphAand EphB While Atype Ephrins preferentially bind to EphAreceptors Btype Ephrins do so to EphB receptors In Xenopusthe streams of NCCs going to the second branchial arch expressEphrinB2 whereas cells reaching the third arch express EphB1disruption of EphEphrin signaling results in aberrant migrationof NCCs causing mixing of the streams in the branchial pouchesSmith Eph receptor functions are best characterizedin the mouse nervous system where they are involved inneuronal development and axon guidance Wilkinson Xuand Henkemeyer migration and proliferation Conover H

Thyroid_Cancer

"Insulin shares a limited physiological concentration range with other endocrine hormones Not onlytoo low but also too high systemic insulin levels are detrimental for body functionsMain body The physiological function and clinical relevance of insulin are usually seen in association with its rolein maintaining glucose homeostasis However insulin is an anabolic hormone which stimulates a large number ofcellular responses Not only too low but also excess insulin concentrations are detrimental to the physiologicalbalance Although the glucoregulatory activity of insulin is mitigated during hyperinsulinemia by dampening theefficiency of insulin signaling œinsulin resistance this is not the case for most other hormonal actions of insulinincluding the promotion of protein synthesis de novo lipogenesis and cell proliferation the inhibition of lipolysisof autophagydependent cellular turnover and of nuclear factor E2related factor2 Nrf2dependent antioxidativeand other defense mechanisms Hence there is no general insulin resistance but selective impairment of insulinsignaling which causes less glucose uptake from the blood and reduced activation of endothelial NO synthaseeNOS Because of the largely unrestricted insulin signaling hyperinsulinemia increases the risk of obesity type diabetes and cardiovascular disease and decreases health span and life expectancy In epidemiological studieshighdose insulin therapy is associated with an increased risk of cardiovascular disease Randomized controlled trialsof insulin treatment did not observe any effect on disease risk but these trials only studied low insulin doses up to IUday Proof for a causal link between elevated insulin levels and cardiovascular disease risk comes fromMendelian randomization studies comparing individuals with genetically controlled low or high insulin productionConclusions The detrimental actions of prolonged high insulin concentrations seen also in cell culture argue infavor of a lifestyle that limits circadian insulin levels The health risks associated with hyperinsulinemia may haveimplications for treatment regimens used in type diabetesKeywords Hyperinsulinemia Insulin resistance Nrf2 Autophagy eNOS Obesity Type diabetes mellitusInflammation Oxidative stress Cardiovascular morbidity and mortalityBackgroundMost endocrine hormones exhibit a window of optimalphysiological concentrations with compromised function of the anism at levels below or above that rangeFor instance subnormal levels of thyroid hormone define the clinical condition of hypothyroidism above normalrepresent hyperthyroidism which usuallyrequires therapy Addison™s disease is characterized bylevels Correspondence kerstinkempfwdgzde2WestGerman Centre of Diabetes and Health Duesseldorf Catholic HospitalGroup Hohensandweg Duesseldorf GermanyFull list of author information is available at the end of the insufficient cortisol production while excess synthesis isseen in Cushing syndromeFor insulin we argue here that not only hypoinsulinemiabut also hyperinsulinemia is detrimental to body functionsHypoinsulinemia causes insulindeficient diabetes and thehormonal actions of insulin are necessary for the life of complex anisms [] On the other hand permanently elevatedlevels of insulin may cause disturbance of normal cellularphysiology and an function We describe the molecularbasis of these undesired insulin actions and consequences ofhyperinsulinemia for healthrelevant endpoints such as obesity or cardiovascular diseases The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKolb BMC Medicine Page of transformingproteins345trisphosphateMain textInsulin signaling pathwaysBinding of insulin to its cognate cell surfacebound receptor causes a conformational change which initiatesa cascade of signaling events Autophosphorylation bythe insulin receptor tyrosine kinase is accompanied bytyrosine phosphorylation of receptor substrates suchas insulin receptor substrate IRS and Src homology domaincontainingSHCproteins Phosphorylation of IRS allows binding ofphosphatidylinositol3kinase PI3K and synthesis ofphosphatidylinositolPIP3which eventually leads to the phosphorylation and activation of the serinethreoninespecific protein kinaseB AKT Upon activation AKT interacts with severalsubstrates which mediate anabolic effects of insulinthese include glucose uptake glycogen synthesis denovo lipogenesis and protein synthesis [] Additionalpathways triggered by the activated insulin receptorcomprise phosphorylation of SHC followed by activathe Rat sarcoma Ras“rapidly acceleratedtion offibrosarcoma Raf“mitogenactivated protein kinasesignalregulated kinasekinaseERK pathway Theamitogenactivated kinase promoting cell proliferationand further cellular activities including protein synthesis [] Another pathway triggered by the engaged insulin receptor involves activation of NADPH oxidase and subsequent hydrogen peroxidemediated inhibition of phosphatase and tensin homolog PTENwhich is an important negative regulator of PI3Ksignaling [] Fig terminal kinase ERK isMEK“extracellularInsulin secretionInsulin secretion by pancreatic islet cells responds tothe level of circulating nutrients such as glucose aminoacids and free fatty acids Sweeteners may further increase carbohydrateinduced insulin secretion A largenumber of endogenous factors contribute to the regulation of cell activity either stimulatory inhibitory orboth contextdependent These include hormones neurotransmitters and immune mediators [“] Insulin isessential for maintaining glucose homeostasis primarilyby facilitating the postmeal uptake of glucose intomuscle and fat cells via translocation of the glucosetransporter [] In the absence of dietary glucose supply and after depletion of glycogen stores glucose in circulation primarily comes from gluconeogenesis in theliver If circulating insulin levels are below the concentrations required for stimulating glucose uptake fromthe blood endogenous stores of fat and protein must beused for energy production For the maintenance of lifein the fasting state circulating insulin levels range between approx and pmoll “ percentile asdetermined for healthy adult persons in the NationalHealth and Nutrition Examination Survey NHANES[] In response to meals with varying carbohydratecontent insulin levels may rise to the range of approx“ pmoll []Insulin promotes obesityAlmost years ago insulin injections were one of theoptions of therapy in nondiabetic persons suffering fromundernutrition in the context of various diseases Insulindoses were in the range of those applied in type Fig Metabolic signaling of insulin is anabolic Insulin signaling through the insulin receptor engages several pathways and results in ananabolic state of metabolism The canonical pathway via phosphokinases PI3K and AKTPKB promotes glucose uptake and glycogen and lipidsyntheses whereas lipolysis is inhibited in adipocytes as well as hepatic gluconeogenesis In addition AKT kinases activate mTORC1 whichsupports de novo lipogenesis and protein synthesis The insulin signaling pathway via SHC and the MAP kinases MEK and ERK promotes cellproliferation and protein synthesis Another insulin signaling pathway involves NOX4 and the inhibition of PTEN an inhibitor of the PI3KAKT pathway 0cKolb BMC Medicine Page of diabetes and led to increased appetite and weight gain[] Indeed one major function of insulin as an anabolic hormone is to favor energy storage over usageThis is reflected by the finding that insulin infusion mUkgmin significantly inhibits lipolysis in the skeletalmuscle about and even more effective in adiposetissue about [] Doubling fasting insulin levelssuffices to inhibit lipolysis by approx and to promote lipogenesis for both mean insulin concentrationfor effect EC50 of approx pmoll [] At thisinsulin level gluconeogenesis is still ongoing For halfmaximal inhibition of gluconeogenesis insulin concentrations must rise to approx pmoll in arterial circulation In order to stimulate glucose uptake to halfmaximum insulin levels must rise to even higher levelsapprox ten times the fasting insulin concentrations “ percentiles for stimulating glucose uptake approx“ pmoll [] Thus a modest rise doubling offasting insulin levels will already substantially inhibit lipolysis and promote lipogenesis while gluconeogenesis isnot yet inhibited Since such small increases of systemicinsulin concentrations are enough for favoring adipogenesis fasting and diurnal insulin levels are a determinantof obesity risk Indeed several data support the obesitypromoting role of insulin for a detailed review see []Fig These include epidemiological studies which foundhigh fasting insulin levels and concomitant insulin resistance in children and adolescents to be associatedwith higher weight gain in later years [] Studies inadults are less consistent [] Pharmaceutical interventions that lower insulin secretion such as treatment withdiazoxide or octreotide led to significant body weightloss [“] This fits with the observation that insulintherapy promotes weight gain [] One probable reasonis that insulin levels in the high normal range are closeto EC50 concentrations for inhibition of lipolysis []In mice modest lowering of circulating insulin concentrations by genetic manipulation ofinsulin genescaused resistance to weight gain despite a highfat diet[] Decreasing insulin gene expression in adult micevia partial gene ablation reversed dietinduced obesity[] In men the Hph1 œT polymorphism in the insulingene region was found to be associated with higher fasting insulin levels and a more rapid weight gain in obesepersons[] A Mendelian randomization analysisshowed that persons with genetically determined higherinsulin secretion to oral glucose exhibited a higher bodymass index BMI [] supporting a causal relationshipbetween insulin and obesity riskTaken together moderate to high normal levels of insulin in metabolic healthy persons appear to be a riskfactor for the development of obesitytransientElevated insulin concentrations impair cellularfunctions”insulin œtoxicityThere is ample evidence thatincreases ofmetabolic or immune mediator levels are benign physiological responses to biochemical challenges such as therise of systemic glucose or cytokines following mealsHowever chronic elevations of such mediators evenwhen modest in amplitude are usually detrimental tocellular functions [] In the case of glucose the termglucose toxicity was coined to describe this phenomenon[] Prolonged conditions of elevated glucose concentrations cause dysfunction of numerous cell types in thebody including beta cells neurons and the endothelium via several pathways including increased oxidative stress and activation of the sorbitol pathway [“] As described below there seems to be a similardetrimental outcome oflongterm elevated insulinconcentrations on cellular functions a correspondingterm would be insulin toxicityFig Insulin promotes obesity Several independent types of observations support the conclusion that insulin promotes adipogenesis andobesity For details see description in the general text 0cKolb BMC Medicine Page of When cells are exposed to continuously elevated insulin levels there is a partial downregulation of insulin signaling The resulting œinsulin resistance is not primarilydue to less insulin receptor expression on the cell surface but due to impaired insulin signal transduction as aresult of receptor dysfunction In response to prolongedhyperinsulinemia there is diminished autophosphorylation of the insulin receptor compared to that observedafter shortterm exposure to insulin and subsequentsteps of the PI3K“AKT signaling pathway are affected[ ] Consequently in muscle and fat cells there isless AKTstimulated translocation of GLUT to the cellsurface Fig Thus insulin resistance can be seen as aprotective mechanism for preventing excess activation ofglucose transport from the blood despite chronically elevated insulin levels for maintaining glucose homeostasisin vivo and for mitigating metabolic and oxidative stressdue to excess glucose influx [“] Limiting glucoseexportfrom the blood does not necessarily requiredampening of insulin signaling During the early weeksof feeding with a high caloric diet mice show decreasedinsulindependent glucose uptake despite unperturbedinsulinstimulated AKT phosphorylation [ ] Fig An interesting aspect is that the partitioning of insulinreceptor isoforms A and B and of hybrid insulininsulinlike growth factor1 receptors among cell types maycontribute to insulin resistance in some tissues but thepathophysiological relevance is unknown []The phenomenon of insulin toxicity partly arises fromthe fact that there are additional cellular responses to elevated insulin levels which are not toned down duringrole ofinsulin resistance Fig These comprise the upregulation of protein synthesis and the accumulation of ubiquitinated or otherwise modified proteins probably dueto insufficient degradation of these polypeptides [] Amajorinsulin signaling via the canonicalmitogenactivated protein MAP kinase pathway Ras“MEK“ERK as well as via activation of NADPH oxidase has been observed [] Even some AKTdependentpathways do not appear to be suppressed by insulin resistance such as de novo lipogenesis in hepatocytes orthe upregulation of mechanistic target of rapamycincomplex mTORC1 [“] Enhanced activity ofmTORC1 leads to increased protein synthesis and to deteriorated cell functions largely because of suppressedautophagy []Hence chronic exposure of cells to high ambient insulin concentrations causes an imbalance of cellular responses because of the downregulation of some insulinsignaling pathways œinsulin resistance but not ofothers The resulting functional state of cells is characterized by an unbalanced anabolic activity of insulin favoring protein synthesis while suppressing autophagyThe latter inhibits autophagic removal and turnover ofproteins and lipids which favors cell senescence [] Inshortterm experiments of exposure to high insulinlevels a protective cellular stress response is observedthe unfolded protein response probably due to the accumulation of derivatized proteins in the absence ofenough disposal In experimentally induced or diabetesassociated chronic insulin resistance and hyperinsulinemiathesuch a protectivestressresponse ofFig Signaling of insulin during insulin resistance During insulin resistance signaling through AKT kinases is partially impaired Not all AKTdependent pathways are affected as well as other signaling pathways indicating that insulin resistance is selective Therefore hyperinsulinemiain the presence of insulin resistance promotes anabolic cell activities via the MEK“ERK pathway and via mTORC1 Although the PI3KAKT pathwayis impaired during insulin resistance and provides only insufficient translocation of GLUT4 for glucose uptake and deficient activation of eNOSthere appears to be a normal activation of mTORC1 In addition to the anabolic consequences of signaling via the MEKERK pathway depicted inthe figure there is enhanced expression of ET1 and PAI1 not shown as well as inhibition of autophagy and of the nuclear factor Nrf2 whichcompromises cell constituent turnover and cell defense mechanisms to radical stress respectively Hyperinsulinemia downregulates glucoseuptake not only via dampening the PI3KAKT pathway œinsulin resistance but also via as yet unknown other pathways 0cKolb BMC Medicine Page of endoplasmic reticulum to high insulin levels is diminished or absent []Another activity of insulin is the suppression of transcription of the nuclear factor Nrf2 via induction of heterogeneous ribonucleoproteins F and K [] Nrf2 is thecentral regulator ofthe protective response of cellsagainst oxidative and other types of electrophile stress[] Suppression of Nrf2 expression is expected to impair the antioxidant and cytoprotective defense capacityof cells Insulin signaling required for Nrf2 inhibition occurs via the MAP kinase pathway and thus is not mitigated by insulin resistance [] Fig It therefore canbe assumed that hyperinsulinemia increases the susceptibility of cells against oxidative or other electrophilestress caused by environmental insults Prolonged exposure of cells to high insulin concentrations can thereforebe regarded as toxic Indeed exposure to nmoll insulin has been found to cause DNA damage in a numberof cell types including human lymphocytes [ ] Atthe only concentration tested nmoll insulin impairs oxygen radical defense and sensitizes apoptosispathways in human islets [] In the brain of micehyperinsulinemia impairs electrophysiological functionsof neurons and protein turnover causing a transition toa senescent cell state and an accompanying cognitive decline [] The direct toxic property of insulin deservesfurther studyChronically elevated insulin concentrations impair bodyfunctionsLongevityThe above list of detrimental cellular responses to highambientinsulin concentrations suggests concomitantfunctional impairments at the level of the anism Thisfits with the observed impact of insulin on longevityStudies in nonvertebrate model systems such as thenematode Caenorhabditis elegans or the fruit fly Drosophila melanogaster find that moderate to high insulinactivity shortens lifespan [ ] A consistent findingfrom mouse model studies is that decreased signaling ofanabolic hormones like insulin insulinlike growth factor or growth hormone results in a prolonged lifespan[] Disruption of the insulinreceptor substrate genecaused insulinresistance with defects in insulin signaling[] and led to an extension of lifespan by “ []A knockout of the insulin receptor in adipose tissue ofmice resulted in an increase of lifespan [] Disruption of the Ins1 gene and one of the two mouse Ins2alleles lowered insulin levels by “ Ins2ˆ’ miceversus Ins2 controls in aged female mice without altering circulating insulinlike growth factorIGF1levels These aged experimental mice exhibited lowerfasting glucose improved insulin sensitivity and “lifespan extension across[]two different dietsConcomitantly the proteome and transcriptome indicated a profile associated with healthy aging An important aspect is that this study selectively addressed insulinOther interventions for promoting longevity or extending healthspan such as caloric restriction not only lowercircadian insulin levels but several additional hormonesincluding IGF1 are also affected []InsulinIGF1 and hybrid insulinIGF1 receptorsshare signaling via PI3K and AKT The subsequent activation of the protein kinase mTORC1 is a major pathway for supporting somatic growth protein synthesisand fertility while impeding autophagy and lifespanSuppression of mTOR signaling by treatment with rapamycin prolongs life in model anisms and mice []In humans hyperinsulinemia in pre type diabetes isassociated with increased mTORC1 activity which mayhave a negative impact on beta cell survival healthspanand longevity []In the Leiden Longevity Studyfollowup of nonagenarians for years showed a strongassociation of low insulin and glucose levels with healthyaging []Since both IGF1 and insulin employ PI3K and AKTfor signal transduction it is difficult to disentangle thecontribution of insulin versus IGF1 to the modulationof longevity In animal models selective downregulationof circulating insulin levels improved the lifespan ofmice and in elderly persons of the Leiden LongevityStudy only insulin and glucose but not IGF1 consistently met all four predefined criteria of healthy aging[ ] Therefore it may be concluded that low circulating insulin concentrations are not only a marker oflongevity but are causally involved in promoting healthspan or lifespan extensionDetrimental combination of hyperinsulinemia with insulinresistanceInsulin resistance is defined as an attenuated effect of insulin on blood glucose homeostasis primarily by less efficient export of glucose from the blood into skeletalmuscle adipose and liver tissue Permanently elevatedinsulin concentrations in the blood are often consideredas an attempt to overcome insulin resistance Indeed induction of insulin resistance by genetic disruption of insulin signaling as well as by increased growth hormonelevels or an inflammatory milieu causes hyperinsulinemia [“] The opposite causality is of more relevanceHyperinsulinemia during insulin infusion in humansleads to systemic insulin resistance [] while in vitrohigh ambient insulin concentrations cause an increase ininsulin resistance in isolated adipocytes [] A summaryanalysis of nine studies in rodents and seven trials inhumans confirmed that the first detectable change in thefasting state after feeding a high caloric diet for severaldays is an increase of basal insulin concentrations but 0cKolb BMC Medicine Page of not of blood glucose concentrations or insulin resistance[] Both increased secretion of insulin by ß cells anddecreased insulin clearance in the liver contribute to elevated insulin levels postmeal the latter being of primaryimportance in the case of carbohydraterich food []functionincluding relaxation ofThe combination of hyperinsulinemia and insulin resistance appears to promote hypertension and atherogenesis Fig One important molecule for maintainingvesselthe arterialsmooth muscle layeris nitric oxide NO which isgenerated by endothelial NO synthase eNOS Insulinincreases NO production via posttranslational modification of eNOS via PI3KAKT activity howeverthismechanism is suppressed during insulin resistance [] Decreased local NO production impairs arterialsmooth muscle relaxation and concomitant vasodilatation An important factor in this context is the calciumion homeostasis of vascular smooth muscle cells Underphysiological conditions insulin promotes both calciuminflux into the cytoplasm of smooth muscle cells via several ion channels including Ltype and storeoperatedCa2 channels and counterregulatory NOmediated efflux of Ca2 and K ions which prevents calcium ioninduced myosin lightchain phosphorylation andFig Hyperinsulinemia insulin resistance and cardiovasculardisease High insulin concentrations in the blood may occur due togenetic predisposition overnutrition or highdose insulin treatmentof type diabetes Hyperinsulinemia induces œinsulin resistance as adefense response to maintain glucose homeostasis Converselyinsulin resistance may be directly induced such as by growthhormone or proinflammatory cytokines Hyperinsulinemia andinsulin resistance enhance the risk of cardiovascular disease byinducing endothelial dysfunction suppression of endothelial nitricoxide synthase eNOS and activation and promotion of calcium ioninflux into smooth muscle cells resulting in increased vascular toneenhanced reabsorption of sodium ions in renal tubules adhesion ofmacrophages to the vessel wall and development of arterial lesionswith increased lipoprotein lipase activity and cardiovascular diseaseconcomitant vascular contractility During insulin resistance NO production is impaired while the supportiveeffect of insulin on calcium ion influx via PI3K deltaand possibly the MEK“ERK pathway and vasoconstriction is still present Fig [ ]At the same time insulin signals through the mitogenactivated protein MAP kinase pathway to upregulatethe expression of endothelin1 ET1 plasminogen activator inhibitor1 PAI1 adhesion molecules and proinflammatory cytokines [ ] The reninangiotensinsystem is activated in the context of endothelial dysfunction and contributes together with decreased NO production and increased ET1 secretion to vascularstiffening and upregulation of vascular tone [“] Inthe absence of hyperinsulinemiainsulin resistance thelower insulin levels exert less potential proatherogenicactivities which are counteracted by insulinstimulatedlocal NO production [ ]Elevated insulin levels also increase the risk of hypertension by enhancing renal reabsorption of sodium ionsby several transport systems in different segments of thenephron Fig Signaling of insulin occurs via insulinreceptor substrate IRS2 and is not suppressed duringinsulin resistance while signaling via IRS1 for counterregulatory mechanisms including local NO production isimpaired [ ] These detrimental actions may be mitigated during chronic hyperinsulinemiainsulin resistance [] However a metaanalysis of prospectiveepidemiological studies showed that the pooled relativerisk of hypertension was when comparing the highest to the lowest category of fasting insulin levels and for comparing highest to lowest selective insulinresistance categories calculated as homeostasis modelassessment of insulin resistance HOMAIR []As a consequence of endothelial dysfunction duringprolonged treatment with insulin arterial lesions rich inlipids are formed [] The progression of early fattystreak lesions to plaques is accompanied by the adhesionand proinflammatory activity of macrophages whicheventually develop into foam cells This process is drivenby endothelial and macrophage lipoprotein lipase activity as demonstrated by the observation of less atherosclerosis in mice with inactivated lipoprotein lipase gene[“] Lipoprotein lipase activity in macrophages isenhanced with higher insulin levels in vivo but there isno direct stimulatory effect of insulin on isolated macrophages []The concern that hyperinsulinemia might promote arterial disease in diabetic persons developed in the late1960s due to the steady increase of incidences of atherosclerosis in diabetic persons despite improved glycemiaand decreased risk of ketosis due to insulin therapy []Since then a wealth of data supports the observationthatis ainsulin resistance and hyperinsulinemia 0cKolb BMC Medicine Page of marker of increased risk of cardiovascular disease in thegeneral population and in patients with diabetes [] Although observational studies suggested an approximatelylinear relation between the severity of hyperglycemiaand vascular damage severallarge randomized controlled trials have shown that intense glycemic controlper se does not decrease the risk of macrovascularcardiovascular events [] indeed insulin therapy may evenincrease the risk [ ] However these trials werenot randomized for insulin treatment and treatment ofCVD risk factors was not kept similar between patientsubgroups In the United Kingdom Prospective DiabetesStudy UKPDS hyperinsulinemia and insulin resistancewere not mitigated by insulin treatment and fastingplasma insulin levels even rose [] By contrastinUKPDS and other trials [ “] oral treatmentwith the biguanide metformin reduced the risk of cardiovascular events and in parallel decreased insulin resistance and hyperinsulinemiaIn epidemiological studies of type diabetesit hasbeen consistently observed that the addition of insulin tothe treatment regimen or the intensification of insulintreatment result in a higher rate of cardiovascular events[“] Fig Indeed it has been shown that therisk increases with increasing insulin dosage [ ]These epidemiological studies may suffer from residualFig Hazard ratio of insulin medication versus different reference medications Shown are adjusted hazard ratios HR for each study with confidence interval Moderate insulin exposure high insulin exposure moderate insulin dose to units per day §high insulin dose units per day 0cKolb BMC Medicine Page of confounding since it is difficult to account for the possibly more advanced disease stage of patients receivinginsulin A higher rate of hypoglycemic events may be anadditional confounder However covariates consideredin the statistical analyses cover a broad range of potential risk factors from different categories SupplementTable Large randomized controlled trials such asUKPDS [] or the Outcome Reduction With InitialGlargine Intervention ORIGIN Trial [] did not observe an increased incidence of cardiovascular diseasewith insulin therapy but these trials focused on lowdose insulin therapy of up to a median of IUday or IUkgday respectively Similar randomized trials ofhigherdose insulin therapy as typicalfor realworldconditions have not been conducted Recent studies ofrealworld clinical settings report mean daily basal insulin doses of close to IUkg in the Canadian REALITY Study for insulinexperienced patients with type diabetes [] and of IUkg in a physician survey inNew York [] In the European multicentre EUTREAT Study mean baseline insulin doses were between and U per day depending on the type of insulin therapy regimen applied [] It can be concludedthat under realworld conditions the majority of insulinexperienced patients with type diabetes receive higherinsulin doses per day than those tried in UKPDS orORIGINIn the absence of randomized controlled trials aMendelian randomization is an appropriate approach oftesting for a causal relationship in humans Mendelianrandomization studies made use of the finding that somegenotypes are associated with high or low fasting insulinlevels When comparing individuals carrying ‰¥ allelesthat raise fasting insulin levels with those exhibiting genetically determined low fasting insulin levels an increasedrisk of elevated blood pressure cardiovascular disease andtype diabetes was observed [] In two large recentMendelian randomization studies a genetic profile predicting high insulin levels in the blood after adjustmentfor BMI was also associated with increased systolic bloodpressure and risk of myocardial infarction []ConclusionsAs discussed aboveinsulin signaling engages at leastthree different pathways and modifies a large number ofcellular responses Table Transient elevations of systemic insulin concentrations are physiological responsesto dietary stimuli or other challenges such as environmental toxins [] In case of prolonged upregulationof insulin levels such as in response to overnutritionglucose homeostasis is maintained by mitigating insulinsignaling via PI3KAKT for glucose export from theblood into tissues Consequently insulin resistance hasbeen considered as a defense response in order to avoidTable Key messagescid129 Insulin employs at least three different pathways of signal transductionOne pathway involves phosphorylation steps via IRS“PI3K“AKT anotheris the MAP kinases Ras“MEP“ERK and third leads to the activation ofNOX4cid129 Insulin resistance is selective because it partially mitigates the PI3KAKTpathway for limiting glucose uptake and eNOS activation despitehyperinsulinemia but many other hormonal actions of insulin are notsuppressedcid129 Signaling via mTOR and the MEPERK pathway causes suppression ofautophagy and NRF2 leading to deficient turnover and impaired celldefensecid129 Moderate to high normal insulin levels inhibit lipolysis and promotelipogenesisobesitycid129 Insulin resistance and hyperinsulinemia are interdependent Dietinduced hyperinsulinemia precedes insulin resistancecid129 In epidemiological studies insulin therapy of type diabetes isassociated with a higher risk of cardiovascular events or deathcid129 Randomized trials of insulin therapy and associated risks only studieddosages up to IUdaycid129 Mendelian randomization studies found that genetically determinedhigh insulin levels lead to cardiovascular diseasecid129 Suppression of hyperinsulinemia and concomitant œinsulin resistanceprovides substantial health benefitshypoglycemia [] However other hormonal actions ofinsulin via the MAP kinase MEKERK pathway and inpart via PI3KAKT are no

Thyroid_Cancer

Official Case Reports Journal of the Asian Pacific Society of RespirologyRespirology Case ReportsEndobronchial metastases from a primary embryonalcarcinomaChiKang Teng1ChihYen Tu121Division of Pulmonary and Critical Care Medicine Department of Internal Medicine China Medical University Hospital Taichung Taiwan2School of Medicine China Medical University Taichung Taiwan3Division of Pathology China Medical University Hospital Taichung Taiwan WenChien Cheng12 ChiehLung Chen1 TingHan Chen1 YunShan Lin3 AbstractWe report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung Bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus Cryosurgical excisionwas performed tissue pathology confirmed the diagnosis of metastatic embryonal carcinoma The patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin BEP chemotherapy regimenKeywordsCryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourCorrespondenceWenChien Cheng Division of Pulmonary and Critical Care Medicine Department of Internal MedicineChina Medical University Hospital No YudeRoad North Dis Taichung City TaiwanEmail wcchengdrgmailcomReceived July Revised July Accepted July Associate Editor James HoRespirology Case Reports e00644 101002rcr2644IntroductionLung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases EBMs from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [“] Primary lung cancer is the most common cause of endobronchialtumours Extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [“] Although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma EBMs from embryonal carcinomas are extremely rare [] In this report we present acase of EBM from a primary embryonal carcinomaCase ReportA 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days Upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days He reported no fever chills coldsweats weight loss or decreased appetite A chest radiograph at admission revealed complete collapse of the leftlung Fig 1A Computed tomography CT was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumBronchoscopytumourobstructing theFig 1B Theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung Immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 TTF1negative sallike protein SALL4positive and cluster of differentiation CD30positive These findings were consistentwith a final diagnosis of metastatic embryonal carcinomaFig We checked the levels of tumour markers in thepatient including those of alphafetoprotein AFP betahuman chorionic gonadotropin BhCG and lactate dehydrogenase LDH Each tumour marker was found to be The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of RespirologyThis is an access under the terms of the Creative Commons AttributionNonCommercialNoDerivs License which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modifications or adaptations are made Vol Iss e00644Page 0cEBM from embryonal carcinomaCK Teng et alFigure Chestradiograph and bronchoscopic view of the endobronchial metastasesEBM A Complete collapse of the left lungon chest radiograph B Bronchoscopic viewof the endobronchial tumour within the leftmain bronchusFigure Tumour pathology of metastatic embryonal carcinoma A Embryonal carcinoma with a complex glandular growth pattern The characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magnification — B Immunohistochemical staining with antithyroid transcription factor1 TTF1 highlighting cells in the alveolar space original magnification — C Immunohistochemical staining with antisallikeprotein SALL4 revealed diffuse nuclear staining original magnification — D Immunohistochemical staining with anticluster of differentiation CD30 highlighting diffuse membranous staining original magnification —presentin high levels AFP ngmL BhCG mIUmL and LDH IUL The patientunderwent a right orchiectomy followed by a BEPbleomycin etoposide cisplatin chemotherapy regimenDiscussionWe report here the case of a young man with an EBMfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumoursLikewise manykindsLung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverEBMs from extrapulmonary malignancies are rare [“] Primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith EBM primarily breast colon and renal carcinomas[“] EBMs from testicular seminomas are also extremelyrare The majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of Respirology 0cCK Teng et alEBM from embryonal carcinomaTable Reports of previous cases of EBMsLocationDiagnostic methodPathology–zt¼rk []MoreiraMeyer []Case Case The orifice of right upper lobeRight main bronchusLeft main bronchus main carina andright main bronchus Fibreoptic bronchoscopy Mixed GCTFibreoptic bronchoscopy Mixed GCTVideobronchoscopyEmbryonal carcinoma–zsu []The orifice of the right upper lobeFibreoptic bronchoscopyTesticular seminomaTuran []Varkey []Our caseand right intermediary lobeRight intermediate bronchusLeft main bronchusLeft main bronchusEBM endobronchial metastases GCT germ cell tumourembryonic carcinomas or seminomas “There are only a few published reports of primary testicularembryonic carcinomas resulting in EBMs [“]The mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently Howeversome patients are asymptomatic [] In our patient symptoms on presentation included haemoptysis and shortnessof breathResults from chest radiography in patients with EBMcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation [] Our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyHowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difficult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these findings alone Toconfirm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue The flexible bronchoscopy fibreoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy The former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure [] The patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the final pathology reportconfirmed the diagnosis of metastatic embryonal carcinomaWe had evaluated the presence of AFP BhCG andLDH tumour markers Elevated AFP levels can be secretedby germ cell tumours GCTs including embryonal carcinoma yolk sac tumour or teratoma In GCTs detectableRigid bronchoscopyBronchoscopyFibreoptic bronchoscopyand cryosurgerySomatictype GCTEmbryonal carcinomaEmbryonal carcinomaBhCG elevation is observed in both seminomas and nonseminomas The serum level of LDH was directly correlated with tumour burden in nonseminomatous GCTswhich is also useful for the surveillance of patients withadvanced seminoma [] The tumour markers in ourpatient showed elevated levels of AFP BhCG and LDHThis was compatible with the diagnosis of embryonal carcinoma MoreiraMeyer also evaluated the patienttumour markers and found elevated levels of AFP ngmL and BhCG mIUmL The elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma [] –zsu onlyevaluated the patient™s BhCG level which was found to beelevated mIUmL and the final diagnosis wasmetastatic testicular seminoma []On comparison with previous case reports Table ours was the first case in which the tissue was obtainedusing cryosurgery Other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation APC to control bleedingCryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining Cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree samplesHaemorrhage was observed to be similar during both procedures [] Further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesIn conclusion EBMs from primary GCTs notably thoseassociated with total or partial collapse are extremely rareWe have presented this case to emphasize the importanceof distinguishing EBM from primary lung carcinoma andto report the first case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of Respirology 0cEBM from embryonal carcinomaDisclosure StatementAppropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisReferences –zt¼rk A Aktas¸ Z and Yılmaz A Endobronchialmetastasis of mixed germ cell tumors two cases TuberkToraks “ Lee SH Jung JY Kim DH Endobronchialmetastases from extrathoracic malignancy Yonsei Med J“ Ikemura K Lin DM Martyn CP Endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing Lung “ MoreiraMeyer A BautistaHerrera D Hern¡ndezembryonal EndobronchialGonz¡lez MCK Teng et alcarcinoma“J BronchologyInterv Pulmonol –zsu S Erol MM Oztuna F Endobronchial metastasis from testicular seminoma Med Princ Pract “tumoraltesticular germ cell Turan D Akif –zg¼l M Kirkil GEndobronchial metastasis ofEurasian J Pulmonol “et Varkey B and Heckman MG Diagnosis of a case ofembryonal carcinoma by bronchial biopsy Chest “ Paradis TJ Dixon J and Tieu BH The role of bronchoscopy in the diagnosis of airway disease J Thorac Dis“ Aktas Z Gunay E Hoca NT Endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisAnn Thorac Med “ Barlow LJ Badalato GM and McKiernan JM Serumtumor markers in the evaluation of male germ cell tumorsNat Rev Urol “ The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of Respirology 0c'

Thyroid_Cancer

"Postoperative pain in ambulatory surgery is a multifactorial issue affecting patient satisfaction time ofdischarge and rehospitalization This study evaluated the efficacy and safety of nalbuphine for the treatment ofpostoperative pain after ambulatory surgery relative to tramadolMethods This multicenter randomized double blind and controlled study was conducted at centers Inaccordance with the inclusion criteria ambulatory surgery patients were recruited These patients hadmoderate to severe pain after ambulatory surgery with a visual analogue scale VAS score cm They wererandomly divided into an experimental n or control n group and treated for analgesia with mgkg of nalbuphine or mgkg of tramadol respectively VAS scores adverse events and vital signs of the patientswere recorded before administration baseline T1 and min T2 h T3 h T4 and h T5 after administrationof analgesia A decrease in pain intensity of more than compared with the baseline was used as an indicatorof analgesic efficacy The experimental and control groups were compared with regard to this indicator of efficacyat each timepointResults The VAS scores of the experimental and control groups were statistically comparable at timepoints T1T4At T5 the VAS scores of the experimental group were significantly lower than that of the control The pain intensitywas significantly higher in the experimental group compared with the control at T2 and T3 Adverse events andvital signs were similar for the two groups at each timepointConclusions Nalbuphine can provide effective and safe pain relief in patients after ambulatory surgeryTrial registration The registration number is ChiCTRIOR16010032 the date of registration was Keywords Nalbuphine Tramadol Ambulatory surgery Postoperative analgesia Anesthesia Pain Correspondence qulianguohotmailcom chengzg2004hotmailcom1Department of Anesthesiology Xiangya Hospital of Central South UniversityNo Xiangya Road Changsha Hunan ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cGuan BMC Anesthesiology Page of BackgroundPostoperative pain is a multifactorial issue that may result in patient dissatisfaction delayed discharge and unanticipated hospital admission after ambulatory surgery[] Both delayed discharge and unanticipated hospitaladmission have the undesirable effect ofincreasinghealthcare costs [] In the postoperative period moderate to severe pain are frequently observed during thefirst to h after ambulatory surgery []hassurgerysignificantlyPatient recovery after ambulatory surgery has improvedsince the introduction of the concept of enhanced recovery after surgery a multimodal perioperative care pathwaydesigned to achieve early recovery after surgery []Ambulatoryshortenedhospitalization accelerated turnover and reduced hospitalcosts and rates of nosocomial infections [] However theshortened hospitalization and increased mobility of surgical patients have necessitated the need to improve theefficacy of anesthesia and perioperative managementTherefore postoperative pain and the complicationsarising from its treatment are important considerationsfor patients undergoing ambulatory surgeryVarious drugs have been used to prolong postoperativeanalgesia such as tramadol [] ketorolac [] dexmedetomidine [] ketamine [] and nalbuphine [] Nalbuphine a synthetic opioid agonistantagonist analgesic isprimarily a kappa κ agonist and a partial mu μ antagonist It has a better safety profile with fewer side effectscompared with other opioids because of its agonist andantagonist activities [] Nalbuphine [] exerts its analgesic and hypnotic effects through its κ opioid receptorwhich may reduce μ opioid receptorrelated adverseevents Numerous studies [ ] have reported its advantages in pain managementThere have been few studies in China of nalbuphinefor the treatment of postoperative pain after ambulatorysurgery The present study evaluated the analgesic efficacy and safety of intravenous nalbuphine hydrochloriderelative to tramadol for the treatment of postoperativepain aftera noninferiority control trialambulatoryincludingsurgeryMethodsParticipantsThis study was approved by the Ethics Committee ofXiangya Hospital of Central South University IRB Written informed consent was obtainedfrom all subjects participating in the trial The trial wasregistered prior to patient enrollment at chictrcnChiCTRIOR16010032 Principalinvestigator QulianGuo Date of registration A multicenter prospectiverandomized parallelcontrolled doubleblinded study for pain managementafter ambulatory surgery in adult patients was undertakenin hospitals Patients were screened at each center Thestudy was reported in accordance with the guidelines ofthe Consolidatedof Reporting TrialsCONSORTStandardsThe patient inclusion criteria were as follows to years old ASA American Society of AnesthesiologistsIII with postoperative pain after surgeries of the breastexcept radical surgery for mastocarcinoma or thyroidor hysteroscopy or laparoscopic cholecystectomy operative time h visual analog scale VAS score cm before the surgery and VAS score cm after recoveryfrom anesthesia body mass index BMI “ kgm2and signed informed consentPatients were excluded from this study if they wereallergic to the medication or any of the excipients inthe product Patients with current or histories of anyof the following were also excluded opioid allergyacute or chronic alcoholism or drug addiction neurological disease opioid used within the last monthsparalytic ileus increased intracranial pressure or headinjury chronic opioid use taking opioids for morethan months hypotension hypothyroidism asthmato be avoided during seizure hypertrophy oftheprostate epilepsy coronary heart disease bronchialasthma respiratory insufficiency or respiratory failurePatients taking or who had taken monoamine oxidaseinhibitor or antidepressants within the past dayswere excluded Patients with abnormal preoperativeliver and kidney function were also excluded definedas abnormal alanine aminotransferase ALT asparticaminotransferase AST blood urea nitrogen BUNor creatinine Cr ALT and AST times the normallimit and BUN and Cr higher than the normallimit coronary heart disease bronchial asthma respiratory insufficiency or respiratory failure or poorlycontrolled or difficult hypertension The latter wasdefined as systolic blood pressure SBP ‰¥ mmHgor diastolic pressure DBP ‰¥ mmHg In additionpatients with any ofthe following were excludedpregnancy abnormal coagulation function participation in another medication trial within the previous days unable to express their intention correctlypoor compliance unable to complete the study program or anyone the researchers considered inappropriate to participateTrial designPatients were randomly assigned to receive either the experimental group E or control group C treatment inthe postoperative period Group E was treated with nalbuphine hydrochloride Yichang HumanwellPharmaceutical diluted with saline to mgL Group Cwas administered tramadol hydrochloride diluted withsaline to mgL 0cGuan BMC Anesthesiology Page of The study medication was selected and prepared according to a random number list nalbuphine hydrochloride or tramadol hydrochloride The study wasblinded by excluding the researcher who prepared thepostoperative medications from participating in test observations and followups The researchers involved inobservation and evaluation of the experiment and patients and doctors were blinded throughout the studybeforeinductionofdexamethasoneInterventionsAll patients were administered intravenously with mgofgeneralanesthesia and mg of ondansetron at the time of surgery completion to prevent postoperative nausea andvomiting The bispectralindex BIS value was maintained between and during the operationAnesthesia induction was performed using sufentanil μgkg and propofol “ mgkg with cisatracurium “ mgkg given when necessary Anesthesiawas maintained by simultaneous infusion of propofol“ μgkgmin An additionaland remifentanil mgkg of cisatracurium was added intraoperativelywhen required Intraoperative fluid infusion and otheranesthetic management were performed routinelyAfter the surgery patients who were fully awake andfeeling pain for the first time were assessed for painwhile at rest using the VAS If the VAS score was cm the patients were included in the study and the painscore was used as the baseline T1 The test medicationsnalbuphine hydrochloride or tramadol hydrochloridewere administered at mLkg The VAS at rest wasused to evaluate the efficacy of the medications and wasrecorded before administration T1 and after administration at min T2 h T3 h T4 and h T5The following vital signs were recorded at each timepoint SBP DBP mean arterial pressure MAP heartrate and respiratory rate Adverse events and any medications used were also recordedWithin h after administration of the medications ifthe VAS score was cm it was deemed that the analgesic effect was invalid and the patient was discontinuedfrom the trial One hundred milligrams of flurbiprofenaxetil was infused intravenously as a rescue analgesiaand the name and dose were recorded The use of otheranalgesics aside from those involved in the study suchas opioidstranquilizers anesthetics and antiemeticswere prohibited during the study period If other analgesics were required to control the pain the patient wasdiscontinuedOutcomesPrimary outcomeThe pain intensity was measured using the VAS A decrease in VAS score of more than compared with thebaseline was used as an indicator of analgesic efficacy []The VAS score was also compared between groups E andC at all timepoints to determine any differences in the efficacy and duration of the analgesic effectsSecondary outcomeThe vital signs SBP DBP respiratory rate and heartrate were measured and used as safety indicators Thevital signs were also compared between groups E and Cand within each group at each timepoint Any differences observed could be used as a secondary indicatorto determine analgesic efficacyAdverse eventsAdverse events such as medication extravasation dizziness nausea vomiting and hidrosis were recorded during the study The rates of adverse events was comparedbetween groups E and C to determine the effects of thetreatmentsSample sizeSample size was calculated by VAS at rest at each timepoint Based on a previous report [] a single intravenous injection of tramadol was administered to patientswith postoperative pain after day surgery and the VASscore was cm at min after administration Assuming that the analgesic effect of nalbuphine was betterthan tramadol with α and β the VAS scoredifference between the two groups μA ˆ’ μB would be and the standard deviation σ The samplesizen was calculated using the formula [] 14 12n ¼ α zþ z1 ˆ’ ⠈’ α2 13 15Þð μˆ’ μBAEach group required subjects and with consideration of the estimated dropout rate patients wereincluded in each group Therefore patients were recruited in this study with patients in each centerStatistical methodsDescriptive statistics were used to describe all demographic data The ttest was applied to analyze thechanges in VAS scores between the two treatmentgroups at each timepoint and at different timepointsrelative to the baseline The Wilcoxon test was used toanalyze the pain classification of patients at each observation timepoint The pain intensity between the twogroups was compared using the chisquared χ2 testP was considered statistically significant The incidence of adverse events changes in blood pressure respiratory rate and heart rate relative to the baseline at 0cGuan BMC Anesthesiology Page of each timepoint and differences between the groupswere analyzed using the ttestResultsParticipantsThe study population comprised randomly codedpatients recruited from centers Fig However patients were excluded as they did not meet theeligibility criteria of a VAS score cm Thusthetrial consisted of patients in group E and in group CBaseline dataThe differences in age and gender between groups Eand C were not statistically significant Table Theresults of the preoperative test physical examinationand medical histories of the two groups were relatively similar with no statistical difference Therewere no statistically significant differences in the typesof surgery between the two groups Table Therewere also no differences in the use of opioids including sufentanil and remifentanil between the twogroups during surgeryDuring the observation period and patients in groups E and C respectively weretreated with rescue analgesic medication consistingof mg of flurbiprofen axetil There was no statistically significant difference between the two groupsTable Patient demographics of groups E and CaSubjects nGender n MaleFemaleAge yBMI kgm2Respiration rpmHeart rate bpmHeart rhythm n NormalAbnormalSBP mmHgDBP mmHgGroup E Group C ± ± ± ± ± ± ± ± ± ± ± ± P ± MAP mmHgaGroup E was treated with nalbuphine hydrochloride diluted with saline to mgL Group C was administered tramadol hydrochloride diluted with salineto mgL ± with regard to the percentage using rescue analgesicmedication χ2 P There was no significant deviation from the regimen for all concomitantnostatistically significant difference between the twogroupscombination medicationsandandFig CONSORT flow diagram of progress through the phases of a randomized doubleblinded parallel controlled trial of the groups 0cGuan BMC Anesthesiology Page of Table Types of surgery n aGroup ETotal nBreast Thyroid Hysteroscopy LC Others Group CaReported as n unless indicated otherwise Group E was treated with nalbuphine hydrochloride diluted with saline to mgL Group C was administeredtramadol hydrochloride diluted with saline to mgL Other surgeries included lumbar disc exploration laparoscopic gastric perforation repair endoscopic sinussurgery surgical removal of internal fixation of fractured bonesLC laparoscopic cholecystectomy OutcomesPrimary outcomeA pairwise comparison of the VASs determined at rest atdifferent timepoints between groups E and C revealed nodifference between the VAS scores at T1 T2 T3 or T4 respectively However at T5 the VAS at rest of group E wassignificantly lower than that of group C Fig A decreasein pain intensity of more than compared with the baseline T1 was used as an indicator of analgesic efficacyTable The analgesic efficacy experienced by group E atT2 and T3 was significantly higher than that of group CAdverse eventsAdverse events occurred in subjects in group Eand subjects in group C with no serious adverse events or deaths occurring in either group Thenumber of adverse events was higher in group E compared with group C but the difference was not statistically significant Table Secondary outcomeThe vital signs SBP DBP respiratory rate and heart rateof groups E and C at all timepoints were statisticallyFig The VAS at rest in the experimental Group E and controlgroup Group C Time points T1 before administration T2 afteradministration at min T3 after administration at h T4 afteradministration at h T5 after administration at h aAfteradministration the VAS of Group E was lower than that of Group Cfrom T2T5 bthere was a statistically significant difference in VASbetween the groups at T5 Data are expressed asmean ± standard deviationsimilar Table For both groups the mean SBP DBPand heart rate at each of the timepoints T2 T3 T4 and T5were significantly lower than at T1 However the bloodpressures at T2 to T5 were comparable to that at admission T0 and there was no significant difference in respiratory ratesDiscussionIn this prospective multicenter study patientswere randomized to receive either nalbuphine groupE or tramadol group C to treat pain after ambulatory surgery Group E experienced significantly longerduration of analgesia compared with group C Ateach timepoint the vital signs SBP DBP respiratoryrate and heart rate of the groups were statisticallycomparable However within each group there weresignificant differences in SBP DBP and heart rate atT2 T3 T4 and T5 relative to T1 Overall the analgesic effect of nalbuphine was comparable to that oftramadol with nalbuphine having a longer durationof analgesiaIn China the number of day surgeries is increasingdue to improvements in surgery and anesthesia withshorter recovery time and patients discharged within h after surgery Therefore there is a higher demand forTable Pain reduction when compared to baseline T1 n adT2eT3fT4gT5EffectiveGroup Eb Noneffective Group Cc χ2PEffective Noneffective Effective Noneffective Effective Noneffective aEffective pain reduction is defined as a decrease in pain intensity compared with the baseline T1 Noneffective is defined as a decrease in painintensity compared with the baseline T1bGroup E n was treated with nalbuphine hydrochloride diluted withsaline to mgLcGroup C n was administered tramadol hydrochloride diluted withsaline to mgLdT2 after administration at mineT3 after administration at hfT4 after administration at hgT5 after administration at h 0cGuan BMC Anesthesiology Page of Table Patients experiencing adverse events nTotal subjects experiencing adverse eventsVasculitis medication extravasationDizziness nausea vomitingHidrosisGroup EGroup Canesthesia and a need to improve the quality of analgesics While achieving rapid recovery patients also needto avoid complications related to surgery and anesthesiasuch as pain nausea and vomiting Numerous studies[ ] have shown that after day surgery nearly ofpatients experience pain Postoperative pain not only affects patients™ rehabilitation and prolongs hospitalizationit can also result in progression from acute to chronicTable Vital signs at each timepoint aT0T1T2T3T4T5Vital signsSBPDBPGroup Eb ± ± Respiratory rate ± Heart rate ± Group Cc ± ± ± ± SBPDBP ± ± ± ± Respiratory rate ± ± Heart rate ± ± SBPDBP ± ± ± ± Respiratory rate ± ± Heart rate ± ± SBPDBP ± ± ± ± Respiratory rate ± ± Heart rate ± ± SBPDBP ± ± ± ± Respiratory rate ± ± Heart rate ± ± SBPDBP ± ± ± ± Respiratory rate ± ± Heart rate ± ± PaT0 at admission T1 before administration T2 after administration at minT3 after administration at h T4 after administration at h T5 afteradministration at hbGroup E n was treated with nalbuphine hydrochloride diluted withsaline to mgLcGroup C n was administered tramadol hydrochloride diluted withsaline to mgLDifference is statistically significant compared with T1 difference isstatistically significant compared with T0pain which is the main cause of readmission after daysurgery []According to the Chinese Society of Anesthesiology[] systemic opioids given to patients undergoing ambulatory surgery with general anesthesia activate opioidreceptors and stimulate various ans This often results in nausea and vomiting pruritus urinary retentionexcessive sedation and respiratory inhibition Thusinprinciple systemic opioids are not used for postoperativepain relief after day surgery The analgesic and adversereactions of mixed agonistantagonist opioids such asnalbuphine and dezocine also exhibit a ceiling effectImplementation of multimodal analgesia using NSAIDscan significantly reduce the dose of opioid and adversereactions and can be used postoperatively to managemoderate pain after ambulatory surgeryNalbuphine a mixed agonistantagonist opioid is associated with milder μ receptorrelated side effects Itsplasma halflife is h and in clinical studies the durationof analgesic activity ranges from to h [] In ourstudy the VAS at rest of group E was less than pointsand the difference was statistically significant comparedwith the VAS at rest before administration This indicates that nalbuphine could effectively relieve pain afterambulatory surgery Similar results were also observedin animal studies that showed amelioration of somaticand visceral pain in mice after treatment with nalbuphine []In the present study the VAS at rest at timepoints T1to T4 of the nalbuphine group group E did not differfrom that of the control At T5 the VAS at rest of thenalbuphine group was significantly lower than that ofthe tramadol group This indicates that the duration ofnalbuphine for pain relief after ambulatory surgery waslonger than that of tramadol There were cases of adverse reactions in the nalbuphine group which wasnot significantly different from the cases in thetramadol groupThe incidence of adverse reactions associated with nalbuphine is relatively low compared with other opioidmedications A metaanalysis of randomized controlledtrials by Zeng [] showed that nalbuphine hassimilar analgesic effects compared to morphine and abetter drug safety profile with a low incidence of postoperative pruritusrespiratory inhibition nausea andvomiting In addition studies have reported that antagonism of the μ receptor by nalbuphine could reduce theadverse reactions of other opioids as seen in the combination of morphine and nalbuphine in patientcontrolled analgesia or patientcontrolled epidural analgesia [ ] and the rate of adverse effects such asurinary retention related to morphine pruritus and nausea was significantly less Nalbuphine with sufentanilused in patientcontrolled analgesia could reduce the 0cGuan BMC Anesthesiology Page of incidence of opioidrelated nausea and vomiting and improved patients™ satisfaction with analgesia [ ]In the present study the difference in respiratoryrates before and after administration in both the nalbuphine and tramadol groups was not statistically significant and no respiratory depression was observedMany studies have reported that respiratory depression caused by nalbuphine is small and has a ceilingeffect [ ] In one study a neonate was wronglyadministered a tenfold higher dose than required ofnalbuphine and it resulted in only prolonged sedationwith no respiratory failure []studyIn the presentStudies have shown that preanesthetic injections ofnalbuphine could reduce stress responses and fluctuations in blood pressure and heart rate during intubation [ ]the bloodpressures and heart rates of both groups after administration were significantly lower than at T1 althoughstill within normal ranges The blood pressure at T0Inception of the study was compared to the bloodpressure after surgery T1T5 the blood pressure atT1 was significantly higher than at T0 However atthe later timepoints T2 to T5 there were no statistical differences in the blood pressures compared toT0 The decrease in blood pressure after administration T2T5 may have been due to the alleviation ofpain If sothen the lowered blood pressure couldalso indicate the analgesic efficacy of nalbuphineThere are several limitations in this study First a limited number of parameters VAS score adverse eventsand change of vital signs were observed within the halflife of the medication Secondly the VAS scores were recorded at rest and not during movement Finally due toethical issues a placebo control group was not possibleTherefore we were not able to assess the effectiveness ofnalbuphine or tramadol at and h after administration Fortunately none of the patients dropped out during the or h after administration of medication forpain However the present results warrant further experiments to determine comprehensively the effectiveness and safety of nalbuphine for the treatment of painafter ambulatory surgeryConclusionThis study indicates that nalbuphine at a recommendeddose of mgkg is safe and effective for pain management after ambulatory surgeryAcknowledgementsNot applicableAuthors™ contributionsGYJ This author contributed to the data collection contributed to the dataanalysis and wrote the manuscript WL This author contributed to the datacollection and analysis LQ This author contributed to the data collectionand analysis FQW This author contributed to the data collection andanalysis HN This author contributed to the data collection and analysis HCFThis author contributed to the data collection and analysis MCH This authorcontributed to the data collection and analysis LCJ This author contributedto the data collection and analysis WHB This author contributed to the datacollection and analysis CH This author contributed to the data collectionand analysis GQL This author designed most of the research plancontributed to the data collection CZG This author designed most of theresearch plan contributed to the data collection All of the authors haveread and approved the manuscriptFundingNot applicableAvailability of data and materialsThe datasets generated and analyzed during the present study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThis study was approved by the Ethics Committee of Xiangya Hospital ofCentral South University IRB Written informed consent wasobtained from all subjects participating in the trial The trial was registeredprior to patient enrollment at chictrcn ChiCTRIOR16010032 Principalinvestigator Qulian Guo Date of registration Consent for publicationAll data published here are under the consent for publication Writteninformed consent was obtained from all individual participants included inthe studyCompeting interestsThe authors declare that they have no conflict of interestAuthor details1Department of Anesthesiology Xiangya Hospital of Central South UniversityNo Xiangya Road Changsha Hunan China 2Department ofAnesthesiology Hunan Provincial People™s Hospital Changsha Hunan China3Department of Anesthesiology Third Xiangya Hospital of Central SouthUniversity Changsha Hunan China 4Department of Anesthesiology PainMedicine Critical Care Medicine Aviation General Hospital of ChinaMedical University Beijing Institute of Translational Medicine ChineseAcademy of Sciences Beijing China 5Department of Anesthesiology PekingUniversity Shougang Hospital Beijing China 6Department of AnesthesiologyShanxi Academy of Medical Sciences Shanxi Dayi Hospital Shanxi China7Department of Anesthesiology Fudan University Shanghai Cancer CenterShanghai China 8Department of Anesthesiology Third Affiliated Hospital ofSun YatSen University Guangzhou Guangdong China 9Department ofAnesthesiology First People™s Hospital of Foshan Foshan Guangdong China10Department of Anesthesiology Beijing Ditan Hospital Capital MedicalUniversity Beijing ChinaReceived March Accepted August AbbreviationsALT alanine aminotransferase ASA American Society of AnesthesiologistsAST Aspartic aminotransferase BIS Bispectral index BMI Body mass indexBUN Blood urea nitrogen C Control CONSORT Consolidated Standards ofReporting Trials Cr Creatinine DBP Diastolic pressure E ExperimentalMAP Mean arterial pressure SBP Systolic blood pressure VAS Visual analogscaleReferencesShirakami G Teratani Y Namba T Hirakata H TazukeNishimura M FukudaK Delayed discharge and acceptability of ambulatory surgery in adultoutpatients receiving general anesthesia J Anesth “ httpsdoi101007s0054000402976Tong D Chung F Postoperative pain control in ambulatory surgery SurgClin North Am “Rawal N Postoperative pain treatment for ambulatory surgery Best PractRes Clin Anaesthesiol “ 0cGuan BMC Anesthesiology Page of decreases incidence of opioidrelated side effects J Formos Med Assoc“ httpsdoi101016S0929664609603727 Zhang S Clinical observation of lowdose nalbuphine combinedwithsufentanil in PCIA Acta Acad Med WeiFang “ Niu NYT nalbuphine combined with sufentanil for clinical observation ofanalgesia afteradult laparoscopic surgery World Med Inform Digest “ Gupta M Gupta P Nalbuphine pretreatment for prevention of etomidateinduced myoclonus A prospective randomized and doubleblind study JAnaesthesiol Clin Pharmacol “ httpsdoi104103joacpJOACP_210_16 Romagnoli A Keats AS Ceiling effect for respiratory depression bynalbuphine Clin Pharmacol Ther “SchultzMachata AM Becke K Weiss M Nalbuphine in pediatric anesthesiaAnaesthesist “ httpsdoi101007s001010142293zTariq AM Z Iqbal and Qadirullah Efficacy of nalbuphine in preventinghaemodynamic response to laryngoscopy and intubation J Postgrad MedInst “ Chawda PM Pareek MK Mehta KD Effect of nalbuphine on haemodynamicresponse to orotracheal intubation J Anaesthesiol Clin Pharmacol “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations Melnyk M Casey RG Black P Koupparis AJ Enhanced recovery after surgeryERAS protocols Time to change practice Can Urol Assoc J “httpsdoi105489cuaj11002Lee JH Anesthesia for ambulatory surgery Korean J Anesthesiol “ httpsdoi104097kjae2017704398Acalovschi I Cristea T Margarit S Gavrus R Tramadol added to lidocaine forintravenous regional anesthesia Anesth Analg “Jankovic RJ Visnjic MM Milic DJ Stojanovic MP Djordjevic DR Pavlovic MSDoes the addition of ketorolac and dexamethasone to lidocaineintravenous regional anesthesia improve postoperative analgesia andtourniquet tolerance for ambulatory hand surgery Minerva Anestesiol“Kumar A Sharma D Datta B Addition of ketamine or dexmedetomidine tolignocaine in intravenous regional anesthesia A randomized controlledstudy J Anaesthesiol Clin Pharmacol “ httpsdoi104103AbdelGhaffar HS Kalefa MA Imbaby AS Efficacy of ketamine as an adjunctto lidocaine in intravenous regional anesthesia Reg Anesth Pain Med “ httpsdoi101097AAP0000000000000128 Youssef MIEN Lidocainenalbuphine versus lidocainetramadol forintravenous regional anesthesia AinShams J Anesthesiol “ Bakri MH Ismail EA AbdElshafy SK Analgesic effect of Nalbuphine whenadded to intravenous regional anesthesia a randomized control trial PainPhysician “ Vilsbøll T The effects of glucagonlike peptide1 on the beta cell DiabetesObes Metab Suppl “ httpsdoi101111j14631326200901073xShin D Kim S Kim CS Kim HS Postoperative pain management usingintravenous patientcontrolled analgesia for pediatric patients J CraniofacSurg “ Mukherjee A Pal A Agrawal J Mehrotra A Dawar N Intrathecal nalbuphineas an adjuvant to subarachnoid block What is the most effective doseAnesth Essays Res “ httpsdoi1041030259116294759 Hua X Chen LM Zhu Q Hu W Lin C Long ZQ Efficacy of controlledrelease oxycodone for reducing pain due to oral mucositis innasopharyngeal carcinoma patients treated with concurrentchemoradiotherapy a prospective clinical trial Support Care Cancer “ httpsdoi101007s0052001946435 Ali M Khan FA Comparison of analgesic effect of tramadol alone and acombination of tramadol and paracetamol in daycare laparoscopic surgeryEur J Anaesthesiol “ httpsdoi101097EJA0b013e328324b747 Chow SC Shao J Wang H Sample size calculations in clinical research 2nded Boca Raton Chapman HallCRC Apfelbaum JL Chen C Mehta SS Gan TJ Postoperative pain experienceresults from a national survey suggest postoperative pain continues to beundermanaged Anesth Analg “ table of contents Aubrun F Ecoffey C Benhamou D Jouffroy L Diemunsch P Skaare K et alPerioperative pain and postoperative nausea and vomiting PONVmanagement after daycase surgery The SFAROPERA national study AnaesthCrit Care Pain Med httpsdoi101016jaccpm201808004 Xu J Expert consen

Thyroid_Cancer

cancer is still one of the most prevalent and highmortality diseases summing more than million deaths in This has motivated researchers to study the application of machine learningbased solutionsfor cancer detection to accelerate its diagnosis and help its prevention Among several approaches one is toautomatically classify tumor samples through their gene expression analysisMethodsstomach breast and lung To do so we have adopted a previously described methodology with which we comparethe performance of different autoencoders AEs used as a deep neural network weight initialization technique Ourexperiments consist in assessing two different approaches when training the classification model ” fixing theweights after pretraining the AEs or allowing finetuning of the entire network ” and two different strategies forembedding the AEs into the classification network namely by only importing the encoding layers or by inserting thecomplete AE We then study how varying the number of layers in the first strategy the AEs latent vector dimensionand the imputation technique in the data preprocessing step impacts the network™s overall classification performanceFinally with the goal of assessing how well does this pipeline generalize we apply the same methodology to twoadditional datasets that include features extracted from images of malaria thin blood smears and breast masses cellnuclei We also discard the possibility of overfitting by using heldout test sets in the images datasetsResults The methodology attained good overall results for both RNASeq and image extracted data Weoutperformed the established baseline for all the considered datasets achieving an average F1 score of Continued on next pageCorrespondence mafaldafferreirafeuppt1Faculty of Engineering University of Porto Rua Dr Roberto Frias sn Porto Portugal2INESC TEC Institute for Systems and Computer Engineering Technology andScience Porto Portugal The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes weremade The images or other third party material in this are included in the ™s Creative Commons licence unlessindicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 The CreativeCommons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of Continued from previous pageand and an MCC of and for the RNASeq when detecting thyroid cancer the Malaria and theWisconsin Breast Cancer data respectivelyConclusions We observed that the approach of finetuning the weights of the top layers imported from the AEreached higher results for all the presented experiences and all the considered datasets We outperformed all theprevious reported results when comparing to the established baselinesKeywords Cancer Classification Deep learning Autoencoders Gene expression analysisBackgroundCancer is a label for a group of diseases that is characterized by abnormal and continuous cell growth with thepotential to spread through its surrounding tissues andother body parts [] During cancer was the secondleading cause of death globally accountable for milliondeaths where around were in developing countries[] Throughout the years and given the evolution of techniques technology and treatments in medicine cancersurvival rates have been improving [] However there arestill some types that have survival rates of under suchas pancreatic esophagus and liver cancers Its prevalencemakes it more crucial to correctly and accurately classify such diseases For tackling this need many researchgroups have been trying to help on accelerating cancerdiagnosis by experimenting and studying the applicationof machine learning algorithms to this problem []When automatically classifying tumor samples oneapproach is to analyze the samples derived molecularinformation which is its gene expression signatures Geneexpression is the phenotypic manifestation of a gene enes by the processes of genetic transcription and translation [] By studying it this gene map can help to betterunderstand cancer™s molecular basis which can have adirect influence on this disease™s life cycle prognosis diagnosis and treatment There are two main cancer genomicsprojects ” The Cancer Genome Atlas TCGA [] andThe International Cancer Genome Consortium ICGC[] ” that aim to translate gene expression systematizing thousands of samples across different types of cancersWith this elevated number of features each representing a particular gene one may find genomewide geneexpression assays datasets in these projects However thistype of data presents some challenges because of alow number of samples an unbalanced class distribution with few examples of healthy samples and a high potential of underlying noise and errors due toeventual technical and biological covariates [] This difficulty in gathering data accurately is underlying for everydataset creation The equipment used to collect the datahas intrinsic errors associated mechanical of acquisitionand others hence the dataset will reflect these errorsSeveral authors have chosen the previously mentionedapproach of analyzing the gene expression of tumor samples Many of the developed methodologies in this scopeuse straightforward supervised training especially whenusing deep neural networks DNNs relying on theirdepth to produce the best results Gao [] proposedDeepCC a supervised deep cancer subtype classificationframework based on deep learning of functional spectra quantifying activities of biological pathways robust tomissing data The authors conducted two studies eachwith a different cancer detection colorectal and breastcancer data The authors claimed that the describedmethod achieved overall higher sensitivity specificity andaccuracy compared with other classical machine learningmethods widely used for this kind of task namely randomforests support vector machine SVM gradient boostingmachine and multinomial logistic regression algorithmswith an accuracy higher than Sun [] proposed Genome Deep Learning GDLa methodology aiming to study the relationship betweengenomic variations and traits based on DNNs Thisstudy analyzed over six thousand samples of Whole ExonSequencing WES mutations files from different cancer types from TCGA and nearly two thousand healthyWES samples from the one thousand genomes projectsThe main goal of GDL was to distinguish cancerous fromhealthy samples The authors built models to identify each type of cancer separately a totalspecific modelable to detect healthy and cancerous samples and a mixedmodel to distinguish between all types of cancerbasedon GDL All the experiments were evaluated througha three performance metrics ” accuracy sensitivityand specificity ” and b Receiver Operating Characteristic curves with the respective Area Under the CurveROCAUC This methodology achieved a mean accuracy of on the specific models on mixturemodels and on total specific models for canceridentificationIn [] Kim compared the performances of a neural network a linear SVM a radial basisfunctionkernel SVM a knearest neighbors and arandom forest when identifying types of cancers and 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of healthy tissues The classifiers were trained with RNAseq and scRNAseq data from TCGA where they selectedup to the most significant genes expressed for eachof the cancer variations To determine the optimal number of genes for each classifier™s binary classification taskthe methods mentioned above were trained with different sizes of gene expression datasets from to genes When learning with genes the neural network the linear SVM and the radial basis functionkernelSVM models achieved their best performance with a witha Matthews Correlation Coefficient MCC of and respectively The knearest neighbors and random forest models achieved an MCC of and accordingly when using genes Furthermore theauthors identified classes with an accuracy of over and achieved a mean MCC of and a mean accuracy of with the neural network classifierHowever many DNNs besides the known challenges regarding their training setting [] have a highertendency to overfit which one can detect when applying the same architecture to unseen data or to a heldouttest Thus our motivation focuses on exploring unsupervised pretraining methods based on a lowerdimensionallatent representation with the usage of an autoencoderAE This approach is grounded in the hypothesis thata there is unessential information in high dimensionality datasets and b the acquisition and processing errorspotentially present in the dataset are discarded contributing to a lower probability of overfitting [] Furthermorepretraining AEs and using the learned weights as priorsof the supervised classification task not just improves themodel initialization but also often leads to better generalization and performance [] This may be one of thereasons why AEs are found to be the most predominantstrategy when analyzing RNASeq data []To support our motivation and choices we presentsome works that include unsupervised training in theirmethodologies In [] the authors designed a solution by combining a Multilayer Perceptron and StackedDenoising Autoencoder MLPSAE aiming to predicthow good genetic variants can be a factor in gene expression changes This model is composed of layers inputtwo hidden layers from the AEs and output and trained itto minimize the chosen loss function the Mean SquaredError MSE The authors started by training the AEs witha stochastic gradient descent algorithm to later use themon the multilayer perceptron training phase as weight initialization crossvalidation was used to select the bestmodel The performance of the chosen model was compared with the Lasso and Random Forest methods andevaluated on predicting gene expression values for a different dataset The authors concluded that their approach outperformed both the Lasso and Random Forest algorithms with an MSE of versus and respectively and was able to capture the change ingene expression quantificationThe authors in [] described a study of four different methods of unsupervised feature learning ” PrincipalComponent Analysis PCA Kernel Principal Component Analysis KPCA Denoising AE DAE and StackedDenoising AE ” combined with distinct sampling methods when tackling a classification task The authorsfocused on assessing how influential the input nodes areon the reconstructed data of the AE™s output when feeding these combinations to a shallow artificial networktrained to distinguish papillary thyroid carcinoma fromhealthy samples The authors highlighted two differentresults in their 5fold cross validation experiment thecombination of a SMOTE [] with Tomek links and aKPCA was the one with the best overall performancewith a mean F1 score of while the usage of a DAEachieved a mean F1 score of In [] presented a stacked sparse autoencoder SSAEsemisupervised deep learning pipeline applied to cancer detection using RNASeq data By employing layerwise pretraining and a sparsity penalty this approachhelps to capture more significant information from theknown high dimensionality of RNASeq datasets usingthe filtered information to the sequent classification taskThe SSAE model was tested on three different TCGARNASeq datasets ” corresponding to lung stomach andbreast cancers ” with healthy and cancerous samplesand compared it to four others classification methodsan SVM a Random Forest a neural network supervisedlearning only and a vanilla AE The authors performed5fold cross validation and evaluated the model™s performance through four metrics accuracy precision recalland F1 score The results show that the semisuperviseddeep learning approach achieved superior performanceover the other considered methods with an average F1score of across the three used datasetsThe authors in [] developed a methodology for detecting papillary thyroid carcinoma They analyzed how theusage of AEs as a weight initialization method affectedthe performance of a DNN Six types of AEs were considered Basic AE Denoising AE Sparse AE DenoisingSparse AE Deep AE and Deep Sparse Denoising AEBefore being integrated into the classifier architecture allAEs were trained to minimize the reconstruction errorSubsequently they were used to initialize the weights ofthe first layers of classification neural network meaningthat the AE layers become the top layers of the wholeclassification architecture using two different strategieswhen importing the weights just the encoding layersand all the pretrained AE Moreover in the training phase the authors studied two different approacheswhen building the classifier a fixing the weights ofthe AE and b allowing subsequent finetuning of all 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of the network™s weights The authors used stratified 5foldcrossvalidation and evaluated the model through distinct metrics Loss Accuracy Precision Recall and F1score The authors reported that the overall best resultwas achieved through a combination of Denoising AEfollowed by its complete import into the classification network and by allowing subsequent finetuning throughsupervised training yielding an F1 score of in which the main goalIn [] the authors present a transfer learning methodologyis to explore whetherleveraging the information extracted from a large RNASeq data repository with multiple cancer types leadsto extract important latent features that can help complex and specific prediction tasks such as identifyingbreast cancer neoplasia The authors used the TCGAPanCancer dataset which is composed of approximately RNASeq gene expression examples of distincttumor types This data was split into two sets breast cancer and nonbreast cancer data The nonbreast data isfirstly used to train the three selected architectures forthis study a sparse AE a deep sparse AE and a deepsparse denoising AE models Then the breast data isused to finetune the resulting AEs After pretrainingthese models the authors aim to predict the breast tumorintrinsicsubtypes which is given by the PAM50 subtype information included in the clinical data included inthe PanCancer data The extracted features from the AEbased architectures are then fed as input to three differentmachine learning classifiers namely Logistic RegressionSupport Vector Machine and a shallow Neural NetworkTo assess the deep AEs performance as feature extractionmethods the authors compared them to other classical feature extraction methods combining them with theclassification algorithms previously mentioned ANOVAMutual Information ChiSquared and PCA A 10foldcross validation was performed and all the combinationswere compared through the accuracy metric The resultsshowed the deep sparse denoising AE performs best whenusing the AE extracted features where the combinationwith a shallow neural network leads to the best overall of ±In [] Ferreira used the same methodologydescribed in [] to discriminate different types of cancerinstead of distinguishing cancerous samples from healthyones In this case they aimed to identify thyroid skin andstomach cancer correctly Given that a Denoising AE wasthe AE that lead to the best results in previous studiesthe authors chose to single it out instead of the original The rest of the experiments remained the same strategies for importing the pretrained AE into the top layersof the classifier two approaches when training the classifier to detect different types of cancer same evaluation ofthe obtained results Although in a different domain thebest outcome was reached with a combination of the samestrategy and the same approach in the previous work []with an F1 score of when identifying thyroid cancerMethodsWe extend the previously described work in [] byassembling three different types of experiments dividedinto two main parts where we use three different AEsand five types of cancer samples In the first one we analyze the performance of a deep neural network DNNusing the same pipeline to identify different types of cancer In the second part we choose one of the used AEsto assess how the variance of its latent vector dimension impacts the essential information capture and therefore possibly influencing the classifier™s performance and different data imputation strategies can influence theoverall performance in the classification task Moreoverwe study if the network architecture is correlated withits overall performance and how the model reacts whentraining with a different data type dataset We built thispipeline in Python using the Numpy [] and Pandas []packages for the data preprocessing step the Keras deeplearning library [] running on top of TensorFlow andthe ScikitLearn [] package to train and evaluate themodels and the Matplotlib [] library for visualizationAdditionally we used an NVIDIA GeForce RTX TiGPU on a Ubuntu operating systemThis section is anized as follows œThe data subsection describes the used data and its inherent preprocessing œAutoencoders subsection overviews the AEs considered to this study œMethodology subsection outlinesthe pipeline for each of the referred experiments œEvaluation subsection details how we evaluate the results toprovide statistical evidence Finally œBaseline subsectionpresents the established baseline results for all the useddatasetsThe dataIn our experiments we use two different types of datawhich are described in the subsections that followRNASeq dataWe used five different RNASeq datasets from The Cancer Genomes Atlas TCGA [] each representing a typeTable Five instances of the thyroid RNASeq dataset we have usedTPTEP1 AKR1C6PUBE2Q2P2 HMGB1P1 LOC155060 ZZZ3 NANANANANANANANANANA NA NA NA NA NAThe first line the header contains the genes names and the column valuesrepresent its expression samplewise except for the first column which is thesample ID NA stands for missing value for a particular gene and sample 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of of cancer thyroid skin stomach breast and lung Onecan find a sample of the described data in Table The datasets were downloaded from the cBioPortal []which gathers cancerrelated data from different projectsincluding TCGA To train DNNs we need as many data aswe can get Ergo our first criterion was to choose cancertypes that had the highest number of examples Additionally we decided to gice priority to cancer types withhigh mortality and high incidence rates We use the samethyroid skin and stomach datasets presented in []alongside the lung and breast datasets The data filteringprocess in the cBioPortal comprised searching with thekeywords PanCancer sorting the obtained results fromhighest to lowest RNASeq examples and finally selectingthe thyroid skin stomach breast and lung datasetsAll five datasets are composed of approximately thousand features Each column feature in these datasetsrepresents a specific gene and the cell values for each column are the expression of that gene in a particular sampleAll the RNASeq data were normalized according to thedistribution based on all samples The expression distribution of a gene is estimated by calculating the mean andvariance of all samples with expression values and discarding zero™s and nonnumeric values such as NA Nullor NaN which are substituted by NA [] With the fivedatasets we gathered examples of thyroid cancer of skin cancer of stomach cancer of breast cancer and of lung cancer We would like to emphasizethat this dataset is only a toy dataset since the data doesnot fairly reflect the immense difficulty associated withidentifying cancer in a real scenarioThe preprocessing pipeline was executed for each RNASeq dataset separately Firstly we removed the columnsthat had only one value throughout all samples Whena value is constant for all the examples there is noentropic value with no value variation one cannot inferany information In total and columns were removed on the thyroid skin stomach breast and lung datasets respectively By default weattributed the remaining missing values represented byNA in the dataset as observable in Table with the meanvalue of the column where the missing value is [] Further normalization was not applied in the data Finally weadded the Label column to link the instances to their typeof cancer when training the classifierSince we aim to distinguish several cancer variations wetest all cancers against each other assigning the positivevalue one to the class of interest and zero to the remainingones When detecting thyroid cancer all thyroid examplesare labeled as one and the skin stomach breast and lunginstances as zero and henceforwardAfter processing all the datasets it is improbable thatthe preprocessing phase removed the same columns in allof them To guarantee the same features describe all thesamples we intersect all the datasets and use the resultas our final dataset Also given that the breast cancerdatasets had almost the double of instances we applydownsampling and randomly select breast cancerexamples to keep the final dataset as evenly distributedfor all the cancers as possible In the end the resultingdataset has approximately instances and more than thousand genesData of features extracted from imagesWe use two datasets of two different diseases composedof features extracted from images malaria and breast cancer Since we aim to evaluate how well this methodologygeneralizes by using distinct types of data we are nowable to gather evidence supporting this premiseThe malaria dataset was created by the FraunhoferAICOS institution through the MalariaScope project[] Their main goal is to develop lowcost solutions thatcan provide fast reliable and accurate results on detecting such disease particularly in developing countries In[] the authors thoroughly describe the feature extraction process from thin blood smear images exclusivelyacquired with smartphones The resulting dataset is composed of samples and features These featureswere normalized between [ˆ’ ] via scaling and groupedinto three main groups geometry color and textureFrom all the examples approximately contain malariaparasites Due to the high unbalance between Malariaand NonMalaria labels we performed downsampling onthe NonMalaria class where we randomly selected examples We decided to choose instead of dueto a wide variety of nonparasite artifacts Once the samples were selected and similarly to the preprocessing stepof the RNASeq data we verify if there are features withconstant values and remove them if that is the case Ourworking malaria dataset has instances negativeand positive and feature columnsThe Wisconsin Breast Cancer dataset [] from the UCIMachine Learning Repository is composed of examples and features These features are computed from afine needle aspirate digitized image of a breast mass anddescribe the cell nuclei characteristics present in thoseimages such as texture area concavity and symmetryFrom the examples approximately are benignsamples and are malign ones No under or oversampling techniques were applied since we do not find it to beneeded As performed in the malaria data we checked ifthere were columns with constant values for which therewere not The data was used as is with the proportionsand characteristics described aboveAutoencodersAn autoencoder AE [] is an unsupervised featurelearning neural network that aims to copy its input based 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of on a lower dimensional representation This type of architecture is able to extract features by reducing the dimension of its hidden layer [] which helps the AE to focuson capturing the essential features that best represent thedataLet the encoding and decoding functions of the AE be fand g parameterized on θe and θd respectively where θ θeˆª θd L being the loss function and J the cost function tobe minimized When learning the AE aims to find value θthatargminθJθ X LX gθdfθe Xpenalizing the reconstruction of the input given by ˆX fθe X the more distinct ˆX is the bigger the appliedgθdpenalty When training an AE we use Mean Squared ErrorMSE as the loss function and the Rectified Linear Unitsactivation function ReLU [] for all its layers Currentlyusing ReLU as activation is the default recommendationwhen training neural networks [] Similarly using MSEas the loss function is a fairly common practice present inthe literature when training AEs [ “]We use the AEs as a weight initialization technique[] since evidence supports that using œunsupervised pretraining guides the learning towards basins of attractionof minima that support better generalization from thetraining dataset [] Thus we pretrained them beforeimporting the encoding part or all their layers to theclassification neural networkBasic autoencoder AEThe simplest AE has only one hidden layer This type ofAE learns through the optimization cost function presented in Eq With the combination of linear activationsReLU and the MSE loss function these AEs behave similarly to the Principle Component Analysis PCA method” when trained with an MSE an AE learns the principalsubspace of the training data consequentially []Denoising autoencoder DAEA Denoising AE DAE [] aims not just to reproduce theinput but also to keep its information intact to undo theeffect of an intentional corruption process applied to theoriginal data Its cost function can be described byFig Overall pipeline of our experiments This figure illustrates the chosen metodology for our work Firstly we pretrain the autoencoders AEsbefore embedding them to the top layers of the classification network fullfilling either Strategy import only the encoding layers from the AE orStrategy import the complete AE Each of the full assembled architectures is then trained to detect one of the cancer types in the input dataThe training process can follow two different approaches regarding the imported weights of the AEs A fixing them or B allowing subsequentfinetune I represents the input layer E the encoding layerˆI the output layer of the AE at the classification region of the network D represents thefully connected layer and O the output of the classifer 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of θfθe ˜XJθ X LX gθdargminwhere ˜X is a copy of the input X intentionally corruptedby a sort of noise [] To simulate a form of BernoulliNoise [] we apply a Dropout layer immediately after theinput layer where of the connections are randomly cutSparse autoencoderSimilarly to a DAE a Sparse AE SAE learning processalso has two main goals minimizing the reconstruction error when aiming to copy the input data and applying a sparsity penatly represented by 01 to theparameters involved in the encoding partJθ X LX gθdfθe X λ · 01θeargminθAlthough it also tries to reproduce X an SAE canaddress unique statistical features of the dataset it hasbeen trained on [ ] To deliver that sparsity elementwe use an L1 penalty with a λ of ˆ’MethodologyWe have adopted the methodology described in []which was also used in [] Our experiments consist ofan analysis of the performance of a DNN trained to classify different cancer types studying how three differentfactors may impact the network performance The top layers where we use three different AEs asweight initialization The dimension of the latent vector of the AEs thatmeans the encoding layer size The imputation technique to replace missing datawhen preprocessing the datasetsBesides the top layers imported from the AE the classification part of the full architecture is composed of aBatch Normalization layer [] followed by two FullyConnected layers with a ReLU [] activation Since weaim to detect one type of cancer at the time the last layer” the predictive one ” is a single neuron layer with aSigmoid nonlinearity [] This activation considers thatif the probability of the classification is lower than the sample is classified as negative that is not having thedisease otherwise the sample is classified as positiveTo assess the following experiments we decided to onlyuse the AE that achieved the best results in the firstexperiments For points and we try three different dimensions and For the data imputationstudy we use three strategies replacing the data witha the mean column value used as default a constantvalue in this case zero and b with the most frequentvalueFurthermore we want to study if when using Strategy importing the complete AE into the classification network the model yields better results just because it hasone more layer and therefore more parameters to trainTo observe if the classifier is better only by being deeperwe pretrained the AE and at the embedding step forStrategy we add a decoder layer with all its weightsrandomized guaranteeing that there are no discrepanciesconcerning the network™s topological complexity for bothstrategiesFinally we want to assess how the pipeline behaveswhen dealing with different data types besides RNAseq entries Hence we apply the same methodologyto the image extracted features datasets described inœThe data section to assess if the model can adapt andgeneralize well to these data characteristicsFor all these we follow the same pipeline see Fig Foreach experience we start by pretraining a different AE tominimize the reconstruction error before importing theminto the top of the classification architecture When doingso we choose one of the two strategies considered for thisstudy add just the encoding layers or add all thepretrained AE After the embedding of the AE to the toplayers we consider two different approaches in the training process A fixing the imported weights of the AElayers and B by allowing them to be finetuned duringthe model training for the classification taskWith the complete architectures AE as the top part ofthe classification network assembled we train each oneto distinguish¢ The RNASeq input data as one of cancers namely¢ The malaria input data as Malaria or NonMalaria¢ The breast masses input data as Malign or Benignthyroid skin stomach breast and lungEvaluationWe use stratified 10fold crossvalidation to ensure andprovide statistical evidence The AEs are trained during epochs and the classifier during with a batchsize of The classification model is trained with thebinary crossentropy loss function [] and with an Adamoptimizer [] Furthermore we assess the overall performance of the model in the training and validation setsby analyzing five more metrics Accuracy Matthews Correlation Coefficient MCC [] Precision Recall and F1score and provide the Receiving Operator Curve with therespective Area Under the Curve ROCAUC and thePrecisionRecall CurveFurthermore to study how the model generalizes tounseen data during the training phase we evaluate theperformance of the best architecture combination on aheldout test set for the Malaria and the Wisconsin BreastCancer datasets For both and separately we use a ratio ofone third to create two new splits Therefore 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of Table Baseline results for cancer detection using a Fully Connected Neural Network the classification architecture without the AEas top layersThyroidSkinStom

Thyroid_Cancer

"Many cancerassociated single nucleotide polymorphisms SNPs are located in the genomic regionsof long noncoding RNAs lncRNAs Mechanisms of these SNPs in connection to cancer risk are not fullyunderstoodMethods Association of SNP rs140618127 in lncRNA LOC146880 with nonsmall cell lung cancer NSCLC wasevaluated in a casecontrol study of individuals The mechanism of the SNP™s biologic influence was exploredwith in vitro and in vivo experiments including plasmid transfection siRNA knockdown flow cytometry assessmentand assays of cell proliferation migration invasion and colony formationResults Association analysis showed that A allele of SNP rs140618127 was associated with low risk of NSCLC in theChinese population Lab experiments indicated that SNP rs140618127 contained a binding site for miR5395p andthe binding between miR5395p and LOC146880 resulted in declined phosphorylation of an oncogene ENO1 Thereduced phosphorylation of ENO1 led to decreased phosphorylation of PI3K and Akt which is further linked to thedecline in cell proliferation and tumor progressionConclusion The study demonstrates that SNP rs140618127 in lncRNA loc146880 provides an alternate binding sitefor microRNA miR5395p which affects the phosphorylation of ENO1 and activation of the PI3K and Akt pathwayKeywords NSCLC lncRNA SNP miRNA ENO1BackgroundLung cancer is the most commonly diagnosed cancer of the total cases and the leading cause of cancerdeath of the total cancer deaths in the world []The majority oflungcancer NSCLC which accounts for around of alllung cancer is nonsmall cell Correspondence qianbiyunsjtueducn Tienan Feng and Nannan Feng contributed equally to this work1Hongqiao International Institute of Medicine Shanghai Tongren HospitalClinical Research Institute Shanghai Jiao Tong University School of MedicineShanghai China7Second Affiliated hospital of Chengdu Medical College China NationalNuclear Corporation Hospital Chengdu Sichuan ChinaFull list of author information is available at the end of the lung cancer cases Genetic factors may play an importantrole in an individual™s susceptibility to NSCLC Longnoncoding RNAs lncRNAs are a class of noncodingtranscripts with nucleotides or more Increasingevidence suggests thatlncRNAs are involved in theoccurrence oflung cancer due to their functions asoncogenes or tumor suppressors [] Our previous studies indicated that a lncRNA on chromosome 17q243named LOC146880 was expressed higher in tumortissues than in adjacent normal tissues and high expression was associated with poor prognosis of NSCLC []Singlenucleotide polymorphisms SNPs in the noncoding regions of the genome have been shown to affect The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cFeng Journal of Experimental Clinical Cancer Research Page of rs140618127 the ˜A™ allelecancer risk via regulating the transcription andor changing the structure of lncRNA [“] A previous studyidentified SNPs in more than humanlncRNAs and a large number of SNPs were predicted tohave a potentialimpact on the microRNA miRNAlncRNA interaction [] Here we report the identificationof SNP rs140618127 in LOC146880 as a new susceptiblelocus to NSCLC Bioinformatics analysis predicts thatin LOC146880variantprovides an altered secondary structure which maycreate a binding site for microRNA miR5395p []sequestering its action on other molecules Shiraishi conducted a GWAS on lung adenocarcinoma andidentified SNP rs7216064 in BPTF 17q243 in association with the cancer risk OR p 7e11 []Seow confirmed that SNP rs7216064 was associated with the risk of lung cancer based on a GWASstudy of Asian female nonsmokers [] We found thatSNP rs140618127 was in strong linkage disequilibriumwith SNP rs7216064 LD r2 and this lncRNASNP was associated with lung cancer risk OR p in our casecontrol study of individualsToSNPrs140618127 in NSCLC development and progressionweconsequence ofLOC146880 and miR5395p interaction and found thatthe microRNA behaved like a tumor suppressor []which prevented LOC146880 from interacting withprotein ENO1 an oncogene product [] reducing itsphosphorylation As a resultthe phosphorylation ofPI3KAKT was also reduced after the suppression ofENO1 phosphorylation [] which further inhibitedtumor growth and metastasisleading to a betterprognosis of NSCLCthe molecular mechanism ofexploreevaluated thatthe biologicalMaterials and methodsStudy populationsSuspected NSCLC individuals had histopathological orcytologically confirmed diagnosis according to theWorld Health anization classification These studysubjects including suspected individuals diagnosed withlung cancer or normal were recruited from the ChinaMedical University CMU Distributions of the basiccharacteristics ofthe study subjects are provided inTable At recruitment an informed consent wasIlluminated Only ifthe subject agreed heshe wasincluded This study was approved by the InstitutionalReview Board at CMUSNP selection and genotypingSNPs with r2 were considered to be in the same LDblock With this criterion one SNP was selected in eachLD block and genotyped using the TaqMan genotypingmethod in the ABI RealTime PCR systemApplied Biosystems For quality control we implemented several measures in our genotyping assays including each plate contained both case and controlsamples technicians were blinded to the casecontrolstatus of the samples both positive and negativecontrol no DNA template samples were included ineach 384well plate and nearly of the sampleswere assayed in duplicate and the concordances werebetween and Cell linesHuman NSCLC cell lines A549 and PC9 and humanlung epithelial BEAS2B cells were purchased from theCell Bank of Type Culture Collection at the ChineseAcademy of Sciences Shanghai Institute of Biochemistryand Cell Biology These celllines were passaged forfewer than months All the cells were tested for mycoplasma and were found to be free from infection Thecells were maintained in DMEM supplemented with FBS and grown without antibiotics in an atmosphere of CO2 and relative humidity at °C² and ² RACE and coding prediction of LOC146880We used ² and ² RACE to determine the transcripinitiation and termination sites of LOC146880tionalwith a SMARTe RACE cDNA Amplification kit Clontech The Alignment File of a fulllength sequence ofLOC146880 obtained from ² and ² RACE is availableupon requestConstruction of reporter plasmids transient transfectionsand luciferase assaysA reporter plasmid in the psiCHECK2 vector Promegawas created which contains a 1000bp LOC146880 exonregion flanking rs140618127 [G] or rs140618127 [A]with the restriction enzymes XhoI and NotI FermentasA549 and PC9 cells were seeded at — cells per wellin 24well plates and ng of the reporter plasmid and pmol of miR5395p mimic Ambion were cotransfected into the cells h later using Lipofectamine Invitrogen These cells were collected h aftertransfection Renilla luciferase activity was measured andused to normalize the efficiency of transfectionRNA extraction and qRTPCR analysisTotal RNA from the NSCLC tissue specimens and celllines used in this study was extracted using the TRIzolreagent Firststrand cDNA was synthesized using theSuperScriptInvitrogenRelative RNA levels determined by qPCR were measuredon an ABI sequence detection system AppliedBiosystems using the SYBR Green method Βetaactinwas employed as an internal control for the quantification of LOC146880 and the mRNA levels of other genesreverse transcriptase kitII 0cFeng Journal of Experimental Clinical Cancer Research Page of For miRNA quantification small nuclear RNA U6 wasused as an endogenous control The relative expressionof RNA was calculated using the comparative CtmethodSubcellular fractionationCytosolic and nuclear fractions of A549 and BEAS2Bcells were prepared and collected according to theinstructions ofthe NuclearCytoplasmic Isolation kitBiovision LOC146880 was mainly detected in thenuclear fraction although it was also present in the cytoplasm Fig S1belowBrieflydescribedRNA pulldown and mass spectrometry analysisRNA pulldown assays were performed following theprotocolbiotinylatedLOC146880 or antisense LOC146880 was incubated withcellular protein extracts from A549 cells and streptavidin beads were then added Recovered proteins associated with LOC146880 or antisense LOC146880 wereexcised and proteomics screening was accomplished bymass spectrometry analysis on a MALDITOF instrumentIn vitro transcription ofLOC146880 and its deletion fragments were analyzedwith primers containing the T7 promoter sequenceBruker DaltonicsPlasmid construction and transfectionTo construct a lentiviral vector expressing humanLOC146880 NR_026899 a fulllength of LOC146880cDNA containing rs140618127 [G] or rs140618127 [A]was commercially synthesized GeneChem and subcloned into the AgeI and NheI sites of the GV367IRESPuromycin lentiviral expression vector GeneChem Toproduce lentivirus containing LOC146880 T cellswere cotransfected with the vector described above andlentiviral vector packaging system GeneChem using Lipofectamine Infectious lentiviruses were collectedat h after transfection and filtered through 045μmPVDF filters for analysis of genotype After conformation these lentiviruses were designated to LOC146880[G] or LOC146880 [A] We used the GV367IRESPuromycin empty vector as a negative control Theviruscontaining pellet was dissolved in DMEM and aliquots of the solution were stored at ˆ’ °C A549 andPC9 cells were infected with concentrated virus in thepresence of polybrene SigmaAldrich The supernatantwas replaced with complete culture medium after hfollowed by selection with puromycin and the expression of LOC146880 in infected cells was verified byqPCRRNA immunoprecipitation assaysRIP experiments were performed using the Magna RIPProteinRNABindingkitMillipore Antibodiesagainst ENO1 Abcam orcontrol proteins were diluted at Total RNA inputcontrol and precipitation with the isotype control IgGfor each antibody were assayed simultaneously The coprecipitated RNAs were detected by RTqPCRImmunoprecipitationCell lysis and immunoprecipitationCells were homogenized in — RIPA buffer supplemented with ProteasePhosphatase Inhibitor CocktailPierce Cell lysates were centrifuged and the supernatants were prepared for immunoblotting or immunoprecipitation withbelowImmunoblot signal was detected using Clarity WesternECL Substrate Thermo FisherantibodiesdescribedtheTable Characteristics of lung cancer patients and healthy controlsVariablesP valueControlN NSCLCN Age at dx year ‰¥ GendermalefemaleSmoking statusnoyesSNP rs140618127G alleleA allele adjusted by smokinggenderage OR valueP valueOR value “ “ “ “ “ “ “ “ 0cFeng Journal of Experimental Clinical Cancer Research Page of from cells orImmunoblot assaysProtein extractsimmunoprecipitationsamples were prepared using detergentcontaining lysisbuffer Total protein μg was subjected to SDSPAGE and transferred to PVDF membrane MilliporeAntibodies against ENO1 Abcam ab155102 ENO1phosphorylated at Cterminalinhibitory site Tyr44StressMarq Bioscience spc965D PI3K CST 13666SPI3K phosphorylated at Tyr458 CST 4228S AKTCST 2938S AKT phosphorylated at Ser473 CST9018S PCNA CST 2586S NFkB CST 8242SVimentin Abcam ab92547 βCatenin CST 8480S Ecadherin CST 14472S NCadherin Abcam ab18203and βActin SigmaAldrich A1978200UL were usedMembranes were incubated overnight at °C withprimary antibody diluted and proteins were detected with the Odyssey near infrared dualcolor laserimaging system LICORAnalysis of cell proliferation migration invasion cellcycle and colony formationCells were seeded in 96well flatbottom plates with cells in μl cell suspension in each well Afterculture cell viability was measured with the CCK8assay Each experiment with six replicates was repeatedthree times For cell cycle analysis cells were collectedand fixed in ethanol overnight at °C Singlecellsuspensions were labeled with μgml Propidium Iodide Sigma and analyzed by flow cytometry BeckmanCoulter For colony formation cells were seededin 65mm culture dishes and allowed to grow until visible colonies formed in complete growth medium weeks Cell colonies were fixed with methanol stainedwith crystal violet and counted Invasion assays wereperformed in Millicell chambers in triplicate The 8μmpore inserts were coated with μg of Matrigel BD Biosciences Cells — were added to the coated filtersin serumfree medium PMI1640 medium containing FBS was filled in the lower chambers as a chemo attractant After h at °C in an incubator suppliedwith CO2 cells that migrated through the filters werefixed with methanol and stained with crystal violet Cellnumbers in three random fields were counted The migration assay was conducted in a similar fashion withoutcoating the filters with MatrigelExperiments on xenograft animalsTen male BALBc mice weeks old were kept in aspecific pathogenfree grade environment All animalexperiments were approved by the Animal Care and UseCommittee of Shanghai Jiao Tong University School ofMedicine Shanghai China All applicable guidelines ofthe Animal Care and Use Committee of Shanghai JiaoTong University School of Medicine for the care and usethe hind flank regions ofof animals were followed PC9 cells of rs140618127 [A]and rs140618127 [G] type were collected and resuspended in PBS at a concentration of — cellsmLand mixed with Matrigel® at a ratio of respectivelyThe mixture mL was subcutaneously injected intotwo sides ofthe micers140618127 [A] and rs140618127 [G] cells in the samemouse Tumor size was measured once every daysusing a Vernier caliper across its two perpendicular diameters and tumor volume was calculated using the following formula V 12ab2 where V is the tumorvolume a is the largest diameter and b is the smallestdiameter After weeks oftreatment all mice weresacrificed and their tumors were collected and weighedHistological evaluation ofsamples wasperformedthe tumorHistopathological analysesTumor tissues from the animals were fixed in paraformaldehyde BOSTER Wuhan Chinafor h atroom temperature The fixed tissues were then dehydrated in a graded series of alcohol cleaned in xyleneand embedded in paraffin A rotary microtome was usedto section paraffin the blocks into 4μm thick sectionsThe sections were deparaffinized and stained withhematoxylin and eosin HE A light microscopeOlympus was used to examine the stained tissuesectionsStatistical analysisThe association between SNP rs140618127 and NSCLCrisk was analyzed under an additive model using the unconditionallogistic regression model adjusted for agesex and smoking status Results of laboratory experiments were presented as Means ± SD Student™s t testwas used to compare means between two groups andANOVA was employed for comparison of more thantwo groups Repeated ANOVA was employed for comparison of more than two groups which contained repeated measure data All the statistical analyses wereperformed using Statistical Product and Service Solutions SPSS software version and GraphPad PrismVersion GraphPad Software San Diego CA USAResultsSNP rs140618127 G A in LOC146880 and NSCLC riskSNP in LOC146880 rs140618127 is in strong linkagedisequilibrium with SNP rs7216064 r2 which is aGWASdiscovered risk allele for NSCLC We found thatSNP rs140618127 G A in the exon of LOC146880chr17 was associated with the risk of NSCLC the ˜A™ allele compared to ˜G™ had an adjusted oddsratio OR “ in a casecontrol study of subjects Table Stratified analyses suggested 0cFeng Journal of Experimental Clinical Cancer Research Page of that this effect was more evidence in those who were ‰¥female and nonsmokers Supplemental years oldTable S1 The minorfrequency of SNPrs140618127 is low globally but can be high as in some American populations see Suppl alleleEffects of LOC146880 with rs140618127 [a] on cellproliferation and behaviorsWe examined the effects of LOC146880 on cell proliferation by its allele at rs140618127 and found that overexpression of rs140618127 [A] in the NSCLC celllinesA549 and PC9 both with the G allele at rs140618127substantially reduced the rate of cell proliferation whencompared with rs140618127 [G]Fig 1A Colonyformation ability in both A549 and PC9 cells wasmarkedly suppressed by rs140618127 [A] when compared with rs140618127 [G] Fig 1B Overexpression ofrs140618127 [A] significantly suppressed the invasionand migration of NSCLC cells Fig 1C 1D Tumorsize in a xenograft animal model of PC9 was decreasedin both genotype groups but the decline in tumor sizewas greater for rs140618127 [A] than for rs140618127[G] P There was no significant difference intumor size between the vector control group and wildtype rs140618127 [G] Fig S2 HE staining showedthat tumors of rs140618127 [A] possessed less malignantmorphology Fig 1E Together these results indicatethat rs140618127 [A] can inhibit the growth of lung cancer more in vitro and in vivo compared to rs140618127[G]such a possibilityInteraction between LOC146880 and miR5395pEvidence suggests that SNPs in lncRNAs may generatenew interacting sites between lncRNAs and other transcripts such as miRNAs [] Using an online softwarelncRNASNP httpbioinfolifehusteducnlncRNASNP[] we found that several SNPs in LOC146880 werepredicted to haveand SNPrs140618127 was indicated to lie within a putative binding site for miR5395p The G A mutation atrs140618127 was predicted to change the local foldingstructures and free energy of LOC146880 which mightcreate a binding site for miR5395p Following this prediction we investigated whether miR5395p interactswith LOC146880 based on its genotype at rs140618127Luciferase reporter assays showed that in comparison tothe construct containing rs140618127 [G] the constructwith the ˜A™ allele had significantly reduced luciferase activity in the presence of miR5395p suggesting moreinteraction of miR5395p with LOC146880 [A] thanwith LOC146880 [G] Fig 2A The interaction betweenmiR5395p and LOC146880 [A] could be blocked bythe miR5395p inhibitor miR5395p is constitutivelyexpressed in both A549 and PC9 cells In cells stablyoverexpressing LOC146880 miR5395p only decreasedthe levels of LOC146880 with rs140618127 [A] not alleleG indicating that allele A is a target of miR5395p Fig2Bthe RNA pulldown assay weInteraction between LOC146880 and ENO1isolated aUsingLOC146880 withrs140618127[G]protein complexMass spectrometry analysis showed that there were threeproteins in this complex and the most abundant onecompared to antisense one was ENO1 Fig 3A Wethen selected ENO1 for validation detecting ENO1 inthree independent RNA pulldown assays RNA immunoprecipitation RIP assays also showed enrichment ofLOC146880 in the complexes precipitated with ENO1antibody as compared with IgG or another irrelevantantibody indicating that ENO1 may be a key target protein of LOC146880 Fig 3b Next we evaluated the consequences of the interaction between LOC146880 andENO1 We found that ENO1 mRNA expression andprotein level were not significantly different P see Fig S3 in the cells overexpressing LOC146880 withrs140618127[A] or rs140618127[G] in the presence ofmiR5395p Fig 3C 3D However HE staining ofxenograft tumors in mice showed that phosphorylatedENO1 was higher in rs140618127 [G] than in [A] Fig3E The expression of CMYC a downstream target ofENO1 was decreased remarkably when the cells weretransfected with a siRNA against ENO1 Fig 3FRegulation of PI3KAKT signal by LOC146880 via ENO1phosphorylationWe found that ENO1 phosphorylation was markedly decreased in cells overexpressing rs140618127 [A] as compared with those overexpressing rs140618127 [G] in thepresence of miR5395p mimics Fig 4A Fig S4 andFig S5 Using insilico prediction tools [ ] weidentified a phosphorylation site at Tyr44 in the proteinbased on the PDB database Fig S5 [] We next investigated the impact of altered LOC146880 levels on thedownstream signal of ENO1 Since our results describedaboveoverexpressionincreased cell proliferation migration and invasion wefocused our investigation on the PI3KAKTNFkB signaling The total amount of PI3K and AKT proteins wasnot significantly different between cells overexpressingrs140618127 [A] and [G] However we observed thatprotein phosphorylation levels affected the expression ofdownstream molecules in the PI3KAKT signaling inA549 Fig S4 Cells overexpressing LOC146880 withrs140618127 [A] showed substantial decreases in NFkB PCNA Vemintin and Ncadherin levels while theirβcatenin and Ecadherin levels weresignificantlyincreased when compared withthesamecellsLOC146880indicatedthat 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig rs140618127[A] inhibits NSCLC cell proliferation and EMT process A proliferation assay of different samples which were compared byrepeated ANOVA B clone formation ability of different samples which were compared by ANOVA C wound healing assay of different sampleswhich were compared by ANOVA D cell invasion assay of different samples which were compared by ANOVA e cancer protective effect ofrs140618127[A] in xenograft animals which were illuminated by repeated ANOVA and ttest 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig rs14061812[G][A] expression of different condition in A549 and PC cell lines a luciferase activity of different conditions which wereilluminated by ANOVA b LOC146880 expression level of different conditions which were compared by ttestoverexpressing LOC146880 with rs140618127 [G] Fig4B Fig 4C Fig S7 and Fig S8 Immunohistochemical staining of xenograft tumors showed that pPI3KpAKT TWIST NCadh and SNAIL were all significantly higher in rs140618127 [G] than in [A] Fig 4Dand Fig S9DiscussionIn this study we found that SNP rs140618127 inLOC144680 contained a binding site for miR5395pand the binding between miR5395p and LOC146880resulted in declined phosphorylation of an oncogeneENO1 which was found to be a downstream target ofLOC146880 Furthermore the reduced phosphorylationof ENO1 led to decreased phosphorylation of PI3K andAkt which was linked to the decline in tumor cell proliferation and progress The entire process of how SNPrs140618127 influences NSCLC is depicted in Fig Our casecontrol study supports the notion that SNPrs140618127 genotype [A] may have a protective effecton NSCLC compared to genotype [G] In a previousstudy we found that LOC146880 expression was significantly higher in NSCLC tumors than adjacent normaltissuesforLOC146880 []a possible oncogenicsuggestingroleThere has been an increasing interest in understandingthe mechanisms of rare genetic variants in lncRNAs inrelation to the complex traits and diseases [ ] Ingle suggested that genetic polymorphisms in lncRNAMIR2052HG offer a pharmacogenomic basis for the response of breast cancer patients to aromatase inhibitortherapy [] Tang indicated that SNP rs9839776in SOX2OT was significantly associated with breast cancer possibly via influencing the expression of SOX2OT[] Redis demonstrated that the GWASidentifiedSNP rs6983267 on 8q24 is in a lncRNA gene calledCCAT2 which regulates cancer cell metabolism in anallelespecific manner through binding to the cleavagefactor I complex This complex is implicated in anallelespecific regulatory mechanism of cancer metabolism orchestrated by alleles ofthe lncRNA [ ]Russell et alidentified a neuroblastoma susceptibilitylocus rs9295534 located in the upstream enhancer of atumor suppressor CASC15S The SNP could decreasethe transcriptional activity of CASC15S and be associated with the disease outcome [] Wang foundthat SNP rs965513 a locus on 9q22 in the FOXE1 geneand lncPTCSC2 was associated with the risk of papillary thyroid carcinoma []In this study we found that a lncRNA could regulatethe function of a protein via its phosphorylation with 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig LOC146880 promotes ENO1 activation in a variantspecific manner A pulldown assaymass spectrum of identificationsilver staining BRIP assay with A549 and PC9 cell lines which were compared by ANOVA C D ENO1 mRNA expression and protein level of rs140618127[G][A]by overexpression plasmid transfection which were compared by ttest E HE staining of ENO1 phosphorylation in vivo which were comparedby ttest F ENO1 LOC146880 and cMYC expression level after ENO1siRNA transfection which were compared by ttestlittle influence on gene expression or protein concentration Similar findings have been reported before in whichthe phosphorylation site of a protein can be blocked by alncRNA leading to decreased phosphorylation For example NFkB can be inhibited by a long noncodingRNA which directly blocks IKB phosphorylation inbreast cancer [] LncRNA can also bind to proteinsincreasing or decreasing their phosphorylation viaanother protein LncRNA DANCR and PANDAR influence the phosphorylation of serine in RXRA and SFRS2via GSK3β and P53 in breast and ovarian respectively[ ] GSK3β™s phosphorylation in breast cancer wasreported to be reduced by lncRNA NLIPMT [] Thephosphorylation of ULK1 can be suppressed by lncRNAHOTAIR in NSCLC [] LINC00675 enhances the phosphorylation of vimentin on Ser83 to suppress gastriccancer progression [] Our finding of a lncRNA™s impact on the phosphorylation of a protein was quiteunique and interesting because it is achieved by a microRNA through a polymorphic site in LOC146880In our study we found that a G to A transition atrs140618127 in LOC146880 could turn into a bindingsite for a microRNA and miR5395p was indeed the target Interestingly the wildtype of LOC146880 had no 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig LOC146880 regulates PI3KAKT signaling via ENO1 A549 PC9 A ENO1pENO1 PI3KpPI3K AKTpAKT protein level usingrs140618127[G][A] overexpression plasmid transfection B PCNA NFkB protein level using rs140618127[G][A] overexpression plasmidtransfection C βCatenin Vimentin NCadherin ECadherin protein level using rs140618127[G][A] overexpression plasmid transfection D HEstaining of pPI3K pAkt TWIST NCadhersin and SNAIL 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig Diagrammatic sketch of rs14061812mediated NSCLC tumorigenesis LOC146880 rs14061812[G] increases ENO1™s phosphorylationresulting in activating PI3KAKT signaling pathway and NSCLC tumorigenesis while LOC146880rs14061812[A] binds to miR5395p decreasesENO1™s phosphorylation resulting in deactivating PI3KAKT signaling pathway and NSCLC tumorigenesisinteraction with the microRNA at all This SNP has notbeen reported before in any studies [] However miR is known to be a tumor suppressor [ ] Ourfinding of miR539™s binding to loc146880 provided newinsights into a possible mechanism that explains the biologic function of miR539 as a tumor suppressor Thiseffect takes place when a microRNA and lncRNA interact through a polymorphic site which results in changesin phosphorylation in a protein ENO1 that the lncRNAmay target on Low levels of LOC146880 did not influence the mRNA expression or protein levels of ENO1but suppressed the phosphorylation of ENO1 ENO1 is ametabolic enzyme involved in the synthesis of pyruvateIt also acts as a plasminogen receptor and mediates theactivation of plasmin and extracellular matrix degradation In tumor cells ENO1 is upregulated and supportsthe Warburg effect The protein is located on the cellsurface where it promotes cancerinvasion and issubjected to substantial posttranslational modificationsnamely acetylation methylation and phosphorylation[] Reduced phosphorylation of ENO1 lowers thePI3KAkt signal which results in slower cell migrationor invasion of NSCLCThe SNPbased interaction between miRNA andlncRNA in regulation of protein function has beenhypothesized and predicted by YaRu but fewstudied have provided evidence [] Our study was thefirst to show the interaction between LOC146880 andmir5395p in the NSCLC and to elucidate the downstream mechanism involving tumor growth and metastasis The modulation model of the lncRNA and miRNA isnot the classical competing endogenous RNAs ceRNAHow LOC146880 interacts with ENO1 to regulate itsphosphorylation and downstream signals remains to beelucidated Although the ˜A™ allele of rs140618127 is lowin general some racial groups still have a relatively highfrequency In some Caucasian populations the ˜A™ allelefrequency is close to ConclusionsWe found in a casecontrol study of Chinese thatSNP rs140618127 in LOC146880 was associated with therisk of NSCLC People with the G allele of rs140618127had higher risk than those with the A allele Our in vitroand in vivo experiments demonstrated that LOC146880was an oncogene and the G allele of rs140618127 hadstronger oncogenic effects on lung cancer cells than theA allele in LOC146880 This differential effect appearedto come from the binding of a microRNA miR539 tothe A allele but not the G allele at rs140618127 ThemicroRNA binding prevented the lncRNA™s interactionwith its downstream target ENO1 which led to the reduction of ENO1 phosphorylation and suppression ofthe PI3KAKT signaling resulting in lower tumor cellproliferation and less aggressive cell behaviors 0cFeng Journal of Experimental Clinical Cancer Research Page of Supplementary informationSupplementary information accompanies this paper at httpsdoi101186s13046020016525Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestAdditional file Fig S1 Locations of LOC146880 A549 BEAS2BLOC146880 locals mainly in cytoplasm The comparison between twogroups using ttestAdditional file Fig S2 Comparison of tumor size between vectorcontrol group and the wide type There was no significant difference oftumor size between vector control group and the wide typers14061812[G]Additional file Fig S3 Comparison of ENO1 protein level ofrs140618127[G][A] by overexpression plasmid transfection which werecompared by ttestAdditional file Fig S4 Comparison of ENO1pENO1 PI3KpPI3KAKTpAKT protein level of A549 cell lines using rs140618127[G][A]overexpression plasmid transfection which were compared by ttestAdditional file Fig S5 Comparison of ENO1pENO1 PI3KpPI3KAKTpAKT protein level of PC9 cell lines using rs140618127[G][A]overexpression plasmid transfection which were compared by ttestAdditional file Fig S6 Predicting site of phosphorylation of ENO1A predicting phosphorylation site of ENO1ENO1 chain A usingNetPhos B predicting phosphorylation site of ENO1ENO1 chain Ausing PhosphoELM BLAST C The predicting phosphorylation site ofENO1 chain A using data of PDB databaseAdditional file Fig S7 Comparison of PCNA and NHkB protein levelof using rs140618127[G][A] overexpression plasmid transfection whichwere compared by ttest A results of A549 cell lines B result of PC9cell linesAdditional file Fig S8 Comparison of βCatenin Vimentin NCadherin ECadherin protein level using rs140618127[G][A] overexpression plasmid transfection which were compared by ttest A results ofA549 cell lines B result of PC9 cell linesAdditional file Fig S9 Comparison of the HE staining of pPI3K pAkt TWIST NCadhersin and SNAIL which were compared by ttestAdditional file Distribution of

Thyroid_Cancer

case of a 14year old boy with tumorassociated refractory epilepsy Positron emission tomography imaging demonstrated a region with heterogeneous high 11Cmethionine uptake and a region with homogenous low 18Ffluorodeoxyglucose uptake within the tumor Histopathological and genomic analyses confirmed the tumor as BRAF V600Emutated polymorphous lowgrade neuroepithelial tumor of the young PLNTY Within the highmethionineuptake region we observed increased protein levels of Ltype amino acid transporter LAT1 a major transporter of methionine cMyc and constituents of the mitogenactivated protein kinase MAPK pathway We also found that LAT1 expression was linked to the BRAF V600E mutation and subsequent activation of MAPK signaling and cMyc Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 expression in BRAF V600Emutated PLNTY and glioblastoma cells The BRAF inhibitor dabrafenib moderately suppressed cell viability in PLNTY Collectively our results indicate that BRAF V600E mutationactivated MAPK signaling and downstream cMyc induces specific metabolic alterations in PLNTY and may represent an attractive target in the treatment of the diseaseKeywords PLNTY BRAF V600E mutation Methionine PET LAT1IntroductionPediatric lowgrade neuroepithelial tumors PLGNTs encompass a group of central nervous system neoplasms that longterm epilepsyassociated tumors LEATs such as ganglioglioma and dysembryoplastic neuroepithelial tumor DNT PLGNTs have different characteristics than their adult counterparts and includes Correspondence ktate12yokohamacuacjp Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama JapanFull list of author information is available at the end of the are commonly driven by genomic alterations in the Rasmitogenactivated protein kinase MAPK pathway such as mutations in BRAF and NF1 [ ] Recent largescale genomic studies and genomewide methylation analyses allowed a thorough characterization of PLGNTs [] and cIMPACTNOW the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy currently classifies P“LGNTs as distinct disease entities [ ] In Huse et a0al described ten cases of polymorphous lowgrade neuroepithelial tumor of the young PLNTY which were histologically characterized by oligodendrogliomalike cellular components with intense CD34 immunopositivity According to previous publications PLNTYs are indolent tumors The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cTateishi a0et a0al acta neuropathol commun Page of that generally exhibit a benign clinical course and harbor either a BRAF V600E mutation or FGFR2FGFR3 fusion [] Based on its histological and genomic profiles cIMPACTNOW Update recommends PLNTY as a possible future classification for pediatrictype glialglioneuronal tumors However because of their rare etiology only a few PLNTYs have been described to date [ ] and it is unclear how genomic alterations promote the pathogenesis of the disease Herein we present a case of PLNTY with unique metabolic imaging features Using positron emission tomography PET we found regions of heterogeneous high 11Cmethionine uptake and homogenous low 18Ffluorodeoxyglucose FDG uptake within the tumor Activation of the MAPK pathway cMyc and expression of Ltype amino acid transporter LAT1 were increased in the highmethionineuptake area compared with the surrounding cortex lowmethionineuptake Glycolytic metabolites were expressed only weakly in tumor cells Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 and inhibited tumor cell viability suggesting that MAPK signaling and downstream cMyc activates methionine metabolism and inhibition of this pathway induces therapeutic vulnerability in PLNTYMaterials and a0methodsCell viability analysisAM38 and normal human astrocytes was purchased from JCRB Cell Bank and ScienCell Research Laboratories respectively Tumorsphere lines were cultured in serumfree neural stem cell medium as previously described [] Normal human astrocytes were cultured with astrocyte medium ScienCell To assess cell viability primary cultured cells were dissociated into single cells and seeded into 96well plates at a density of cellswell After a0h dabrafenib Selleck and trametinib Selleck were serially diluted and added to the wells Cell viability was measured using the CellTiterGlo Promega assay at day and the results were indicated as viability of the DMSO controlshRNA cell line generationTo knockdown BRAF 293T cells were transfected with lentiviral vector packaging plasmid DNA containing a0 μg of Human BRAF shRNA TRCN0000381693 GP and a0μg of a0pVSVgRev a0with Lipofectamine„¢ TRCN0000196844 Sigma Aldrich a0μg of a0pHIVThermo Fisher Scientific YMG62 and AM38 cells were infected with lentivirus in polybrene a0μgmL for a0h Two days later the cells were selected with puromycin a0μgmL for a0days and used for experiments GIPZ nonsilencing lentiviral shRNA Control RHS4348 Horizon Discovery was used as a nonsilencing NS controlImmunohistochemistryTumor tissue specimens were fixed in neutral buffered formalin and embedded in paraffin Hematoxylin and eosin staining was performed using standard procedures For immunohistochemical analysis 5µmthick sections were deparaffinized treated with H2O2 in methanol rehydrated and heated for a0min for antigen retrieval After blocking with serum tissue sections were incubated with primary antibodies against CD34 Novus Biologicals LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology phosphoERK Bethyl Laboratories and cMyc Cell Signaling Technology at a0°C overnight The next day sections were washed with PBS incubated with biotinylated secondary antibody for a0 min at room temperature and then incubated with ABC solution PK6101 PK6102 Vector laboratories for a0 min at room temperature Finally the sections were incubated with DAB Dako and counterstained with hematoxylinWestern blottingcOmplete„¢ Mini EDTAfree Protease Inhibitor Cocktail Cells were lysed in RIPA buffer SigmaAldrich with a Roche Fifty micrograms of protein was separated by SDSPAGE gel and transferred to polyvinylidene difluoride membranes Millipore by electroblotting After blocking with or nonfat dry milk in TBST a0mM Tris [pH ] a0 mM NaCl Tween20 membranes were incubated at a0 °C overnight with primary antibodies After washing and incubation with horseradish peroxidaseconjugated secondary antibodies Cell Signaling Technology blots were washed and signals were visualized with chemiluminescent HRP substrate Millipore Primary antibodies against BRAF Gene Tex cMyc Cell Signaling Technology GAPDH Gene Tex LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology a0phosphoERK Bethyl Laboratories and Vinculin Novus Biologicals were used for western blottingCase presentationThis study was performed in accordance with declaration of Helsinki and was approved by the Institutional Review Board Yokohama City University [YCU Yokohama Japan] IRB numbers A1711300006 and B190600002 Written informed consent was obtained from the patient and parents A 14year old boy presented with chronic medial temporal lobe epilepsy for a year Magnetic resonance imaging MRI indicated 0cTateishi a0et a0al acta neuropathol commun Page of See figure on next pageFig Characteristics of a patient with PLNTY a T2weighted left T1weighted middle and contrastenhanced right MR images b Computed tomography CT left 18FfluorodeoxyglucosePETCT middle and 11CmethioninePETCT right images c Video electroencephalography indicating ictal onset in the left temporal lobe with spread to the contralateral temporal lobe d PETCT and MRI merged intraoperative navigation image left and surgical image right showing the highmethionineuptake region and surrounding abnormal lesion on MRIhypointensity on T2weighted images and hyperintensity on T1weighted images with a cystic component in the left temporal lobe Contrastenhanced MRI showed no significant enhancement in the lesion Fig a01a while computed tomography revealed heavy calcification FDGPET showed lower FDG uptake in the tumor while 11CmethioninePET demonstrated increased methionine uptake in the same lesion SUVmax tumornormal tissue ratio Fig a01b Videoelectroencephalographic EEG monitoring indicated ictal onset in the left temporal lobe with subsequent spread to the contralateral temporal lobe Fig a01c We speculated that this abnormal lesion was a LEAT Since we considered this tumor to be completely resectable the patient underwent craniotomy and resection of the neoplasm including the highmethionineuptake region Fig a01d To achieve epileptic control electrocorticography was performed intraoperatively After removal of the highmethionineuptake and T2 hyperintense lesions the surrounding tissue was resected until interictal epileptiform discharge could no longer be detected by electrocorticography The patient became epilepsyfree after lesion removal and MRI indicated complete remission a0months after the surgeryTissue samples of the highmethionineuptake region and surrounding cortex low methionine uptake were collected Hematoxylin and eosin staining indicated diffusely infiltrating growth patterns and presence of oligodendroglialike cellular components Fig a02a Astrocytic and highgrade features were absent with a Ki67 index of less than Chicken wirelike branching capillaries and microcalcification were also found in region Despite lower cellularity oligodendroglialike cells were present in the surrounding tissue Immunohistochemistry revealed extensive CD34 expression and peripherally associated ramified neural elements in the tumor cells Fig a0 2a Targeted DNA sequencing identified a BRAF V600E mutation in the tumor without recurrent mutations in IDH1 IDH2 TERT promoter FGFR1 H3F3A or HIST3H1B Fig a0 2b Chromosome 1p19q codeletion was absent Fig a02c The above histological and genetic features fulfilled the diagnostic criteria for PLNTYTo assess the mechanisms underlying the methionineFDG uptake mismatch indicated by PET we compared the expression of LAT1 glucose transporter GLUT and hexokinase2 HK2 between tissue regions and Notably LAT1 which is a major methionine transporter was more highly expressed in than in Fig a0 3a In contrast GLUT1 and HK2 which is correlated with FDG uptake and lactate dehydrogenase A LDHA expression were weak in either region Additional file a0 Fig a0 S1 LAT1 expression is mediated by cMyc activation and BRAF V600E mutation activates the MAPK pathway and downstream cMyc [ ] Therefore we hypothesized that BRAF V600E mutation promotes LAT1 expression through MAPK signaling and consequent cMyc activation a0 in PLNTY Levels of phosphoMEK phosphoERK and cMyc were higher in tissue region than in Fig a03a suggesting activation of the MAPK pathway and cMyc within the highmethionineuptake lesion To verify whether the BRAF V600E mutation can induce the expression of LAT1 we exposed primary cultured YMG83 PLNTY cells to a BRAF inhibitor dabrafenib As expected the expression of phosphoMEK phosphoERK cMyc and LAT1 was suppressed after dabrafenib treatment in YMG83 cells Fig a0 3b Notably BRAF inhibitor dabrafenibtreated YMG83 cells had lower cell viability compared to normal human astrocytes Fig a03c To confirm the reproducibility of these molecular features we used patientderived YMG62 cells epithelioid glioblastoma with the BRAF V600E mutation which exhibited high 11Cmethionine uptake by PET imaging Additional file a0 Fig a0 S2 and AM38 glioblastoma cells BRAF V600E mutant We found that dabrafenib and a MEK inhibitor trametinib inhibited the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03d and 3e Similarly BRAF knockdown suppressed the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03f Collectively these findings indicated that activation of the MAPK pathway by the BRAF V600E mutation deregulates cMyc and promotes LAT1 expression This oncogenic signaling pathway increases methionine metabolism and tumor maintenance in PLNTYDiscussionThirty cases of PLNTY have been described to date with the first ten reported by Huse et a0al in [ ] BRAF V600E mutation was seen in of the patients and BRAF fusion in patient These BRAF alterations were mutually exclusive with other genomic events including FGFR3TACC3 fusion FGFR3 amplification FGFR2CTNNA3 fusion FGFR2INA fusion 0cTateishi a0et a0al acta neuropathol commun Page of 0cTateishi a0et a0al acta neuropathol commun Page of Fig Histopathologic and genomic features of a patient with PLNTY a Hematoxylin and eosin HE staining top and CD34 immunohistochemistry bottom in the highmethionineuptake and lowmethionineuptake region within tumor tissue Bars μm b Sanger sequencing for detection of BRAF V600E arrow left and IDH1 R132H arrow right mutations c Fluorescence in situ hybridization for detection of 1p311q25 left and 19q1319p13 right chromosomal deletionsFGFR2 KIAA1598 fusion FGFR2 rearrangement and NTRK2 disruption suggesting that the vast majority of PLNTYs are induced by BRAF mutation or FGFR fusion and subsequent MAPK activation Therefore targeting MAPK signaling may become a potential therapeutic strategy in PLNTY Indeed BRAF V600Emutated PLNTY cells were relatively vulnerable to dabrafenib and trametinib in the present study Thus targeted molecular therapy for the MAPK pathway may be particularly useful in PLNTY located in surgically unresectable regions In addition Koh et a0 al reported that the BRAF V600E mutation contributes to the intrinsic epileptogenicity in pediatric brain tumors and that inhibition of BRAF suppressed epileptic seizures [] Thus BRAFMEK inhibitors could exert antiepileptic as well as antitumor effects in PLNTYPET imaging revealed a region with increased methionine uptake and low FDG uptake within tumor tissue in our patient Consistent with this finding previous case reports demonstrated increased methionine uptake but only mild FDG uptake in patients with BRAF V600Emutated PLNTY [ ] Thus excessive 0cTateishi a0et a0al acta neuropathol commun Page of Fig Activating the MAPK pathway induces LAT1 expression in a patient with PLNTY a Immunohistochemistry of indicated proteins in the highmethionineuptake and lowmethionineuptake regions within tumor tissue Bars μm b Western blot analysis of phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG83 PLNTY left cells treated with DMSO and μM BRAF inhibitor BRAFi dabrafenib for h GAPDH loading control c Relative cell viability of dabrafenibtreated left and trametinibtreated right YMG83 cells and immortalized normal human astrocytes NHA P DMSO versus dabrafenib left and trametinib right d Western blot analysis for indicated proteins in YMG62 epithelioid glioblastoma left and AM38 glioblastoma right cells treated with DMSO μM BRAF inhibitor BRAFi dabrafenib and μM MEK inhibitor MEKi trametinib for h GAPDH loading control e Western blot analysis of BRAF phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG62 left and AM38 right cells treated with DMSO and dabrafenib at indicated concentrations Vinculin loading control f Western blot analysis for indicated proteins in nonsilencing NS and BRAF and transduced YMG62 and AM38 cells GAPDH loading controlmethionine uptake and low FDG uptake may be imaging features specific to PLNTY A preclinical study has demonstrated that high uptake of 18FFDG was correlated with increased Glut1 and HK2 expression in human cancers [] Although the diagnostic accuracy is insufficient FDGPET imaging is useful to differentiate highgrade from lowgrade gliomas [] In the present case low FDG uptake and weak expression of Glut1 HK2 and LDHA were observed in tumor tissue suggesting low glycolytic activity in PLNTY On the other hand due to a high signaltonoise ratio 11Cmethionine PET imaging is practical for brain tumors [ ] Several PET imaging studies have demonstrated that methionine uptake was higher in 0cTateishi a0et a0al acta neuropathol commun Page of highgrade adult gliomas than in lowergrade gliomas [ ] In epileptogenic brain tumors however all gangliogliomas and “ of DNT had increased methionine uptake although these tumors are classified as WHO grade I [ ] implying that methionine uptake may be irrespective of tumor grade in LEATsPrevious studies have reported that methionine uptake was correlated with LAT1 in gliomas [ ] LAT1 plays a major role in the transport of neutral essential amino acids including methionine and is driven by several cancerrelated genes such as MYC [] It has been demonstrated that cMyc which is partly mediated by the MAPK pathway regulates LAT1 expression and MEK inhibitor suppresses LAT1 SLC7A5 transcription [ ] thereby indicating a role of the MAPK pathway and cMyc in the regulation of LAT1 Since RASMAPK pathwayassociated genomic alterations are common in LEATs [] and that the BRAF V600E mutation has been identified in “ and of gangliogliomas and DNTs respectively [ ] there is a possibility that the BRAF V600E mutation and MAPK pathwayrelated genomic alterations may activate methionine metabolism in LEATs To investigate this hypothesis we evaluated the protein expression of LAT1 and the molecules that are involved in the MAPK pathway As expected levels of phosphoMEK phosphoERK cMyc and LAT1 were higher in the highmethionineuptake area than in the lowmethionineuptake area We also found that genetic andor pharmacological BRAF inhibition suppressed MAPK pathway activation and attenuated LAT1 expression in BRAF V600EmutatedPLNTY cells and glioblastoma cell lines These findings support the hypothesis that the BRAF V600E mutation may upregulate LAT1 and methionine metabolism through cMyc activation for cell survival In addition to LAT1 methionine uptake was correlated with microvascular density MVD in gliomas [] PLNTYs are considered benign brain neoplasms proposed as WHO grade I however in the present case a chicken wirelike MVD which is one of the histopathological characteristics of oligodendroglioma was also observed in the highmethionineuptake tissue region Intriguingly methionine uptake has been reported to be relatively higher in oligodendrogliomas than in astrocytomas [] Thus PLNTY which has an oligodendrogliomalike microvascular structure might show unique metabolic imaging features Further studies are warranted to validate this hypothesis Nonetheless our data indicated that the BRAF V600E mutation induced MAPK pathway activation and downstream cMyc promoted LAT1 expression and methionine metabolism with little effect on glycolytic pathway activation These findings may explain the unique metabolic imaging features of FDGmethionine mismatch in PLNTYSupplementary informationSupplementary information accompanies this paper at https doi101186s4047 Additional file a0 a0Figure S1 Low glycolysis activation in a patient with PLNTY Immunohistochemistry for glucose transporter hexokinase and lactate dehydrogenase A in the highmethionineuptake upper and lowmethionineuptake lower region within tumor tissue A Bars μm Figure S2 Images of the patient™s glioblastoma with the BRAF V600E mutation Contrastenhanced magnetic resonance left and 11Cmethionine positron emission tomography right images of the YMG62 patientAcknowledgementsWe thank Mrs Emi Hirata and Yasuko Tanaka YCU for technical and administrative assistance We also would like to thank Editage wwweditagecom for English language editingAuthors™ contributionsKT led the study collected samples designed experiments performed experiments interpreted data and wrote the manuscript JS TH and YM performed experiments NI HM provided tumor samples and associated clinical details TO RM and DU interrupted PET and MRI studies NU and SY performed the histological classification of tumor samples TY designed experiments and interpreted data All authors read and approved the final manuscriptFundingThis work was supported by GrantAid for Scientific Research 19K09488 Princess Takamatsu Cancer Research Fund Takeda Science Foundation SGH Cancer foundation Yokohama Foundation for Advancement of Medical Science and BristolMyers Squibb FoundationCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama Japan Department of Pathology Yokohama City University Hospital Yokohama Japan Department of Radiology Graduate School of Medicine Yokohama City University Yokohama Japan Departmento of Radiology Division of Nuclear Medicine National Center for Global Health and Medicine Tokyo Japan Received June Accepted August References Borbely K Nyary I Toth M Ericson K Gulyas B Optimization of semiquantification in metabolic PET studies with 18Ffluorodeoxyglucose and 11Cmethionine in the determination of malignancy of gliomas J Neurol Sci “ https doi101016jjns200602015 Chappe C Padovani L Scavarda D Forest F NanniMetellus I Loundou A Mercurio S Fina F Lena G Colin C et al Dysembryoplastic neuroepithelial tumors share with pleomorphic xanthoastrocytomas and gangliogliomas BRAFV600E mutation and expression Brain Pathol “ https doi101111bpa12048 Chen Y Tian T Guo X Zhang F Fan M Jin H Liu D Polymorphous lowgrade neuroepithelial tumor of the young case report and review focus on the radiological features and genetic alterations BMC Neurol https doi101186s1288 Ellison DW Hawkins C Jones DTW OnarThomas A Pfister SM Reifenberger G Louis DN cIMPACTNOW update diffuse gliomas 0cTateishi a0et a0al acta neuropathol commun Page of characterized by MYB MYBL1 or FGFR1 alterations or BRAFV600E mutation Acta Neuropathol “ https doi101007s0040 Gupta VR Giller C Kolhe R Forseen SE Sharma S Polymorphous lowgrade neuroepithelial tumor of the young a case report with genomic findings World Neurosurg “ https doi101016jwneu201908221 Hafliger P Graff J Rubin M Stooss A Dettmer MS Altmann KH Gertsch J Charles RP The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully 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doi101007s0023 y Kato T Shinoda J Oka N Miwa K Nakayama N Yano H Maruyama T Muragaki Y Iwama T Analysis of 11Cmethionine uptake in lowgrade gliomas and correlation with proliferative activity AJNR Am J Neuroradiol “ https doi103174ajnrA1242 Katsanos AH Alexiou GA Fotopoulos AD Jabbour P Kyritsis AP Sioka C Performance of 18FFDG 11Cmethionine and 18FFET PET for glioma grading a metaanalysis Clin Nucl Med “ https doi101097RLU00000 dysembryoplastic neuroepithelial tumors and other epileptogenic brain neoplasms with [11C]methionine PET Neuro Oncol “ https doi101093neuon cnou02 Riva G Cima L Villanova M Ghimenton C Sina S Riccioni L Munari G Fassan M Giangaspero F Eccher A Lowgrade neuroepithelial tumor unusual presentation in an adult without history of seizures Neuropathology “ https doi101111neup12504 Rosenberg DS Demarquay G Jouvet A Le Bars D Streichenberger N Sindou M Kopp N Mauguiere F Ryvlin P [11C]Methionine PET dysembryoplastic neuroepithelial tumours compared with other 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polymorphous lowgrade neuroepithelial tumor of the young and comparison with oligodendroglioma World Neurosurg “ https doi101016jwneu201903181 Surrey LF Jain P Zhang B Straka J Zhao X Harding BN Resnick AC Storm PB Buccoliero AM Genitori L et al Genomic analysis of dysembryoplastic neuroepithelial tumor spectrum reveals a diversity of molecular alterations dysregulating the MAPK and PI3KmTOR pathways J Neuropathol Exp Neurol “ https doi101093jnennlz10 Kobayashi K Ohnishi A Promsuk J Shimizu S Kanai Y Shiokawa Y Nagane Tateishi K Nakamura T Yamamoto T Molecular genetics and M Enhanced tumor growth elicited by Ltype amino acid transporter in human malignant glioma cells Neurosurgery “ discussion “ https doi10122701neu00003 Koh HY Kim SH Jang J Kim H Han S Lim JS Son G Choi J Park BO Heo WD et al BRAF somatic mutation contributes to intrinsic epileptogenicity in pediatric brain tumors Nat Med “ https doi101038s4159 10180172x Kracht LW Friese M Herholz K Schroeder R Bauer B Jacobs A Heiss WD Methyl[11C] lmethionine uptake as measured by positron emission tomography correlates to microvessel density in patients with glioma Eur J Nucl Med Mol Imaging “ https doi101007s0025 Lelotte J Duprez T Raftopoulos C Michotte A Polymorphous lowgrade neuroepithelial tumor of the young case report of a newly described histopathological entity Acta Neurol Belg “ https doi101007s1376 Louis DN Wesseling P Aldape K Brat DJ Capper D Cree IA Eberhart C FigarellaBranger D Fouladi M Fuller GNet al cIMPACTNOW update new entity and diagnostic principle recommendations of the cIMPACTUtrecht meeting on future CNS tumor classification and grading Brain Pathol https doi101111bpa12832 Okubo S Zhen HN Kawai N Nishiyama Y Haba R Tamiya T Correlation of Lmethyl11Cmethionine MET uptake with Ltype amino acid transporter in human gliomas J Neurooncol “ https doi101007s1106 Ong LC Jin Y Song IC Yu S Zhang K Chow PK [18F]2deoxydglucose FDG uptake in human tumor cells is related to the expression of GLUT1 and hexokinase II Acta Radiol “ https doi10108002841 Rheims S Rubi S Bouvard S Bernard E Streichenberger N Guenot M Le Bars D Hammers A Ryvlin P Accuracy of distinguishing between therapeutic targets of pediatric lowgrade gliomas Brain Tumor Pathol “ https doi101007s1001 Tateishi K Tateishi U Nakanowatari S Ohtake M Minamimoto R Suenaga J Murata H Kubota K Inoue T Kawahara N 62Cudiacetylbis N4methylthiosemicarbazone PET in human gliomas comparative study with [18F]fluorodeoxyglucose and Lmethyl[11C]methionine PET AJNR Am J Neuroradiol “ https doi103174ajnrA3679 Wakimoto H Kesari S Farrell CJ Curry WT Jr Zaupa C Aghi M Kuroda T StemmerRachamimov A Shah K Liu TC et al Human glioblastomaderived cancer stem cells establishment of invasive glioma models and treatment with oncolytic herpes simplex virus vectors Cancer Res “ https doi10115800085472CAN083886 Yue M Jiang J Gao P Liu H Qing G Oncogenic MYC activates a feedforward regulatory loop promoting essential amino acid metabolism and 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Thyroid_Cancer

Inconsistency of the results regarding the genetic variability within genes coding for receptor activator of nuclear factor κB RANK and its ligand RANKL in rheumatoid arthritis RA prompted us to study the RANK and RANKL polymorphisms as potential biomarkers associated with disease predisposition and response to antiTNF treatment in a group of Polish patients with RA This study enrolled RA patients and controls RANK rs8086340 C G rs1805034 C T and RANKL rs7325635 G A rs7988338 G A alleles were determined by realtime PCR with melting curve analysis and related with clinical parameters In addition RANKL serum levels were measured by ELISA The RANK rs8086340G allele was overrepresented among patients as compared to controls OD p Creactive protein CRP levels were significantly p associated with RANK rs8086340 polymorphism and were higher in the CChomozygotes at the baseline while lower in the GGcarriers at the 12th week of the treatment At the latter time point RANKL rs7325635GGpositive patients also showed significantly lower CRP concentrations Higher alkaline phosphatase levels before induction of antiTNF therapy were observed in RANK rs8086340 and RANK rs1805034 CC homozygotes p and p respectively The GG homozygosity of both RANKL single nucleotide polymorphisms was significantly associated with the number of swollen joints rs7988338 and rs7325635 before and at the 12th week of therapy respectively p in both cases These results imply that polymorphisms within the RANK and RANKL genes affect RA susceptibility and antiTNF treatment outcomeKeywords RANK a0· RANKL a0· Polymorphism a0· AntiTNF therapy a0· Rheumatoid arthritiskatarzynaboguniakubikhirszfeldpl Katarzyna BoguniaKubik Laboratory of a0Clinical Immunogenetics and a0Pharmacogenetics Hirszfeld Institute of a0Immunology and a0Experimental Therapy Polish Academy of a0Sciences Wroclaw Poland Department of a0Rheumatology and a0Connective Tissue Diseases Jan Biziel University Hospital No Bydgoszcz Poland Department of a0Rheumatology and a0Internal Medicine Wroclaw Medical University Wroclaw Poland Ludwik Rydygier Collegium Medicum in a0Bydgoszcz Nicolaus Copernicus University Torun PolandIntroductionRheumatoid arthritis RA is an autoimmune disorder that is present in approximately of the Caucasian population mostly women It is identified by inflammation of joint synovial membrane leading to the progression of cartilage and bone tissue damage eventually prompting disability RA is caused by a combination of environmental genetic and stochastic factors Smolen et a0al Introduction of the tumor necrosis factor TNF inhibitors to RA treatment had a positive effect on the quality of patients™ lives Biologic agents successfully suppress disease symptoms as well as stop further bone damage and reduce disability Geiler et a0al Our previous studies showed that some genetic features may influence disease susceptibility or antiTNF therapy outcome in RA patients BoguniaKubik et a0al Vol01234567891 0c Page of Archivum Immunologiae et Therapiae Experimentalis Gębura et a0al Iwaszko et a0al Świerkot et a0al 2015aWellcharacterized risk factors and early diagnosis are crucial for preventing bone loss and achieving therapeutic success However molecular mechanisms behind osteoporosis in RA have not been fully elucidated It is known that glucocorticoid therapy inflammatory cytokines reduced physical activity and low calcium intake are involved in RArelated osteoporosis Corrado et a0al Pathak et a0al demonstrated that sera from patients with active disease enhanced osteoblast proliferation and differentiation via receptor activator of nuclear factor NFκB ligand RANKL thus leading to bone loss Dysregulation of balance between bone resorption and formation was previously observed in RA Boyce and Xing Under chronic inflammation conditions and in presence of proinflammatory cytokines such as interleukin IL TNFα IL6 and IL17 RANKL is expressed by T cells fibroblastlike synoviocytes bone marrow stromal cells Braun and Zwerina Schett and B cells thereby promoting osteoclastdependent bone resorption Meednu et a0al Yeo et a0al RANKL plays a pivotal role in osteoclast activation development and survival Nemeth et a0al Nevertheless binding to its receptor RANK is required for the process to begin RANK is a transmembrane protein belonging to the TNF receptor superfamily TNFRSF Schett et a0al and is mainly expressed on the surface of the macrophagemonocyte lineage cells Besides that it is widely present on the surface of osteoclast progenitors and mature osteoclasts Liu and Zhang Typically those molecules do not have kinase activity and must engage factors able to activate signaling pathways Walsh and Choi During osteoclastogenesis the TNF receptorassociated factors are recruited and NFκB an essential transcription factor is activated Leibbrandt and Penninger The nature of the relationship between osteoclasts and TNFfamily proteins has been reported in numerous studies Kitaura et a0al Ono and Nakashima In our study we focused on the genetic variability of the RANK TNFRSF11A and RANKL TNFSF11 genes in RA patients subjected to antiTNF treatmentThe significance of chosen RANK and RANKL single nucleotide polymorphisms SNPs have been described before The role of RANK rs1805034 polymorphism has been analyzed in regard to RA Mohamed et a0al Yang et a0al cancer Yin et a0al Zhang et a0al hip osteoporotic fractures Zhang et a0al knee osteoarthritis Wang et a0al age at menarche Duan et a0al Pan et a0al and in Paget™s disease Chung et a0al The RANK rs8086340 was also previously documented in RA patients as well as with respect to age at natural menopause Lu et a0al It has been reported that RANKL rs7325635 may be a genetic marker in heart failure patients Schmitz et a0al and RANKL rs7988338 was significantly associated with femoral neck compression strength index Dong et a0al However according to our knowledge those polymorphisms has never been studied as potential markers associated with disease predisposition and response to treatment with TNF inhibitors in an independent cohort of Polish RA patientsMaterials and a0MethodsPatients and a0ControlsThreehundredeighteen Polish patients with diagnosed rheumatoid arthritis and qualified for treatment with antiTNF agents were enrolled in this study Patients were classified according to the American College of Rheumatology criteria as well as by the presence of active disease represented by the Disease Activity Score in joints [DAS28] ‰¥ prior to initiating biologic agent therapy The following inclusion criteria were applied age over a0years a complete medical history and physical examination of patients and resistance to treatment with at least two diseasemodifying antirheumatic drugs The exclusion criteria included clinically significant impairment of hepatic and renal function the coexistence of other systemic diseases of connective tissue besides RA infection with hepatotropic viruses infections resistant to therapy ongoing history of cancer or uncontrolled diabetes alcohol abuse pregnancy or breastfeeding insufficient clinical records and unwillingness or inability to cooperate Data collected from the patients comprise level of Creactive protein CRP presence of rheumatoid factor RF presence of anticyclic citrullinated peptide antiCCP antibodies disease activity score DAS28 and erythrocyte sedimentation rate ESR painful and swollen joint counts global health assessments by a patient and a physician and visual Table Characteristics of RA patientsNumber of RA patientsFemalesmales of femalesAge years [mean ± SD]Disease onset years [mean ± SD]Disease duration years [mean ± SD]DAS28 baseline [mean ± SD]CRP baseline [mean ± SD]RFpositive []AntiCCP positive []DAS28 disease activity score CRP Creactive protein RF rheumatoid factor antiCCP anticyclic citrullinated peptide antibodies ± ± ± ± ± 0cArchivum Immunologiae et Therapiae Experimentalis Page of analogue scale VAS of pain value The study was approved by the Wrocław Medical University Ethics Committee Identification Code KB6252016 and written informed consent was obtained from all participants Baseline characteristics of the patients are summarized in Table a0 The control group consisted of Polish unrelated healthy blood donors with no personal history of autoimmune diseasesA subgroup of patients was characterized by the following additional parameters levels of calcium alkaline phosphatase ALP vitamin D3 and thyroidstimulatinghormone TSH that together with the number of painful and swollen joints and VAS score might correlate with bone conditionAnti‘TNF Therapy OutcomeDisease activity in RA patients was estimated using the DAS28 score based on four components number of swollen and tender joints levels of CRP and ESR patients™ global assessment of general health expressed on the visual analogue scale The level of disease activity was interpreted as either low DAS28 ‰¤ moderate DAS28 ‰¤ or high DAS28 Clinical response was assessed according to the European League Against Rheumatism EULAR criteria at the 12th week after initiation of antiTNF treatment The patients were divided into three groups according to their response to treatment good moderate or nonresponders A good response was defined as improvement in the DAS28 score specifically ΔDAS28 and DAS28 at endpoint ‰¤ at the endpoint The moderate response was defined as either ΔDAS28 and DAS28 at endpoint or ΔDAS28 ‰¤ and DAS28 at endpoint ‰¤ A lack of response was defined as ΔDAS28 ‰¤ or ΔDAS28 ‰¤ and DAS28 at endpoint as previously described Iwaszko et a0al SNP Selection and a0GenotypingFor the present study the genetic variants of the TNFRSF11A and TNFSF11 genes were selected based on available literature analysis as well as search results from NCBI Database of Short Genetic Variations dbSNP Information regarding predicted functional consequences of SNPs was obtained using the SNPinfo Web Server Xu and Taylor The RANK gene is located on chromosome rs8086340 C G is placed within intron and rs1805034 C T missense substitution in exon leads to an amino acid change from alanine to valine The RANKL gene is located on chromosome and both rs7325635 G A and rs7988338 G A are situated in intron in possible transcription factor binding sites The minor allele frequencies MAF of all the selected SNPs were higher than Genomic DNA was isolated from peripheral blood of RA patients and controls using QIAamp DNA Blood Midi Kit Qiagen Hilden Germany according to recommendations of the manufacturer RANK rs8086340 rs1805034 and RANKL rs7325635 rs7988338 alleles were determined by realtime polymerase chain reaction PCR amplification and meltingcurve analysis using LightSNiP assay TIB MOLBIOL Berlin Germany on the LightCycler RealTime PCR system Roche Diagnostics Rotkreuz SwitzerlandRANKL Serum Level AnalysisSerum concentration of RANKL was measured by commercial ELISA kits DY626 RD Systems Minneapolis MN USA according to protocols provided by the manufacturer The analyses were performed for a subgroup of RA patients consisting of individuals before antiTNF treatment and controls The absorbance was measured in a Tecan Sunrise absorbance reader and Magellan software Tecan Trading AG Switzerland The optical density of each well run in duplicate was determined by microplate reader set to a0nm with wavelength correction set to a0nm Peptide concentration in the samples was measured by comparing the optical density of the sample with a computergenerated four parameters logistic curvefit standard curveStatistical AnalysisThe Hardy“Weinberg equilibrium was tested in patients and controls for each SNP Potential associations between examined SNPs and clinical parameters of RA patients were analyzed applying the Mann“Whitney U test for quantitative data and the Fisher™s exact test for parametric values The frequencies of respective genotype groups among RA patients in relation to the antiTNF outcome were investigated by comparing the EULAR scores using Fisher™s exact test Fisher™s exact test was also used to compare genotype variation distribution within patients and controls p values less than were considered statistically significant All statistical calculations were performed in the GraphPad7 Prism softwareResultsDistribution of a0RANK and a0RANKL Genotypes in a0Patients and a0ControlsGenotype distribution of all the studied SNPs in both groups patients and controls are presented in Table a0 MAF values were as follows RANK rs8086340 C patients vs controls RANK rs1805034 C vs 0c Page of Archivum Immunologiae et Therapiae Experimentalis Table Distribution of RANK rs8086340 C G rs1805034 C T and RANKL rs7325635 G A rs7988338 G A genotypes in RA patients and controlsGeneRANKrs8086340GenotypeCCCGGGCCCTTTGGGAAAGGGAAARA patients N 117a 489b Controls N RANKrs1805034RANKLrs7325635RANKLrs7988338N number of individuals with a given genotypea OR CI “ p b OR CI “ p RANKL rs7325635 A vs RANKL s7988338 A vs Genotype frequencies of the RANK rs8086340 polymorphism in RA patients were different from those in the control group Table a0 Both the presence of the G variant as well as CG heterozygosity were more frequent among patients than controls RANK heterozygous genotype was present in of patients and of controls OR CI “ p The RANK rs8086340 G allele was detected in of RA patients as compared to of controls CG GG vs CC OR CI “ p These results imply that the RANK rs8086340 SNP may affect disease susceptibilityThere were no differences in genotype distribution of RANK rs1805034 RANKL rs7325635 RANKL rs7988338 between patients and controlsRelationship between a0Patients™ Genotypes and a0Clinical ParametersAmong the patients included in this study the mean ± SD of the CRP concentration in RA patients™ blood was ± and the disease activity score DAS28 level was ± After a0weeks of antiTNF treatment both parameters significantly decreased to ± and ± respectively p in both casesExcept for CRP levels none of the clinical parameters such as RF antiCCP or DAS28 was found to be associated with any of the polymorphisms studied For details please see Table a0 Significant differences were observed between levels of CRP and RANK rs8086340 and RANKL rs7325635 SNPs In general both before and after treatment with TNF inhibitors CRP concentrations were lower in the RANK rs8086340 GG and RANKL rs7325635 GG homozygotes as compared to patients carrying other RANK rs8086340 or RANKL rs7325635 genotypes respectively These differences were especially noticeable after the 12th week of antiTNF administration RANK rs8086340 GG vs CG CC p RANKL rs7325635 GG vs GA AA p RANK rs8086340RANK rs1805034Table Distribution of the RANK and RANKL genotypes with respect to selected clinical parameters at baseline and at the 12th week of the therapyGeneCCPDAS28 at Baseline DAS28 at 12th week CRP at Baseline CRP at 12th week RFMean ± SDCC ± CG ± GG ± ± CCCT ± ± TTRANKL rs7325635 GG ± ± ± RANKL rs7988338 GG ± ± ± Mean ± SD311a ± ± ± ± ± ± ± ± ± ± ± ± Mean ± SD ± ± 715b ± ± ± ± 717c ± ± ± ± ± ± Mean ± SD ± ± ± ± ± ± ± ± ± ± ± ± Number Number GAAAGAAAa p b p c p 0cArchivum Immunologiae et Therapiae Experimentalis Page of Additionally RANK rs8086340 CC carriers had significantly higher CRP levels before treatment than carriers of other genotypes of this SNP CG GG vs CC p Response to a0Anti‘TNF Therapystudied polymorphisms were not found to affect the outcome of biological treatmentRelationship between a0Patients™ Genotypes and a0Bone Parameters or a0Thyroid DysfunctionResponse to antiTNF therapy after a0weeks was evaluated using EULAR criteria In general a good and moderate response was achieved by and of patients respectively while the remaining of RA patients investigated did not respond to treatment The We were able to analyze and compare the distribution of selected SNPs analyzed in the present study with regard to various bone parameters such as calcium and alkaline phosphatase levels vitamin D TSH levels Table a0 number of painful and swollen joints and VAS score Table a0Table Distribution of the RANK and RANKL genotypes with regard to biochemical parameters level presented as mean ± SDGeneRANK rs8086340GenotypeCCCGGGCCCTTTGGGAAAGGGAAACalcium [mgdl] ± ± ± ± ± ± ± ± ± ± ± ± Alkaline phosphatase [Ul] ± 659a ± ± ± 325b ± ± ± ± ± ± ± ± Vitamin D3 [ngml] ± ± ± ± ± ± ± ± ± ± ± ± TSH [ulUml] ± ± ± ± ± ± 115c ± ± ± ± ± ± RANK rs1805034RANKL rs7325635RANKL rs7988338a p b p c p Table Distribution of the RANK and RANKL genotypes with respect to the number of painful or swollen joints and visual analogue scale VAS for pain score presented as mean ± SD at baseline and at the 12th week of the therapyGeneVAS [mm] at baselineVAS [mm] at 12th weekNumber of painful joints at baselineNumber of painful joints at 12th week ± ± ± ± ± ± ± ± ± ± ± ± Number of swollen joints at baseline ± ± ± ± ± ± ± ± ± ± 559b ± ± Number of swollen joints at 12th week ± ± ± ± ± ± ± 641a ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± RANK rs8086340 CC ± CG ± GG ± RANK rs1805034 CC ± CT ± TT ± GG ± GA ± AA ± GG ± GA ± AA ± rs7325635RANKL RANKL rs7988338a p b p 0c Page of Archivum Immunologiae et Therapiae Experimentalis Analyses revealed that both polymorphisms in the RANK gene rs8086340 and rs1805034 are associated with alkaline phosphatase level Table a0 whereas RANKL variants correlated with the number of swollen joints before and after therapy Table a0 Please note that both numbers of painful ± and swollen joints ± as well as VAS scores ± significantly decreased following the biological treatment to ± and ± as well as ± respectively p in all casesThe RANK rs8086340 and rs1805034 CC homozygotes were characterized with higher alkaline phosphatase levels The statistically significant relationship was observed for rs1805034 CC vs CT TT p while a strong tendency was seen in the RANK rs8086340 SNP CC vs CG GG p The RANKL rs7988338 GG homozygotes were identified with a smaller number of swollen joints compared to patients carrying the A allele GG vs AG AA p Also a significant correlation between a number of swollen joints and RANKL rs7325635 was observed at the 12th week of the antiTNF therapy Interestingly RANKL rs7325635 GG homozygotes in this polymorphism had also more swollen joints after treatment GG vs AG AA p In addition RANK rs8086340 C allele was more commonly observed in a group of patients with higher TSH levels TT vs CT CC p although more TT homozygous patients had thyroid dysfunction TSH level below or above the norm “ ulUml vs p RANKL Serum ConcentrationSerum concentrations of RANKL were assessed in RA patients before antiTNF treatment patients after a0weeks of treatment and controls It appeared that only patients at baseline after treatment and controls presented with RANKL concentrations exceeding the minimum standard curve point a0pgml No statistically significant difference was observed between RANKL serum levels of patients and controls However it was noticed that average protein concentration equaled a0pgml and a0pgml for RA patients before induction of antiTNF agents and controls respectively Furthermore no correlation was found between RANKL genotypes and serum levels either in patients or in controlsInterestingly after a0weeks of antiTNF treatment RANKL serum levels decreased to an average serum concentration of a0pgml similar to that observed for the control group a0pgmlDiscussionIn recent years several studies investigated the association between the RANK TNFRSF11A and RANKL TNFSF11 gene polymorphisms and the risk for RA development in different populations However the results coming from these studies were conflicting For example Mohamed et a0al described RANK rs1805034 and RANKL rs9525641 nonsynonymous polymorphisms as potential genetic risk factors for osteoporosis in postmenopausal women with RA In other studies RANKL rs9525641 was found to be associated with younger age at onset of RA disease Tan et a0al Wu et a0al In German patients the major allele of RANKL rs2277438 and a minor variant of RANK rs35211496 both located within intronic regions were described to increase the risk for RA Assmann et a0al Moreover the RANKL rs2277438 G allele was found to be associated with radiographic progression of joint damage Furuya et a0al A very recent study of Yang et a0al documented that RANKL rs2277438 polymorphism increased RA risk and that the RANK rs1805034 SNP was not related to RA risk On the other hand Wang et a0al found the RANK rs1805034 SNP to be associated with susceptibility to knee osteoarthritisOur present study was conducted to evaluate the role of the RANK rs8086340 C G rs1805034 C T and RANKL rs7325635 G A rs7988338 G A SNPs as potential diagnostic biomarkers associated with the RA risk in the Polish population and prognostic biomarkers affecting the outcome of the biological treatment To the best of our knowledge the present study is the first one investigating relationships of these selected SNPs with clinical parameters in patients with RASimilarly to the results of Yang et a0al our study showed that the RANK rs1805034 SNP was not found to be related with RA risk However we observed a higher frequency of RANK rs8086340 heterozygotes and G allele carriers among patients than in controls showing that patients possessing the RANK rs8086340 G allele were more prone to RA development Such an association between RANK rs8086340 SNP and RA susceptibility was not previously describedRecently published results in French cohorts found the RANK rs8086340 SNP to be correlated with the presence of antiCCP RuyssenWitrand et a0al but we did not observe such a correlation in our patients of Polish origin either with antiCCP or RF Concerning some other clinical parameters we did notice a statistically significant decrease in the level of CRP and DAS28 during therapy but only changes in CRP serum concentrations were related to investigated polymorphisms We observed that GG homozygosity of both RANK rs8086340 and RANKL rs7325635 was 0cArchivum Immunologiae et Therapiae Experimentalis Page of associated with lower CRP levels especially after the 12th week of antiTNF treatment Our observations correspond with earlier studies considering the role of TNF blocker as an effective disease activity reducer Kurz et a0al SNPs investigated in this study may have a regulatory role in gene transcription Polymorphism rs8086340 of the RANK gene is located within an intronic region while rs1805034 results in an amino acid change from alanine to valine RANKL rs7325635 as well as rs7988338 are situated in an intronic area in a transcription factor binding site Therefore we also measured serum RANKL concentration in patients at baseline at the 12th week after induction of antiTNF agents and in the control groupPrevious studies reported significantly increased RANKL levels in RA patients compared to controls and in antiCCPpositive individuals Boman et a0al Higher RANKL concentration was also detected before the onset of RA Johansson et a0al Interestingly in our study we observed some differences in serum concentrations between patients before initiation of biological treatment and controls with a higher average protein concentration in RA patients a0pgml vs a0pgml in controls However this difference did not reach statistical significanceNo significant relationship was detected between RANKL concentration and the both SNPs within the RANKL encoding gene Although an average RANKL concentration was over a0pgml higher at the baseline in patients carrying the RANK rs8086340 G allele found in the present study to be associated with an increased risk for RA development Patients carrying the G allele presented with an average of a0pgml RANKL in serum vs a0pgml for patients lacking this genetic variantAs for RANKL SNPs investigated in our study RuyssenWitrand et a0al described RANKL rs7325635 as significantly associated with antiCCP antibody presence and erosions whereas RANKL rs7988338 was significantly associated with femoral neck compression strength index a phenotypic parameter integrating bone density bone size and body size having significant potential to improve hip fracture risk assessment Dong et a0al In our group of RA patients the GG homozygosity of both RANKL SNPs rs7988338 and rs7325635 was significantly associated with the number of swollen joints while the RANK rs8086340 and rs1805034 CC homozygotes were significantly more frequent among patients with higher ALP levels These relationships have not been previously described Surprisingly a significant difference was also noted with respect to glucocorticosteroid dose The RANKL rs7988338 G allele carriers received a significantly lower dose of this drug than the AA homozygous patients individual data not shown Despite antiinflammatory effects glucocorticosteroids also affect the delayed progression of erosive lesions in the joints Świerkot and Madej This finding is in line with our observation on the lower number of swollen joints before treatment in the GG homozygous patientsWe also observed a decrease in RANKL serum level during the therapy At the 12th week of the treatment the average protein concentration equaled a0pgml which corresponds to RANKL level of the control group This observation may reflect effectiveness of the antiTNF treatment which is in line with the results of GonzálezAlvaro et a0al They also showed a decrease in RANKL serum concentration during antiTNF therapy and further suggested that serum level of RANKL may be helpful in predicting outcome in patients receiving biologic agents GonzálezAlvaro et a0al Also Świerkot et a0al 2015b demonstrated decreasing RANKL concentration in the subgroup of RA patients with goodmoderate MTX therapy response after months of treatmentThe results of our study indicate a possible role of the RANK TNFRSF11A and RANKL TNFSF11 gene polymorphisms as diagnostic andor prognostic factors in Polish patients with RA They imply that RANK rs8086340 SNP may affect disease susceptibility Furthermore RANK rs8086340 and RANKL rs7325635 polymorphisms may have a prognostic role in modulating the clinical outcome of antiTNF therapyNevertheless further genetic studies on larger groups of patients of various origins subjected to biological treatment are necessary to confirm these findingsAcknowledgements This work was supported by a grant from the National Science Centre Poland 201621BNZ501901 The authors thank the Regional Centre of Transfusion Medicine and Blood Bank in Wroclaw for providing control samples We are also grateful to Piotr Łacina for his assistanceCompliance with ethical standards Conflict of interest The authors declare that they have no competing interestOpen Access This article is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this article are included in the article™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the article™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons orglicen sesby40 0c Page of ReferencesAssmann G Koenig J Pfreundschuh M et a0al Genetic variations in genes encoding RANK RANKL and OPG in rheumatoid arthritis a casecontrol study J Rheumatol “BoguniaKubik K Świerkot J Malak A et a0al IL17A IL17F and IL23R gene polymorphisms in Polish patients with rheumatoid arthritis Arch Immunol Ther Exp “Boman A Kokkonen H Ärlestig L et a0al Receptor activator of nuclear factor kappaB ligand RANKL but not sclerostin or gene polymorphisms is related to joint destruction in early rheumatoid arthritis Clin Rheumatol “Boyce BF Xing L Functions of RANKLRANKOPG in bone modeling and remodeling Arch Biochem Biophys “Braun T Zwerina J Positive regulators of osteoclastogenesis and bone resorption in rheumatoid arthritis Arthritis Res Ther Chung PY Beyens G Riches PL Genetic variation in the TNFRSF11A gene encoding RANK is associated with susceptibility to Paget™s disease of bone J Bone Miner Res “Corrado A Maruotti N Cantatore FP Osteoblast role in rheumatic diseases Int J Mol Sci Dong S Liu X Chen Y et a0al Association analyses of RANKLRANKOPG gene polymorphisms with femoral neck compression strength index variation in Caucasians Calcif Tissue Int “Duan P Wang ZM Liu J et a0al Gene polymorphisms in RANKLRANKOPG pathway are associated with ages at menarche and natural menopause in Chinese women BMC Womens Health Furuya T Hakoda M Ichikawa N et a0al Associations between HLADRB1 RANK RANKL OPG and IL17 genotypes and disease severity phenotypes in Japanese patients with early rheumatoid arthritis Clin Rheumatol “Gębura K Świerkot J Wysoczańska B et a0al Polymorphisms within genes involved in regulation of the NFκB pathway in patients with rheumatoid arthritis Int J Mol Sci Geiler J Buch M McDermott MF AntiTNF treatment in rheumatoid Arthritis Curr Pharm Des “GonzálezAlvaro I Ortiz AM Tomero EG et a0al Baseline serum RANKL levels may serve to predict remission in rheumatoid arthritis patients treated with TNF antagonists Ann Rheum Dis “Iwaszko M Świerkot J Kolossa K et a0al Influence of NKG2D genetic variants on response to antiTNF agents in patients with rheumatoid arthritis Genes Johansson L Ärlestig L Kokkonen H et a0al An increased concentration of receptor activator of nuclear factor kappaB ligand predates the onset of rheumatoid arthritis Rheumatology “Kitaura H Kimura K Ishida M et a0al Immunological reaction in TNFαmediated osteoclast formation and bone resorption in a0vitro and in a0vivo Clin Dev Immunol Kurz K Herold M Russe E et a0al Effects of antitumor necrosis factor therapy on osteoprotegerin neopterin and sRANKL concentrations in patients with rheumatoid arthritis Dis Mark https doiorg101155201527696 Leibbrandt A Penninger JM RANKRANKL regulators of immune responses and bone physiology Ann N Y Acad Sci “Liu W Zhang X Receptor activator of nuclear factorκB ligand RANKLRANKosteoprotegerin system in bone and other tissues Review Mol Med Rep “Lu Y Liu P Recker RR et a0al TNFRSF11A and TNFSF11 are associated with age at menarche and natural menopause in white women Menopause “Archivum Immunologiae et Therapiae Experimentalis Meednu N Zhang H Owen T et a0al Production of RANKL by memory B cells a link between B cells and bone erosion in rheumatoid arthritis Arthritis Rheumatol “Mohamed RH Mohamed RH ElShahawy EE Relationship between RANK and RANKL gene polymorphisms with osteoporosis in rheumatoid arthritis patients Genet Test Mol Biomark “Nemeth K Schoppet M AlFakhri N et a0al The role of osteoclastassociated receptor in osteoimmunology J Immunol “Ono T Nakashima T Recent advances in osteoclast biology Histochem Cell Biol “Pan R Liu YZ Deng HW et a0al Association analyses suggest the effects of RANK and RANKL on age at menarche in Chinese women Climacteric “Pathak JL Bravenboer N Verschueren P et a0al Inflammatory factors in the circulation of patients with active rheumatoid arthritis stimulate osteoclastogenesis via endogenous cytokine production by osteoblasts Osteoporos Int “RuyssenWitrand A Degboé Y Cantagrel A et a0al Association between RANK RANKL and OPG polymorphisms with ACPA and erosions in rheumatoid arthritis results from a metaanalysis involving three French cohorts RMD Open 2e000226Schett G Effects of inflammatory and antiinflammatory cytokines on the bone Eur J Clin Invest “Schett G Ha

Thyroid_Cancer

"Sintilimab blocks the interaction between programmed death1 PD1 and its ligands The safety andefficacy of sintilimab combined with oxaliplatincapecitabine CapeOx as firstline treatment were evaluated inpatients with gastric Ggastroesophageal junction GEJ adenocarcinoma in a phase Ib clinical trialMethods Patients with locally advanced or metastatic GGEJ adenocarcinoma without previous systemic treatmentwere enrolled as one cohort of a multicohort study Sintilimab was administered at a dose of mg intravenously IV incombination with CapeOx mgm2 capecitabine orally bid D1“ and mgm2 oxaliplatin IV D1 every daysfor up to cycles After combination treatment patients continued to receive sintilimab mg at weekly intervals asmaintenance therapy until progressive disease PD unacceptable toxicity withdrawal of informed consent or for up to months Adverse events AEs were monitored to assess safety in terms of their frequency intensity and causality Theefficacy endpoints included the objective response rate ORR disease control rate DCR progressionfree survival PFSand overall survival OS Tumor mutation burden TMB was evaluated for its association with clinical responseResults A total of patients were enrolled and received sintilimab plus CapeOx All patients reported treatmentrelatedAEs TRAEs Grade “ TRAEs were found in patients Seventeen patients obtained partial response and theORR was CI “ Three had stable disease and DCR was CI “ Asdata cutoff of May the median followup was months The median PFS was months CI “ andmedian OS had not been reached The OS rates at months and months were and No association wasobserved between TMB and efficacyContinued on next page Correspondence Nongxuzhjphoutlookcom1Department of Medical Oncology The First Affiliated Hospital School ofMedicine Zhejiang University No Qingchun Road Hangzhou ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cJiang BMC Cancer Page of Continued from previous pageConclusions Sintilimab combined with CapeOx as firstline treatment demonstrated acceptable safety andpromising efficacyTrial registration ClinicalTrialsgov NCT02937116 Registered October Keywords Sintilimab Capecitabine Oxaliplatin Gastricgastroesophageal junction adenocarcinoma Tumormutation burdenBackgroundThe fifth most commonly diagnosed cancer worldwide isgastric cancerGC accounting for about ofcancerrelated deaths globally and the third most common cancer in China with almost half of worldwide newGC cases occurring in China annually [ ] The standard treatments exhibit regional differences among western countriesJapanKorea and China which areconsidered to be associated with different screening andearly detection methods as well as different biologicalbehaviors disease characteristics and ethnicity [“]treatment mainlySurgical resection is the only radical therapy for gastricgastroesophageal junction GGEJ cancer Howeversystemic chemotherapy is an alternative main therapyfor GGEJ cancer because of the high relapse rate afterpostresection surgery and for the many patients diagnosed at an advancedstage For advanced GGEJ cancerinvolves platinumbasedfirstlinechemotherapy using a combination oftwo or threedrugs trastuzumab is given to patients whose tumor ishuman epidermal growth factorreceptor2 HER2positive but the overall survival OS is disappointingsince the maximum OS time has been reported to be months [“] Any potential novel drug that willincrease patient survival times is urgently neededinparticular for firstline treatmentinhibitor treatmentImmune checkpointis a newapproach for tumor immunotherapy [ ] The treatment diminishes the immune system tolerance to tumorcells and improves the effective identification and eradication of tumor cells by blocking T cell inhibition [] Theprogrammed death1 PD1 antibody specifically binds toPD1 thereby inhibiting apoptosis of antigenspecific Tcells and thus reducing regulatory T cell Treg apoptosisby inhibiting the activation of PDL1 [ ]patients with recurrent or advanced GC In the ATTRACTION2 study nivolumab monotherapy improvedOS from to months hazard ratio CI“ P compared with a placebo in advanced GC that was refractory or intolerant to previoustreatment regimens []However between and of patients exhibit noresponse to PD1 blockade which is considered to beassociated with T cell exclusion or exhaustion or inadequate T cell trafficking and many immunosuppressivefactors accumulate in the tumor microenvironment []New therapy regimens that improve the response andlongterm efficacy are desperately needed The efficacy ofantiPD1 therapy in combination with chemotherapy hasbeen confirmed in nonsmallcell lung cancer [ ] Inaddition to direct tumor killing conventional cytotoxicchemotherapy has demonstrated immunoregulatory properties by enhancing tumor antigenicity disturbing immunesuppressive pathways inducing immunogenic cell deathand increasing effector Tcell reactions [] It is safe tohypothesize that antiPD1 antibodies in combination withchemotherapy may further improve the clinical outcomesof patients with advanced GC Sintilimab is a highly selective monoclonal IgG4 antibody that inhibits interactionsbetween PD1 and its ligands with strong antitumorresponse [] A phase 1a study for dose escalation hasdemonstrated the tolerance and pharmacological activity ofsintilimab in patients with advancedstage solid tumors butthere is limited evidence for the efficacy of antibodiesagainst PD1 plus chemotherapy in Chinese GGEJ adenocarcinoma patients Thus the present trial was conductedto investigate the safety and efficacy of sintilimab combinedwith CapeOx as firstline therapy for a cohort of patientswith GGEJ adenocarcinomaThe efficacy of antiPD1 antibodies monotherapy inpatients who had prior chemotherapy for advanced GChas been demonstrated and supported by several trialsIn the KEYNOTE012 and KEYNOTE059 trials pembrolizumab monotherapy showed objective responserates ORR of n [] and n []respectively in PDL1 positive advanced GCpatients after at least two prior systemic therapies Basedon such results the Food and Drug Administration approved pembrolizumab forthirdline treatment ofMethodsStudy design and patientsThe present study was an label multicenter phase Ibstudy to evaluate the safety and efficacy of sintilimab in cohorts of patients with solid tumors Patients age range“ years with cytologically or histologically confirmedunresectable GGEJ adenocarcinoma were enrolled in theGGEJ cohort Tumor nodes and metastases TNM staging has been evaluated according to the Union for International Cancer Control UICC TNM classification 8th 0cJiang BMC Cancer Page of edition [] The patients had received no previoussystemic treatment for advanced disease or had disease progression PD more than months after systemic adjuvant therapy Other major inclusion criteriawere at least one measurable lesion as defined by theResponse Evaluation Criteria in Solid Tumor RECISTversion criteria score or for Eastern TumorCollaborative Group Performance Status ECOGPSadequate an and bone marrow functions and lifeexpectancy ‰¥ weeks Patients with amplification oroverexpression of the HER2 gene were excluded fromthe trial Appendix contains a complete list of allinclusion and exclusion criteriaThe institutional review boards of all centers approvedthe protocols and the study was carried out in strictaccordance with the declaration of Helsinki principlesall participating patients signed consent forms beforetaking partProceduresAccording to NCCN guideline the preferred firstlinechemotherapy regimens for advanced gastric cancer arefluorouracil or capecitabine combined with cisplationor oxaliplatin [] Howeverthe results from theREAL2 study [] revealed significant clinical benefitof the oxaliplatincapecitabine CapeOx regimen whichled to the longest OS time of months comparedwith other regimens Oxaliplatin produces less renaltoxicity there is no requirement for hydration and ithas a lower emetic potential compared to cisplatinwhile capecitabine has no requirement for continuousintravenous IV infusion and is administered orallywhich should ensure an improved quality of life for patients in their homes Therefore a CapeOx regimen hasbeen chosen During the combination phase enrolledpatients were given sintilimab in combination withCapeOx for up to cycles every weeks Each cycleconsisted of intravenous sintilimab mg plus oxaliplatin mgm2 on day and capecitabine mgm2 twice daily orally from day to day Aftercombination treatment patients without PD continuedto receive sintilimab mg at weekly intervals asmaintenance therapy until PD unacceptable toxiceffects withdrawal of informed consent or for up to monthsStudy assessmentsAdverse events AEs were monitored for days after thelast administration of a treatment dose Responses wereassessed by computed tomography CT or magnetic resonance imaging MRI every weeks until PD new treatment initiation withdrawal of informed consent or deathEndpointsSafety was assessed as collected AEs according to theirtype frequency causality and severity grading defined bythe National Cancer Institute Common TerminologyCriteria CTCAE ver The efficacy endpoints werethe ORR disease control rate DCR time to responseTTR duration of response DOR progression freesurvival PFS and OS Efficacy was determined by an investigator according to RECIST v11 guidelinesExploratory endpoints were to evaluate the correlationof tumor mutation burden TMB with clinical efficacyTumor mutation burden analysisThe tumor biopsies and blood samples were collected atbaseline DNA sequences were extracted from biopsiesof tumors with matched blood samples and submittedfor next generation sequencing using a designed gene panel Genecast Beijing China TMB was determined by analysis of the quantity of somatic mutationsper megabase Mb Median TMB was used as a cutoffto define a tumor as highTMB HTMB and lowTMBLTMBStatistical analysisAll patients who received at least one study treatmentwere included in the safety and efficacy analyses AEswere coded following the Medical Dictionary for Regulatory Activity and tabulated by system an class andpreferred terms Causality between AEs and the studytreatment was assessed by the investigator ORR was calculated as the proportion of patients who had achieved acomplete response CR or partial response PR and the CIs were evaluated by the binomial distributionDCR was calculated as the proportion of patients whoobtained PR CR and stable disease SD and data arepresented with the CIs Median DOR TTR PFSOS and the PFS and OS rates at and months weredetermined using the KaplanMeier methodology Fisher™s test was used to compare the ORRs between patientswith HTMB and LTMBResultsFrom Dec to Oct patients werescreened and were enrolled in the GGEJ adenocarcinoma cohort Fig The median interval between initial diagnosis and screening was days range “Most patients had metastatic disease status and had ECOG scores of Table The TNMstage summary is shown in Table and the staging ofeach patient in Supplementary Table At data cutoff on May the median followuptime was months range “ The median treatment duration was months range “ Allpatients received more than cycles of treatment with 0cJiang BMC Cancer Page of Fig Flowchart of the studythe median doses of received sintilimab being range“SafetyAll of the patients reported at least one treatmentrelated adverse event TRAE and the most commonTRAE was platelet count decreased n Grade or treatmentrelated AEs TRAEs occurredin patients the most common also being aplatelet count decreased n Table NoTRAE was fatal and patient discontinued the treatment due to treatmentrelated Grade hepatic functionabnormal Sintilimabrelated AEs occurred in patients Grade “ sintilimabrelated AEs occurred in patients the most common being platelet countdecreased n Supplementary Table Chemotherapyrelated AEs were found in all patientsGrade “ chemotherapyinduced AEs were found in patients the most common being platelet countdecreased n Supplementary Table Fivepatientsadverseevents platelet count decreased n abnormal hepatic function n hypothyroidism n pneumonitis n and autoimmune colitis n reported treatmentrelated seriousEfficacyAll patients experienced a decrease in the sum oftheir target lesions Fig 2a and in the majority the lesions kept smaller than at baseline Fig 2b The medianTTR was months CI “ and the medianDOR was months CI “ According tothe best tumor response following RECIST guidelines patients reached a PR CI “ and patients obtained a confirmedobjective response ie by two continuous PRs at intervalsof weeks In addition patients had SD and DCR was CI “ Table One patient achieved a CR after the primary analysisby May This patient began the study treatmenton October and completed cycles of treatment before CRThe median PFS time was months CI “ and the month PFS rate was Median OSwas not reached and the 6month and 12month OSrates were and respectively Fig 2c dTumor mutation burdenValid results were obtained from patients The median TMB value was Mb The ORR was CI “ in patients with HTMBand CI “ in patients with LTMB No significant difference in clinical responseswere found between HTMB and LTMB patientsP Fig 2eDiscussionIn the present study the results from the GGEJ adenocarcinoma cohort in a Phase Ib study demonstratedmanageable safety and favorable antitumor activity ofsintilimab combined with a CapeOx regimen as firstlinetreatment for unresectable advanced metastatic GGEJadenocarcinoma 0cJiang BMC Cancer Page of Table Demographics and disease characteristicsTable Treatmentrelated adverse events TRAEsAge median range in yearsAll patients N to Gender n MaleFemaleECOG PS n Time since initial diagnosismedian range in daysDisease status n Locally advancedMetastaticLocation of the primary tumor n UpperMiddleLowerTNM staging n T3T4TxM0M1N1N2N3NxHistology n Poorly differentiated adenocarcinomaModerately differentiated adenocarcinomaUnknown differentiated adenocarcinoma “ ECOG Eastern Cooperative Oncology Group T tumor N node M metastasisIn terms of safety the incidence and severity of TRAEswith sintilimab and CapeOx were generally consistentwith those of known toxic effects of conventional chemotherapy [“] and previously reported side effects ofother antiPD1 antibody combined with chemotherapyregimens [ ] Platelet count white blood cell countand neutrophil count decreases were most commonly andmostly grade to reported TRAEs and are expected AEsassociated with CapeOx [“] Only patient reporteddiscontinuation of investigational drug application due toa TRAE abnormal hepatic function No treatmentrelated death occurred in this study and in general theaddition of sintilimab to CapeOx showed a manageablesafety profile and did not bear extra safety risksAll TRAEs nPlatelet count decreasedWhite blood cell count decreasedNeutrophil count decreasedHypothyroidismRashAlanine aminotransferase increasedAspartate aminotransferase increasedAnemiaHepatic function abnormalVomitingNauseaHyperchlorhydriaThyroid function test abnormalHypokalemiaHypesthesiaPyrexiaProteinuriaγglutamyl transferase increasedDiarrheaAutoimmune colitisAll graden Grade “n Pneumonitis Listed are any grade TRAE found in ‰¥ patients and all grade “ TRAEs In the present study after treatment with sintilimabplus CapeOx patients with unresectable GGEJ adenocarcinoma obtained an ORR of CI “ which is higher than that of conventional firstline chemotherapy For GGEJ adenocarcinoma firstline treatment mainly involves platinumbased chemotherapy and fluoropyrimidine[] The ORR ofcapecitabinebased or oxaliplatinbased therapies fGEJ adenocarcinoma was about “ [ ]The ORR for antiPD1 antibodies with a chemotherapy regimen were variableIn the KEYNOTE059study the ORR was CI “ forpembrolizumab plus cisplatin5fluorouracilm 5FUas firstline treatmentIn the KEYNOTE062study ORRs were and in patients with a ‰¥ and ‰¥ combined positive score CPS respectivelyafter they received pembrolizumab plus cisplatin5FUor capecitabine regimen as firstline therapy [] InATTRACTION04 the ORR for nivolumab with S1oxaliplatin was CI “ and theORR for nivolumab with CapeOx was CI“the ORR wasIn another study[][] 0cJiang BMC Cancer Page of Fig Evaluation of efficacy and tumor responses a Maximum change in tumor size from baseline Seventeen of patients obtained PR basedon the percentage changes of the sum of the maximum diameter of the tumor lesion range ˆ’ to ˆ’ b the change of lesiondiameters over time from baseline each line represents the changes in one patient c PFS KaplanMeier curve d OS KaplanMeier curve e theobjective response rate in low and high TMB groups OS overall survival PFS progressionfree survival PR partial response SD stable diseaseTMB tumor mutation burdenreported to be for an antiPD1 antibody toripalimab plus CapeOx treatment []Sintilimab plus CapeOx also showed favorable longterm efficacy Median PFS was months CI “ and the 6month PFS rate was Median OS wasnot reached and the 6month and month OS rateswere and respectively which was higherthan for conventional treatments with a median PFS of months CI “ for capecitabinecisplatinregimen [] and a median OS of months CI“epirubicinoxaliplatincapecitabineregimen [] The median PFS times for antiPD1foran 0cJiang BMC Cancer Page of Table Efficacy evaluation of sintilimabEfficacy evaluationCRnSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1288502007251zAdditional file Table S1 TNM stages of each patient Table S2Sintilimab related adverse events Table S3 Chemotherapyrelated adverse events Appendix Inclusion and exclusion criteriaPRSDPDORR CI CI “ CI “DCR CICI confidence interval CR complete response DCR disease control rate ORRoverall response rate PD progressive disease PR partial response SDstable diseaseAbbreviationsAEs Adverse events CapeOx Oxaliplatincapecitabine CPS Combinedpositive score CR Complete response CT Computed tomographyDCR Disease control rate DOR Duration of response ECOGPS EasternTumor Collaborative Group Performance Status G Gastric GC Gastric cancerGEJ Gastroesophageal junction HER2 Human epidermal growth factorreceptor2 HTMB High tumor mutation burden LTMB Low tumormutation burden Mb Megabase MRI Magnetic resonance imagingNGS Nextgeneration sequencing ORR Objective response rate OS Overallsurvival PD Progressive disease PD1 Programmed death1 PDL1 Proteinprogrammed deathligand PFS Progressionfree survival PR Partialresponse SD Stable disease TMB Tumor mutation burden TNM Tumornodes and metastases TRAEs Treatmentrelated AEs TTR Time to responseUICC Union for International Cancer ControlAcknowledgementsWe thank the patients and their families and the participating study teamsfor making this study possible and Yuan Fang Innovent Biologics IncSuzhou China for assistance with drafting the manuscriptAuthors™ contributionsHPJ NX LS and HZ were responsible for the design of the study HPJ YLZJQ CM XX NL CX HW LST and LS were responsible for acquisition of datafurthermore NX LS HPJ SYW and DLZ were in charge of analysis andinterpretation of data HPJ drafted the manuscript NX LS HZ SYW and DLZrevised and commented the draft BP conducted the biomarker analysis Allauthors read and approved the final manuscriptFundingThe study was sponsored by Innovent Biologics Inc and cofunded by EliLilly and company The study was also supported by the National Health andFamily Planning Commission Research Fund Zhejiang Provincial Medicaland Health major Science and Technology Plan Project Grant No KWJZJ and the Public Welfare Technology Application Research Project ofZhejiang Province Grant No LGF20E030004 Innovent Biologics Inc involved in the design of the study and collection analysis and interpretationof data and in writing the manuscript All remaining funding bodies had norole in the design of the study and collection analysis and interpretation ofdata and in writing the manuscriptAvailability of data and materialsThe datasets generated andor analyzed during the current study are notpublicly available since the new drug is being submitted to the NationalMedical Products Administration for approval but are available from thecorresponding author on reasonable requestEthics approval and consent to participateThe trial protocol was approved by the Institutional Review Boards andEthical Committee of The First Hospital Affiliated to Zhejiang UniversitySchool of Medicine and Beijing Cancer Hospital and the study was carriedout strictly following the declaration of Helsinki principles all participatingpatients signed consent forms before taking part in the trial The trialnational registration number is NCT02937116Consent for publicationNot applicableCompeting interestsHui Zhou Shuyan Wang Donglei Zhu Bo Peng are the staff of InnoventBiologics Inc Suzhou China Suzhou China Lin Shen is the associate editorof BMC Cancer All remaining authors declare that they have no competinginterestsantibodies with a chemotherapy regimen were variableranging from to months a finding which mightbe associated with different populations and diseasestatus [“]Nextgeneration sequencing NGS hasresearcherenabled to perform target capture sequencing which hasbeen proposed as a reliable technique to identifymutated driver genes and for the estimation of TMBsIts use has led to the detection of actionable alterationsin various cancer related genes [] Regarding highTMB and the efficacy of PD1 treatments inconsistentresults have been reported in previous studies Wang suggested that TMB might be associatedwith better efficacy for PD1 monotherapy [] whereasMishima [] did not find a significant relationshipbetween TMB and the response of gastric cancers toPD1 therapy [] The latter data is in accordance withour finding that after treatment with sintilimab in combination with CapeOx no significant difference in theclinical responses was found between HTMB and LTMB patients However using the median TMB as acutoff is difficult to extrapolate to the real world clinicand bias due to the small sample size could not beexcluded in the present study In addition it has beennoted that up to now there is no uniform standard forHTMB [] and further investigations are urgentlyrequiredConclusionsOur results strongly indicate that sintilimab combined withCapeOx is an option for the firstline treatment of patientswith advanced or metastatic GGEJ adenocarcinoma However the sample size was small and it was a singlearmstudy without a comparator The large scale doubleblinded and randomized Phase III clinical trial ORIENT16for previously untreated advanced GGEJ adenocarcinomapatients is being conducted to evaluate the efficacy andsafety of sintilimab combined with CapeOx vs CapeOxalone ClinicalTrialsgov Identifier NCT03745170 0cJiang BMC Cancer Page of Author details1Department of Medical Oncology The First Affiliated Hospital School ofMedicine Zhejiang University No Qingchun Road Hangzhou China 2Department of Surgical Oncology The First Affiliated Hospital Schoolof Medicine Zhejiang University Hangzhou China 3Department of MedicalScience and Strategy Oncology Innovent Biologics Inc Suzhou China4Department of Translational Medicine Innovent Biologics Inc SuzhouChina 5Department of Medical Oncology Beijing Cancer Hospital BeijingChinaReceived March Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin “Chen W Zheng R Baade PD Zhang S Zeng H Bray F Cancer statisticsin China CA Cancer J Clin “Bickenbach K Strong VE Comparisons of gastric Cancer treatments east vsWest J Gastric Cancer “Lui FH Tuan B Swenson SL Wong RJ Ethnic disparities in gastric cancerincidence and survival in the USA an updated analysis of SEERdata Dig Dis Sci “Ye XS Yu C Aggarwal A Reinhard C Genomic alterations and molecularsubtypes of gastric cancers in Asians Chin J Cancer Van Cutsem E Moiseyenko VM Tjulandin S Majlis A Constenla M Boni C Phase III study of docetaxel and cisplatin plus fluorouracil comparedwith cisplatin and fluorouracil as firstline therapy for advanced gastriccancer a report of the V325 study group J Clin Oncol “Shah MA Janjigian YY Stoller R Shibata S Kemeny M Krishnamurthi S et alRandomized multicenter phase II study of modified Docetaxel Cisplatinand fluorouracil DCF versus DCF plus growth factor support in patientswith metastatic gastric adenocarcinoma a study of the US gastric Cancerconsortium J Clin Oncol “AlBatran SE Hartmann JT Probst S Schmalenberg H Hollerbach SHofheinz R Phase III trial in metastatic gastroesophagealadenocarcinoma with fluorouracil leucovorin plus either oxaliplatin orcisplatin a study of the Arbeitsgemeinschaft Internistische Onkologie J ClinOncol “Kang YK Kang WK Shin DB Chen J Xiong J Wang J Capecitabinecisplatin versus 5fluorouracilcisplatin as firstline therapy in patients withadvanced gastric cancer a randomised phase III noninferiority trial AnnOncol “ Bang YJ Van Cutsem E Feyereislova A Chung HC Shen L Sawaki A et alTrastuzumab in combination with chemotherapy versus chemotherapyalone for treatment of HER2positive advanced gastric or gastrooesophageal junction cancer ToGA a phase label randomisedcontrolled trial Lancet “ Wei SC Duffy CR Allison JP Fundamental mechanisms of immunecheckpoint blockade therapy Cancer Discov “ Park YJ Kuen DS Chung Y Future prospects of immune checkpointblockade in cancer from response prediction to overcoming resistance ExpMol Med MarinAcevedo JA Dholaria B Soyano AE Knutson KL Chumsri S Lou YNext generation of immune checkpoint therapy in cancer newdevelopments and challenges J Hematol Oncol Lowther DE Goods BA Lucca LE Lerner BA Raddassi K van Dijk D et alPD1 marks dysfunctional regulatory T cells in malignant gliomas JCIInsight 201615e85935 Dong H Strome SE Salomao DR Tamura H Hirano F Flies DB Tumorassociated B7H1 promotes Tcell apoptosis a potential mechanism ofimmune evasion Nat Med “ Muro K Chung HC Shankaran V Geva R Catenacci D Gupta S et alPembrolizumab for patients with PDL1positive advanced gastric cancerKEYNOTE012 a multicentre label phase 1b trial Lancet Oncol “Fuchs CS Doi T Jang RW Muro K Satoh T Machado M Safety andefficacy of Pembrolizumab Monotherapy in patients with previously treatedadvanced gastric and Gastroesophageal junction Cancer phase clinicalKEYNOTE059 trial JAMA Oncol 201845e180013Kang YK Boku N Satoh T Ryu MH Chao Y Kato K Nivolumab inpatients with advanced gastric or gastrooesophageal junction cancerrefractory to or intolerant of at least two previous chemotherapy regimensONO453812 ATTRACTION2 a randomised doubleblind placebocontrolled phase trial Lancet “Song M Chen X Wang L Zhang Y Future of antiPD1PDL1 applicationscombinations with other therapeutic regimens Chin J Cancer Res Gandhi L RodriguezAbreu D Gadgeel S Esteban E Felip E De Angelis F Pembrolizumab plus chemotherapy in metastatic nonsmallcell lungCancer N Engl J Med “ PazAres L Luft A Vicente D Tafreshi A Gumus M Mazieres J et alPembrolizumab plus chemotherapy for squamous nonsmallcell lungCancer N Engl J Med “ Yan Y Kumar AB Finnes H Markovic SN Park S Dronca RS Combiningimmune checkpoint inhibitors with conventional Cancer therapy FrontImmunol Wang J Fei K Jing H Wu Z Wu W Zhou S Durable blockade of PD1signaling links preclinical efficacy of sintilimab to its clinical benefit MAbs“ Brierley JD Gospodarowicz MK Wittekind C TNM classification of malignanttumours 8th ed Oxford Wiley National Comprehensive Cancer Network Guidelines for Gastric CancerVersion p Cunningham D Starling N Rao S Iveson T Nicolson M Coxon F et alCapecitabine and oxaliplatin for advanced esophagogastric cancer N Engl JMed “Lordick F Kang YK Chung HC Salman P Oh SC Bodoky G et alCapecitabine and cisplatin with or without cetuximab for patients withpreviously untreated advanced gastric cancer EXPAND a randomised label phase trial Lancet Oncol “ Waddell T Chau I Cunningham D Gonzalez D Okines AF Okines C et alEpirubicin oxaliplatin and capecitabine with or without panitumumab forpatients with previously untreated advanced oesophagogastric cancer REAL3a randomised label phase trial Lancet Oncol “ Bang YJ Kang YK Catenacci DV Muro K Fuchs CS Geva R et alPembrolizumab alone or in combination with chemotherapy as firstlinetherapy for patients with advanced gastric or gastroesophageal junctionadenocarcinoma results from the phase II nonrandomized KEYNOTE059study Gastric Cancer “ Boku N Ryu MH Kato K Chung HC Minashi K Lee KW Safety andefficacy of nivolumab in combination with S1capecitabine plus oxaliplatinin patients with previously untreated unresectable advanced or recurrentgastricgastroesophageal junction cancer interim results of a randomizedphase II trial ATTRACTION4 Ann Oncol “Tabernero J Cutsem EV Bang YJ Fuchs CS Wyrwicz L Lee KW et alPembrolizumab with or without chemotherapy versus chemotherapy inadvanced GGEJ adenocarcinoma the phase keynote062 study J ClinOncol 20193718supplLBA4007 Wang F Wei XL Wang FH Xu N Shen L Dai GH Safety efficacy andtumor mutational burden as a biomarker of overall survival benefit inchemorefractory gastric cancer treated with toripalimab a PD1 antibody inphase IbII clinical trial NCT02915432 Ann Oncol “ Cai H Jing C Chang X Ding D Han T Yang J Mutational landscape ofgastric cancer and clinical application of genomic profiling based on targetnextgeneration sequencing J Transl Med Mishima S Kawazoe A Nakamura Y Sasaki A Kotani D Kuboki Y et alClinicopathological and molecular features of responders to nivolumab forpatients with advanced gastric cancer J Immunother Cancer Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Neurosteroids Biosynthesisand Physiological FunctionsShogo Haraguchi and Kazuyoshi Tsutsui Department of Biochemistry Showa University School of Medicine Tokyo Japan Graduate School of Integrated Sciencesfor Life Hiroshima University Hiroshima JapanSimilar to the adrenal glands gonads and placenta vertebrate brains also producevarious steroids which are known as œneurosteroids Neurosteroids are mainlysynthesized in the hippocampus hypothalamus and cerebellum however it has recentlybeen discovered that in birds the pineal gland a photosensitive region in the brainproduces more neurosteroids than other brain regions A series of experiments usingmolecular and biochemical techniques have found that the pineal gland producesvarious neurosteroids including sex steroids de novo from cholesterol For instanceallopregnanolone and 7αhydroxypregnenolone are actively produced in the pinealgland unlike in other brain regions Pineal 7αhydroxypregnenolone an upregulator oflocomotion enhances locomotor activity in response to light stimuli in birds Additionallypineal allopregnanolone acts on Purkinje cells in the cerebellum and prevents neuronalapoptosis within the developing cerebellum in juvenile birds Furthermore exposure tolight during nighttime hours can cause loss of diurnal variations of pineal allopregnanolonesynthesis during early posthatch life eventually leading to cerebellar Purkinje cell deathin juvenile birds In light of these new findings this review summarizes the biosynthesisand physiological functions of pineal neurosteroids Given that the circadian rhythms ofindividuals in modern societies are constantly interrupted by artificial light exposure thesefindings in birds which are excellent model diurnal animals may have direct implicationsfor addressing problems regarding the mental health and brain development of humansKeywords allopregnanolone 7αhydroxypregnenolone neurosteroid pineal gland cerebellum lightINTRODUCTIONSimilar to the gonads and placenta vertebrate brains actively also produce various steroidhormones These steroid hormones produced in the brain are named œneurosteroids Theproduction of neurosteroids was demonstrated firstly in mammals and then in other vertebrates“ Thus neurosteroid production appears to be a universal feature of the brain in vertebratesIt is known that neurosteroids are produced in glial cells and neurons of the centraland peripheral nervous systems However we have demonstrated thatthe pinealgland produces neurosteroids from cholesterol in birds during early posthatch period “Notably allopregnanolone also known as 3α5αtetrahydroprogesterone 3α5αTHP and 7αhydroxypregnenolone are the two major neurosteroids produced in the pineal gland Of thesetwo pineal allopregnanolone prevents the death of developing Purkinje cells and pineal 7αhydroxypregnenolone functions as an upregulator of locomotion regulating locomotor activity inresponse to light stimuli in birds Edited byVance L TrudeauUniversity of Ottawa CanadaReviewed byVincent M CassoneUniversity of Kentucky United StatesMaria Claudia Gonzalez DeniselleCONICET Instituto de Biolog­a yMedicina ExperimentalIBYME ArgentinaCorrespondenceShogo HaraguchishogoharaguchigmailcomSpecialty sectionThis was submitted toNeuroendocrine Sciencea section of the journalFrontiers in EndocrinologyReceived April Accepted July Published August CitationHaraguchi S and Tsutsui K Pineal Neurosteroids Biosynthesisand Physiological FunctionsFront Endocrinol 103389fendo202000549Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsBIOSYNTHESIS OF PINEALNEUROSTEROIDSThe pineal glands of vertebrates respond to light stimuli andfulfillimportant functions in the anization of circadianrhythms The secretion of melatonin a major hormone producedby the pineal gland shows a clear daily rhythm with its peakconcentration occurring at night However it was notknown whether the pineal gland produces neurosteroids untilrecently We have recently demonstrated that the pineal gland isa newly found neurosteroidogenic an producing a variety ofneurosteroids from cholesterol αhydroxypregnenolone and allopregnanolone are activelyreleased Taken together these findings indicate that the pineal gland injuvenile birds produces various neurosteroids from cholesterolAccordingly this is the first demonstration of neurosteroidsynthesis in the pineal gland in a vertebratePHYSIOLOGICAL FUNCTION OF PINEAL7αHYDROXYPREGNENOLONE INLIGHTDEPENDENT LOCOMOTIONthatgene3hydroxysteroidPregnenolone is an anabolic intermediate of most endogenoussteroid hormones and is produced from cholesterol throughthe mitochondrial cholesterol side chain cleavage enzymecytochrome P450scc P450scc encoded by the Cyp11a geneWe have demonstrated by transcriptionpolymerase chainreaction RTPCR thatthe pineal gland in juvenile birdsexpresses P450scc mRNA Figure The proteinproduct of this mRNA is localized in the cells that form thefollicular structures in the pineal glands of birds We havedemonstrated by highperformance liquid chromatography3HHPLC with radioactive flow detector analysischolesterolis converted to radioactive pregnenolone whenincubated with pineal gland extract from juvenile birds This observation has confirmed the presence offunctionalP450scc in the pineal gland Figure which has also beendetected by gas chromatographymass spectrometry GCMS Subsequent RTPCR“based assessment has revealed thatkey steroidogenic enzymes cytochrome P450 7αhydroxylaseP4507α encoded by the Cyp7b gene 3αhydroxysteroiddehydrogenase 01 014isomerase 3αHSD encoded by thedehydrogenase 01 01Hsd3aisomerase 3HSD encoded by the Hsd3b gene 5αreductaseencoded by the Srd5a gene 5reductase encoded by theSrd5b gene cytochrome P450 17αhydroxylasec1720lyaseP45017αlyase encoded by the Cyp17 gene 17hydroxysteroiddehydrogenase 17HSD encoded by the Hsd17b gene andcytochrome P450 aromatase P450arom encoded by the Cyp19gene are expressed in the pineal gland of birds Figure We further demonstrated that steroid hormones are indeedpresent in the pineal gland Incubation of 3Hpregnenolonewith pineal glands from posthatch birds generates 7α andor7hydroxypregnenolone by the action of P4507α foundin the pineal glands Figure In addition to theseneurosteroid isomers progesterone allopregnanolone 3α 5αTHP andor epipregnanolone 3 5THP androstenedionetestosteroneandestradiol17 are also produced Figure These exvivo observations have confirmed that the pineal glands injuvenile birds have the biosynthetic machinery for majorsteroid hormones which have also been verified to be producedas neurosteroidsFigure Although HPLCanalysis has failed to resolve the isomers of these hormonesalloepipregnanolonesuch as 7αhydroxypregnenoloneand 5αdihydrotestosterone several sets ofisomers havebeen successfully isolated by GCMS analysis Especially5dihydrotestosteronein vivo andor5αanalysis hasactivation oftranscriptomicslightinduced transcriptionalfor studiesThe chick pineal gland is used as a modeltheon the lightdependent phaseshifting mechanism ofcircadian clock To search for genes involved in thisa diï¬erential GeneChip analysis has beenmechanismidentifiedperformed Thisthethefullset of genes in the pineal gland involved in cholesterolbiosynthesis When the pineal gland was exposed tolightit produced cholesterol and 7αhydroxypregnenoloneex vivo Interestingly this lightinduced production of 7αhydroxypregnenolone occurred only when the gland wasexposed to light at early night but not atlate night orduring the daytime During early night time the circadianclock is sensitive to light which causes phasedelay of theclock Thusthe lightsensitive pineal production of7αhydroxypregnenolone appearsto be regulated by thecircadian clockactivateslocomotorIn vertebratesan intracerebroventricularinjection of7αhydroxypregnenoloneactivities“ Thusthe intracerebroventricular injection of 7αhydroxypregnenolone was administered in a dosedependentmanner at early night in chicks After the injection chickswere placed individually for locomotor activity measurementin an open field apparatus for min Spontaneous locomotoractivities of chicks were stimulated by the intracerebroventricularinjection of 7αhydroxypregnenolone in a dosedependentmanner Furthermore when chicks are exposed to lightduring early nightreachthe daytime level These results suggest that pineal 7αhydroxypregnenolone reaches the target sites within the brain byvolume transmission upon light exposure at early nightlocomotor activitiestimetheirPHYSIOLOGICAL FUNCTION OF PINEALALLOPREGNANOLONE IN PURKINJECELL SURVIVAL DURING DEVELOPMENT7αHydroxypregnenolone and allopregnanolone are activelyreleased during early posthatch period compared with adulthood Therefore 7αhydroxypregnenolone and allopregnanolonemay play key roles in birds during early posthatch periodIn vertebrates pinealectomy decreases cell number in thedeveloping brain These findings suggest that these majorneurosteroids secreted from the pineal gland are involved in thedevelopment of brain cellsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsFIGURE Biosynthetic pathways of pineal neurosteroids Allopregnanolone and 7αhydroxypregnenolone are the major neurosteroids produced in the pineal glandof birds P450scc cytochrome P450 sidechain cleavage enzyme P4507α cytochrome P450 7αhydroxylase 3HSD 3hydroxysteroiddehydrogenase 01 014isomerase 3αHSD 3αhydroxysteroid dehydrogenase 01 014isomerase 5αreductase 5reductase P45017αlyase cytochrome P45017αhydroxylasec1720lyase 17HSD 17hydroxysteroid dehydrogenase and P450arom cytochrome P450 aromataseFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidstheincreasessupplementation oftheIn chicks pinealectomy decreases the concentration ofallopregnanolone and the number of cerebellar Purkinjeallopregnanolonecells whereasto pinealectomized birdsconcentration ofallopregnanolone and recovers the number of Purkinje cells Thus pineal allopregnanolone is considered to be an essentialfactor for the normal development of cerebellar Purkinje cellsIt thus appears that pineal allopregnanolone functions as anessential factor for Purkinje cells during posthatch periodIn addition pinealectomy in juvenile birds increases theexpression of active caspase3 in Purkinje cells whereasallopregnanolone supplementation decreases the expressionof active caspase3 during posthatch period Thus theneuroprotective action of pineal allopregnanolone on cerebellarPurkinje cells is exerted by suppressing the activation of caspase3Figure Allopregnanolone acts mainly as a ligand ofthe γaminobutyric acid type A GABAA receptor and may alsoact as an agonist of the membrane progesterone receptors αmPRα as well as the mPR and mPRγ “ Thereforeeither mPR siRNA orisoallopregnanolone an antagonistof allopregnanolone was delivered into the cerebellum ofposthatched chicks It was found that the silencing of mPRαincreases the number of Purkinje cells that express active caspase in the cerebellum of chicks Furthermore to uncoverthe mechanism of neuroprotective action of allopregnanoloneFIGURE A schematic model of the effect of pineal allopregnanolone on Purkinje cell survival immediately after hatching under a h lightdark cycle or with hlight exposure during the dark period lightatnight condition Left panel The normal cerebellar development under a h lightdark cycle during the first weekafter hatching Pineal allopregnanolone induces the expression of pituitary adenylate cyclaseactivating polypeptide PACAP a neuroprotective factor through themembrane progestin receptor α mPRα receptor binding mechanism in Purkinje cells Subsequently PACAP inhibits the activation of caspase3 that facilitates theapoptosis of cerebellar Purkinje cells Right panel The abnormal cerebellar development under the lightatnight condition during the first week after hatching Thelightatnight condition disrupts the diurnal rhythm in pineal allopregnanolone synthesis Decreased pineal allopregnanolone synthesis leads to decreased expressionof PACAP in Purkinje cells Consequently the active caspase3 level increases inducing the apoptosis of Purkinje cells in the cerebellumFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidsin cerebellar Purkinje cells allopregnanolone action on theexpression of neuroprotectiveneurotoxic factors “ hasbeen investigated Pinealectomy decreases the mRNA levels ofpituitary adenylate cyclaseactivating polypeptide PACAP aneuroprotective factor in the cerebellum of juvenile birds It has been found that a daily injection of allopregnanolone inpinealectomized juvenile birds upregulates PACAP relative tothe levels in control birds These findings show that PACAPmediates the neuroprotective action of pineal allopregnanolonethrough mPRα receptor binding during cerebellar developmentFigure LIGHTATNIGHT AFFECTS THEDEVELOPMENT OF CEREBELLUMTHROUGH A MECHANISM MEDIATED BYPINEAL ALLOPREGNANOLONE ACTIONIt is known that environmental stimuli aï¬ect the developmentof animals including humans In vertebrate brain development anatural lightdark cycle promotes better brain development thanconstant conditions such as constant light or constant darkness“ Howeverthe molecular mechanisms that controlhow environmental light conditions aï¬ect brain developmentremain unclear The pineal gland is a photosensitive anTo investigate whether light conditions are involved in thesynthesis of allopregnanolone in the pineal gland the birdshave been incubated under either a h lightdark LDcycle or LD cycle with h light exposure during the darkperiod lightatnight Consequently it has been found that theallopregnanolone concentration and synthesis during the darkperiod are higher in the pineal glands of LD birds than in thoseof lightatnight birds Figure Furthermore the numberof cerebellar Purkinje cells is decreased by the lightatnightcondition Figure It is therefore considered that pinealallopregnanolone is a critical metabolite that aï¬ects cerebellardevelopment in vertebrates depending on the environmentallight conditionsREFERENCES Baulieu EE Neurosteroids of the nervous system by the nervous system forthe nervous system Rec Prog Hormone Res “ Tsutsui K Ukena K Takase M Kohchi C Lea RW Neurosteroidbiosynthesis in vertebrate brains Comp Biochem Physiol C “ 101016S0742841399000651 Compagnone NA Mellon SH Neurosteroids biosynthesis and functionthese novel neuromodulators Front Neuroendocrinol “of 101006frne19990188CONCLUSIONSthedatarecentreview summarizedThison pinealneurosteroids Studies have indicated that the pineal glandproduces neurosteroids from cholesterol in birds Pineal 7αhydroxypregnenolone regulates locomotion in response to lightstimuli in birds Pineal allopregnanolone prevents the death ofdeveloping Purkinje cells by suppressing neuronal apoptosisduring development In addition circadian disruption by lightexposure during nighttime leads to cell death of developingPurkinje cellsthrough pineal allopregnanolonedependentmechanisms in juvenile birds These observations suggest thatnighttime artificial light exposure in modern societies may alsoperturb the development of the human brainAlmost all animals have circadian rhythms However modernlife conditions chronically disrupt circadian rhythm throughartificial light exposure The disruption of circadian rhythm isassociated with a decline in mental and physical health “The most potent circadian rhythm disruption is inappropriatelytimed bright light exposure eg lightatnight To investigatethe eï¬ects of chronic circadian disruption in modern societieson mental and physical health which is efficiently modeled bythe lightatnight condition presented here many studies havebeen conducted on mice However it is important for us tobear in mind that laboratory mice are mainly nocturnal animalswhereas humans are diurnal Thus birds are excellent animalmodels to uncover the eï¬ect of lightatnight on diurnal animalsincluding humansAUTHOR CONTRIBUTIONSSH and KT wrote the manuscript All authors contributed to the and approved the submitted versionFUNDINGThis work wasJSPS GrantsinAid forScientific Research KAKENHI Grant Numbers JP15K18571and JP19K09033supported bysterol regulatory elementbinding protein Xboxbinding protein andheat shock factor pathways Proc Natl Acad Sci USA “ 101073pnas1015959108 Haraguchi S Hara S Ubuka T Mita M Tsutsui K Possible role of pinealallopregnanolone in Purkinje cell survival Proc Natl Acad Sci USA “ 101073pnas1210804109al Lightatnight Haraguchi S Kamata M Tokita T Tashiro KI Sato M Nozaki Mthroughaï¬ects brain developmenteLifeetallopregnanolonedependentpineal8e45306 107554eLife45306037mechanismsexposure Tsutsui K Matsunaga M Miyabara H Ukena K Neurosteroid biosynthesis in Reiter RJ Pineal melatonin cell biology of its synthesis and of its physiologicalthe quail brain J Exp Zool 305A733“ 101002jeza302interactions Endocr Rev “ 101210edrv122151 DoRego JL Seong JY Burel D Leprince J LuuThe V Tsutsui Ket al Neurosteroid biosynthesis enzymatic pathways and neuroendocrineregulation by neurotransmitters and neuropeptides Front Neuroendocrinol “ 101016jyfrne200905006 Hatori M Hirota T Iitsuka M Kurabayashi N Haraguchi S KokameK et al Lightdependent and circadian clockregulated activation of Fukada Y Okano T Circadian clock system in the pineal gland Mol Neurobiol “ 101385MN251019 Matsunaga M Ukena K Baulieu EE Tsutsui K 7αHydroxypregnenoloneacts as a neuronal activator to stimulate locomotor activity of breeding newtsby means of the dopaminergic system Proc Natl Acad Sci USA “ 101073pnas0407176101Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroids Tsutsui KInoue K Miyabara H Suzuki S Ogura Y Haraguchi7αHydroxypregnenolone mediates melatonin action underlying“Sdiurnal 101523JNEUROSCI3562072008locomotorNeuroscirhythmsJ Haraguchi S Koyama T Hasunuma I Vaudry H Tsutsui K Prolactin increasesthe synthesis of 7αhydroxypregnenolone a key factor for induction oflocomotor activity in breeding male newts Endocrinology “ 101210en20091229 Haraguchi S Koyama T Hasunuma I Okuyama S Ubuka T Kikuyama S et alAcute stress increases the synthesis of 7αhydroxypregnenolone a new keyneurosteroid stimulating locomotor activity through corticosterone action innewts Endocrinology “ 101210en20111422 Haraguchi S Yamamoto Y Suzuki Y Hyung Chang J Koyama T Sato Met al 7αHydroxypregnenolone a key neuronal modulator of locomotionstimulates upstream migration by means of the dopaminergic system insalmon Sci Rep 101038srep12546 Fillenz M Volume transmission in the brain Novel mechanisms for neuronaltransmission In Fuxe K Agnati LF editors Advances in Neurosciences Vol New York NY Raven Press p “ Kilic E Hermann DM Isenmann S B¤hr M Eï¬ects of pinealectomy andmelatonin on the retrograde degeneration of retinal ganglion cells in a novelmodel of intraorbital optic nerve transection in mice J Pineal Res “ 101034j1600079x20021823x Tun§ AT Turgut M Aslan H Sahin B Yurtseven ME KaplantheS Neonatalcerebellum of“ 101016jbrainres200510011pinealectomythechick a stereologicalstudy Brain ResPurkinjeinduceslosscellin Pang Y Dong J Thomas P Characterization neurosteroid binding and braindistribution of human membrane progesterone receptors δ and ε mPRδand mPRε and mPRδ involvement in neurosteroid inhibition of apoptosisEndocrinology “ 101210en20121772 Schumacher M Mattern C Ghoumari A Oudinet JP Liere P LabombardaF et al Revisiting the roles of progesterone and allopregnanolone in thenervous system resurgence of the progesterone receptors Prog Neurobiol “ 101016jpneurobio201309004 Belelli D Lambert JJ Neurosteroids endogenous regulators of the GABAAreceptor Nat Rev Neurosci “ 101038nrn1703 Bernal J Thyroid hormone receptors in brain development and function NatClin Pract Endocrinol Metabol “ 101038ncpendmet0424 FalluelMorel A Vaudry D Aubert N Galas L Benard M Basille M et alPituitary adenylate cyclaseactivating polypeptide prevents the eï¬ects ofceramides on migration neurite outgrowth and cytoskeleton remodelingProc Natl Acad Sci USA “ 101073pnas04096 Koibuchi N Chin WW ThyroidEndocrinoldevelopment 101016S1043276000002381TrendshormoneMetabactionandbrain“ Vaudry D FalluelMorel A Leuillet S Vaudry H Gonzalez BJ Regulators ofcerebellar granule cell development act through specific signaling pathwaysScience “ 101126science1085260 Sasahara K Shikimi H Haraguchi S Sakamoto H Honda S Harada N et alMode of action and functional significance of estrogeninducing dendriticgrowth Spinogenesis and synaptogenesis in the developing Purkinje cell JNeurosci “ 101523JNEUROSCI0710072007 Bakkum BW Benevento LA Cohen RS Eï¬ects of lightdark and darkrearing on synaptic morphology in the superior colliculus and visualcortex ofJ Neurosci Res “ 101002jnr490280107the postnatal and adult rat Brooks E Waters E Farrington L Canal MM Diï¬erential hypothalamictyrosine hydroxylase distribution and activation by light in adult mice rearedunder diï¬erent light conditions during the suckling period Brain Struct Funct “ 101007s0042901103189 DulcisDSpitzerNCIlluminationcontrolsdopamineof“ 101038nature07569neuronsregulatingbehaviourdiï¬erentiationNature Li Y Komuro Y Fahrion JK Hu T Ohno N Fenner KB et al Lightstimuli control neuronal migration by altering ofinsulinlike growthfactor IGF1 signaling Proc Natl Acad Sci USA “ 101073pnas1111326109 Ohta H Mitchell AC McMahon DG ConstantPediatrthe“ 10120301pdr00002331141840366developing mousebiologicalclocklight disruptsRes Kantermann T Roenneberg TIsfactor or a health risk predictor Chronobiol lightatnightInthealthriska “ Wu J Dauchy RT Tirrell PC Wu SS Lynch DT Jitawatanarat P et alLight at night activates IGF1RPDK1 signaling and accelerates tumrowth in human breast cancer xenografts Cancer Res “ 10115800085472CAN103837 Smarr BL Grant AD Perez L Zucker I Kriegsfeld LJ Maternal andearlylife circadian disruption have longlasting negative consequenceson oï¬spring development and adult behavior in mice Sci Rep 101038s41598017034064Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Haraguchi and Tsutsui This is an openaccess distributedunder the terms of the Creative Commons Attribution License CC BY The usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this journal is cited in accordance with accepted academic practice No usedistribution or reproduction is permitted which does not comply with these termsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer

Lung cancer has high mortality often accompanied with systemic metabolicdisorders The present study aimed at defining values of transnodules crossclinical phenomic and lipidomic network layers in patients with adenocarcinoma ADC squamous cell carcinomas or small cell lung cancer SCLCWe measured plasma lipidomic profiles of lung cancer patients and found thataltered lipid panels and concentrations varied among lung cancer subtypes genders ages stages metastatic status nutritional status and clinical phenomeseverity It was shown that phosphatidylethanolamine elements and were SCLC specific whereas lysophosphatidylcholine and snposition1 and phosphatidylcholine and were ADCspecific There were statistically more lipids declined in male ages latestage metastasis or body mass index Clinical transomics analyses demonstrated that one phenome in lung cancer subtypes might be generated from multiple metabolic pathways and metabolites whereas a metabolic pathway andmetabolite could contribute to different phenomes among subtypes althoughthose needed to be furthermore confirmed by bigger studies including larger population of patients in multicenters Thus our data suggested that transomic profiles between clinical phenomes and lipidomes might have the value to uncoverthe heterogeneity of lipid metabolism among lung cancer subtypes and to screenout phenomebased lipid panels as subtypespecific biomarkersK E Y WO R D Slipidomics lung cancer phenomes subtypes transomicsINTRODUCTIONLung cancer is a systemic and aggressive disease withhigh morbidity and mortality and it is often accompanied with systemic metabolic disorders for exampleup or downregulated expression of mechanismassociatedgenes or activation of metabolismdependent enzymes Forexample metabolismassociated genes of small cell lungThis is an access under the terms of the Creative Commons Attribution License which permits use distribution and reproduction in any medium provided theoriginal work is properly cited The Authors Clinical and Translational Medicine published by John Wiley Sons Australia Ltd on behalf of Shanghai Institute of Clinical BioinformaticsClin Transl Med 202010e151101002ctm2151wileyonlinelibrarycomjournalctm2 of 0c of ZHU et alcancer SCLC cells altered after mitogenactivated proteinkinase MAPK kinase module MEK5ERK5 was blockedaccompanied by dysfunctions of several lipid metabolismpathways like the mevalonate pathway for cholesterolsynthesis1 Lipids mainly including subclasses of phosphatidic acid PA phosphatidylcholines PC phosphatidylethanolamine PE phosphatidylglycerol PGphosphatidylinositol PI and phosphatidylserine PShave multiple important biological functions such asbiomembrane composition vesicular trafficking adhesion migration apoptosis energy storage neurotransmission signal transduction and posttranslational modification They have alterations under circumstance of lungcancer Circulating levels of PCs and PEs in patients withnonsmall cell lung cancer NSCLC differed from thosewith noncancer lung diseases or health and were suggested as diagnostic biomarkers of early NSCLC2 Theheterogeneity of circulating lipidomic profiles was foundto exist among patients with squamous cell carcinomasSCC adenocarcinoma ADC or SCLC and there was aclear correlation between genomic and lipidomic profilesof lipidassociated proteins and enzymes3 As the part ofclinical transomics the lung cancerspecific and subtypespecific lipidomics in the circulation were defined and evidenced by integrating lipidomic data with genomic expression of lipid proteins among lung cancer subtypesClinical transomics is defined as a new subject tointegrate clinical phenomes with molecular multiomicsfor understanding molecular mechanisms of diseases inmultiple dimensions4 Clinical transomics becomes moreimportant as a new and novel approach for the discovery of diseasespecific biomarkers and therapeutic targetsalthough there are still many obstacles to be overcomefor example specificity and decisive role of transnodulesamong multiomic networks for intra and intercellular communication56 Recent studies applied the transomics among phosphorproteomics transcriptomics genesequencing and genomics for new molecular category ofliver cancer to provide a new therapeutic strategy7 As thepart of clinical transomics clinical lipidomics was considered as one of major metabolic profiles for identificationand validation of lung cancerspecific biomarkers by integrating clinical phenomes with lipidomic profiles89 Clinical lipidomics could demonstrate the complexity of thelipidome in metabolic diseases and lung cancer and presented the variation among diseases and subtypes of lungcancer1012Our previous study demonstrated the difference oflipidomic profiles among patients with different lungcancer subtypes and the potential association betweenlipidomic phenotypes and gene expression oflipidmetabolismassociated proteins and enzymes as a conceptevaluation3 The present study furthermore investigatedthe values of transnodules crossclinical phenomic andlipidomic network layers in the recognition of lung cancersubtypes ADC SCC and SCLC in order to understandclinical phenomeassociated lipid changes or lipidomicphenotypeassociated clinical phenomes We also evaluated the differences of lipidomic profiles between maleand female various ages early and late stages with orwithout metastasis body mass index BMI or and digital evaluation scores less or more than METHODS AND MATERIALSChemical agentsThe internal standard cocktails were subscribed fromAvanti Lipids Polar Alabaster AL USA the acetone acetonitrile ammonium bicarbonate dithiothreitol formicacid iodoacetamide and Tris base Analytical Gradefrom SigmaAldrich St Louis MO USA and ammonium acetate NH4OAc hexane isopropyl alcohol IPAmethanol and highperformance liquid chromatographygrade chloroform CHCl3 from Merck Millipore Billerica MA USAPatient populationThe study designed as a casecontrol approach wasapproved by the Ethical Evaluation Committee of Zhongshan Hospital ethical code B2018187 The subjectsgave informed consent for clinical data collection andlipids analysis before all the other procedures The studyincluded lung cancer patients diagnosed according topathology of whom were ADC SCC SCLC and other healthy people The stage and severity of lung cancerwere defined according to the Eighth Edition of TNMClassification for Lung Cancer13 Patients were recruitedduring October to March Healthy controlsparticipated were blood donors in Zhongshan HospitalSubjects with other respiratory diseases or family historyof lung cancer were excluded Fasting blood was drawnfrom healthy controls and lung cancer patients on theday of entering hospital to harvest plasma All the clinicaldata including symptoms signs laboratory tests imagespathologic information and survival status years laterwere collected and followed upDigital evaluation score systemThe Digital Evaluation Score System DESS is a scoreindex system by which clinical descriptive information of 0cZHU of each phenome can be translated into clinical informatics14When the severity of each component was scored as or of which represented the most severe condition whereas indicated normal physiological range Thegross DESS scores ranged from to points the higherthe score the severer the condition A total of clinical phenomes were collected and scored in each of threelung cancer group including histories symptoms signs laboratory measurements image features and pathologic indexes as listed in Table S1spectrometry analysisLipid extraction for massAbout µL plasma was collected into a glass tubeinto which µL internal standard was added and then mL of methanolchloroformformic acid asreported previously315 This mixture was incubated at “—¦C overnight after vigorous shaking Two milliliters ofHajra™s reagent M H3PO4 M KCl were droppedblended and centrifuged at rpm for min Afterstratification chloroform in the lower layer was pipettedto another glass tube and concentrated to µL withthe nitrogen flow where the liquid with isopropyl alcoholhexane100 mM ammonium acetate at the ratio of was added till mL The sample was then centrifugated at rpm at —¦C for min The normalphaseliquid chromatography coupled TripleQuadrupole massspectrometer QTRAP SCIEX Framingham MAUSA was used for lipid extraction by the positive and negative electrospray ionization mode In the multiple reactionQTrap was utilized to scan the precursorproduct ion andexamine the mode operation Each test was repeated threetimes The peak area of each pair was quantified with multiple reaction monitoring data by the software MultiQuantAB SCIEXPurification of plasma lipidsLipid samples were derived through Ultimate SiO2 mm × mm µm Agilent Technologies Santa ClaraCA USA with mLmin flow rate highpurity heliumIn the meanwhile µL was added with the split ratio of at the ignition chamber temperature of —¦C and theinjection port temperature of —¦C It was started at temperature —¦C which gradually increased —¦Cmin to—¦C and kept for min The mass spectrometry was subjected to liquid chromatographymass spectrometer analysis FOCUS DSQTM II Thermo Fisher Scientific mainlyunder the following conditions Electron Ionization EI asionization source ion source temperature at —¦C ionization voltage at eV multiplier voltage at kV minsolvent delaying and amu of scan rangeIdentification of lipidomic profilesLipid extracts were loaded onto an Ultremex silica column mm × mm µm which was fitted with a mm× mm silica guard cartridge Phenomenex TorranceCA USA and then eluted The sample was enriched ata gradient of nLmin In the min™s run B phasewas from to min then rose to from to min linearly ramped for min as to return from to min until the end The QTrap was conductedin the multiplereaction monitoring mode and the different precursorproduct ion pairs were scanned in thepositivenegative electrospray ionization mode Up to lipids of plasma samples were carried out to get possiblelipids chemical structureslipidomic profilesComprehensive analyses ofMultiQuant software AB SCIEX was used to process dataafter lipids were identified by mass spectrometry Further MetaboAnalyst software wwwmetaboanalystcawas utilized for conducting multivariate statistical analysis cluster analysis dimensionality reduction and makingheat mapphenome and lipidome network layersTransnodule analyses crossThe typespecific lipids were identified as more than twotimes elevated or declined significantly compared withother lung cancer subtypes fold change and Pvalue whereas the coexpression lipids were identified as those similarly changed in all lung cancer subtypesas compared with healthy controls The expression quantitative trait locus eQTL model was utilized to evaluatetransnodules between lipidomic profiles and clinical phenomesStatistical analysisData were presented as mean ± SE The means of eachgroup were used for calculation and comparison Statistical significance of differences between two groupsor among multiple groups was determined by Student™sttest or oneway ANOVA test respectively Statistical 0c of ZHU et alsignificance was affirmed when Pvalue We alsoseparately calculated mean values of each phenome™sDESS score in different lung cancer subtypes which wereranked to obtain top clinical phenomes of those threegroups of patients Volcano maps showed the significantlyelevated or declined lipids in ADC SCC or SCLC patientsA VIP plot was further exploited to sort the lipids according to their importance to differentiate the four groups Toexplore the correlation between lipid elements and clinical phenomes we applied the lipidquantitative trait locimodel modified from eQTL model Besides MatrixlQTL Rpackage was used to acquire the significant phenomelipidpairs and corresponding Pvalues Moreover GraphPadPrism was utilized to make the receiver operating characteristic curve to evaluate the earlydiagnostic value andaccuracy of clinical phenomespecific lipid elements inADC SCC or SCLC The present study furthermore analyzed the significant differences of lipids among differentages eg and between female and maleearly and late stage metastasis and nonmetastasis highand low DESS scores ‰¤ and and high and lowBMI ‰¤ and RESULTSwith lung cancer subtypesClinical phenomes of patientsEighteen female and male lung cancer patients wereenrolled in the present study aged from to ± years old including ADC SCC and SCLC The totalscores of DESS were ± ± and ± in patientswith ADC SCC and SCLC respectively The DESS values of SCLC group were significantly higher than those ofhealthy control group P Top clinical phenomesof ADC SCC or SCLC patients as well as patients survivedor nonsurvived during study period were listed in Table Stages at primary diagnosis and recruitment period for thestudy lymphatic metastasis N12 in ipsilateral paratracheal hilum or mediastinum and enhanced images egfocus enhanced in CT or hypermetabolism in PETCTwere shown in all three subtypes of lung cancers In addition thyroid transcription factor1 TTF1 Napsin A keratin and location of tumor were noticed in ADC obscureboundary emphysema tumor size the cycle number offirst line chemotherapy obstructive pneumonia atelectasis and pulmonary nodule in SCC as well as number ofmetastatic lymph nodes in SCLC separately Top clinicalphenomes were similar between survived and nonsurvivedpatients but the total amounts of DESS of nonsurvivedpatients were significantly higher than those of survivedpatients Table Of total clinical phenomes hadthe statistical significance of each two groups inbetweenTable P or lesswith lung cancer subtypesLipidomic profiles of patientsTotal lipid elements of plasma were identified qualitatively and quantitatively mainly including PAs PCs PEs PGs PIs PSs lysophosphatidylcholineslysoPC lysophosphatidylethanolamines lysoPE lysophosphatidylglycerols lysoPG lysophosphatidylinositols lysoPI lysophosphatidylserines lysoPS ninediacylglycerides and triacylglycerols TAG Levels ofsome lipid elements in ADC SCC or SCLC patients weresignificantly higher Table S2 or lower Table S3 as compared with healthy control twofold P The majority of those elevated lipid elements were PC PA and lysoPC in ADC PE PC PS and PG in SCC or PS PE PG lysoPS and lysoPI in SCLC Of those declinedlipid elements were PS in ADC whereas and were PA in SCC and SCLC respectively Table demonstrates lung cancer subtypespecific lipid elements identified by those lipid elements elevated or declined exclusively in each lung cancer subtype for example some oflysoPC and PC in ADC whereas lysoPI lysoPS PE andPA in SCLC By partial least squares discrimination analysis PLSDA analysis top lipid elements were definedon the basis of variable import in project VIP score of eachgroup TAG565 lysoPG182 and PG and increased in ADC lysoPG181 PA140245 PI384180PA and and PE385PE180 increased inSCLC and PI362PI180 and lysoPG182 decreased in SCCdetail in Figure 1A There was a clear distribution oftop lipid elements among lung cancer groups as compared with the healthy control Figure 1B Of those significantly increased lipid elements in patients with lungcancers top six lipids of each group were identified Figure levels of LysoPC sn2 sn1 and sn1 in ADC Figure 2A PS363 in SCC Figure 2B andPA and and PI and inSCLC Figure 2C were significantly higher than in otherthree groups PLSDA component analysis demonstratedthat five principal components selected were and Figure 3A In the atom map whichwas based on the expression of major C atom numbersin various lipid types levels of lipids with carbons and increased whereas those with carbons decreased as compared with healthy control Figure 3BAs compared with the healthy control Figure 4A wenoticed that PI mainly declined in ADC Figure 4B PA 0cZHU of TA B L E carcinoma SCLC as well as lung cancer patients survived or nonsurvivedTop clinical phenomes of patients with adenocarcinoma ADC squamous cell carcinomas SCC or small cell lungPatients with ADCStage at primarydiagnosis ± Stage at recruitmenttime ± TTF1 ± N2 ipsilateralmediastinum ± Napsin A ± Enhanced image ± Location ± N1 ipsilateralparatracheal ± N1 ipsilateral hilum ± CK7Patients with SCCN1 ipsilateral hilum ± Enhanced image ± Stage at recruitmenttime ± Stage at primarydiagnosis ± obscure boundary ± Emphysema ± T tumor ± L1 cycle ± Obstructivepneumoniaatelectasis ± pulmonary nodulePatients with SCLCStage at recruitmenttime ± Stage at primarydiagnosis ± N1 ipsilateralParatracheal ± T tumor ± N1 ipsilateral hilum ± N2 ipsilateralmediastinum ± Enhanced image ± pulmonary nodule ± N LN ± MaintenancetreatmentPatients survivedStage at primarydiagnosis ± N2 ipsilateralmediastinum ± Enhanced image ± Stage at recruitmenttime ± N1 ipsilateral hilum ± TTF1 ± Napsin A ± Location ± N1 ipsilateralparatracheal ± LobularPatientsnonsurvivedStage at recruitmenttime ± Stage at primarydiagnosis ± N1 ipsilateralparatracheal ± N2 ipsilateralmediastinum ± N1 ipsilateral hilum ± Enhanced image ± N2 below carina ± TTF1 ± N LN ± T tumor ± ± Abbreviations N degrees of lung cancer metastasis to lymph nodes of TNM category N1 degree that has metastatic lymph nodes near pulmonary center andside of main bronchia N2 degree that has metastatic lymph nodes in the same side of the mediastinum as lung cancer TTF1 thyroid transcription factor1 asan immunohistochemical biomarker for adenocarcinoma CK7 keratin as an immunohistochemical biomarker for epithelial cells L1 cycle number of the firstline chemotherapy cycles ± ± ± in ADC and SCC Figure 4C and lysoPG in SCLC Figure 4D whereas PG and TAG increased in ADC and SCCPE in SCLC and PC in SCC The volcanic map demonstrated the clear patterns of lipid elements significantlyincreased or declined between heathy controls with ADCFigure 4E SCC Figure 4F or SCLC Figure 4G and varied among different subtypes of lung cancerspatient gendersDifferent lipidomics betweenAbout or lipid elements significantly increased and or declined more than twofold in male or femalelung cancer patients as compared with male or femalehealthy controls respectively Tables S4 and S5 Of thosePC and PE mainly elevated in male and female patientswhereas PA declined in both although the number ofPA in male patients was more than in female patientsTable demonstrates genderspecific lipid elements identified by those lipid elements elevated or declined exclusively in either male or female lung cancer patients forexample some of lysoPS PC and PS elevated in malepatients whereas lysoPI and PE in female patients Therewere about or increased or declined lipid elementsdiffered between male and female lung cancer patientsTable 0c of ZHU et alTA B L E adenocarcinoma ADC squamous cell carcinoma SCC or small cell lung cancer SCLC patientsComparisons of clinical phenomes in increased folds and statistical significance Pvalues between each two groups ofTTF1Napsin ABullaeP40HemoptysisEmphysemaSputumCK7HbCoughEGFRVacuole cavityCEAN2 ipsilateral mediastinumNew metastasisP63Cyfra211Obstructive pneumonia atelectasisSmokingPleural pullThirdlineWBCL1 cyclePDL1 tumorPTBronchiectasisPD1 tumorL2 chemo regimenSynMaintenance treatmentNSEN1 ipsilateral ParatrachealCD56CHGT tumorPD1 interstitialSum of all tumors mmN LNKi67Bronchial stenosisN3 opposite sideBurrNeuL2 cyclePulmonary noduleCK56ADC vs SCCFoldsNANANANANANANANAPvaluesNANASCC vs SCLCFoldsNANANANANANANANANAPvaluesNANANAADC vs SCLCFoldsNANANANANANANANAPvaluesNANANANA 0cZHU of Lung cancer subtypeassociated lipid elements significantly elevated or declined alone in patients with adenocarcinomaFoldsPvalues LipidsFolds PvaluesSquamous cell carcinomad181SoC1P240d181S1PPS363TA B L E squamous cell carcinoma or small cell lung cancer more than twofold as compared with healthy control PvaluesSmall cell lung cancerAdenocarcinomaLipidsLipidsElevated twofoldlysoPC sn2lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPE lysoPG lysoPG lysoPS lysoPS lysoPS PA PA PA PC PC PC 332e PC 161e181PC 352e PC 160e202PC PC lysoPG lysoPI sn1lysoPI sn2lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS PA PA PE PE orFoldsPvaluesPC PC or PC PC PC PC orPE PE Declined twofoldPG PS PS SM240PG PS401PE PE PI PI PI PI PI PI PIP PS PS TAG PA PA PA PA PA PA PA PA PA 0c of ZHU et alF I G U R E Scores of altered lipid elements in variable import in project VIP chart A where top lipid elements were defined amongpatients with adenocarcinoma ADC squamous cell carcinomas SCC small cell lung cancer SCL and healthy controls CON The xaxisrepresents the VIP score and the yaxis represents the lipid elements corresponding to the VIP score The right color grid stands for the relativeconcentration of lipid elements in four groups The degree of altered concentrations increased from green to red The heatmap B describesthe top lipid elements at the high concentration and the degree of lipid elements increased from blue low to brown highF I G U R E less than and respectively as compared with the healthy controlTop six significantly increased lipid elements in patients with ADC A SCC B and SCLC C and stand for the Pvalue 0cZHU of F I G U R E Histography of five component distributions and percentages A measured by partial least squares discrimination analysisPLSDA and the carbon atom map B in healthy controls red and patients with ADC green SCLC orange and SCC blue Each ofselected five principal components represents as the model to interpret that values of abscissa and ordinate represents the distance from thesample nodule to the origin of the center after projecting to a plane in multidimensional space A The atom map describes the expression ofmajor carbon atom number between and in various lipid types BF I G U R E The proportion of main lipid elements of healthy controls A ADC B SCC C and SCLC D and volcanic mapbetween heathy controls with ADC E SCC F or SCLC G respectively The lipid elements were identified on the basis of statistical significance The abscissa represents log values of fold changes where the left side of the first dotted line perpendicular to the abscissa represents fold changes and the right side of the second dotted line represents 2fold changes The vertical coordinate represents “log10 Pvalue Theupper side of the dotted line perpendicular to the ordinate stands for Pvalue less than as compared with healthy controls 0c of ZHU et alFoldsPvaluesPvaluesFemale patients with lung cancerLipidsFoldsTA B L E Genderassociated lipid elements significantly elevated or declined alone in male or female patients with lung cancer morethan twofold as compared with healthy control PvaluesMale patients with lung cancerLipidsElevated twofoldlysoPI sn1lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS PC PC or PC PC PC 375ePC 160e225180e205PC PC PC PC PC PC PC PC PC PC PC PC PC C1P120 MeanC1P160 MeanC1P240 MeanCer120d171Sod180Sa1Pd181S1Pd181SolysoPC sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS PC PC PE 355p PE 160p204PE 356p PE 160p205PE PE PE 376p PE 180p205 orPE PE PG PG PI 311p PI 160p160PS PS PS PS PS PS Declined twofoldlysoPS PA PA PA PA PA PA PA PA PA 181p204 160e226PE 377p PE 160p226PE PE PE PE orPI PI or or PS PS PA PA Continues 0cZHU et alContinuedTA B L E Male patients with lung cancerLipidsPG PG PS PS PS FoldsPvalues of Female patients with lung cancerLipidsFoldsPvaluesDifferent lipidomics among patientages stags metastases and survival statusAbout and lipid elements significantly elevatedor and declined in lung cancer patients at years old respectively as compared with healthycontrols P We noticed that elements of PG andPS mainly increased in lung cancer patients at all agegroups for example and at 60year group and at to 70year group and and at year group lysoPC and PC increased at 60year group and and at 70year group and PEincreased at to 70year group and at 70yeargroup as detailed in Table S6 Elements of PA mainlydeclined in lung cancer patients at all ages Table S7 Ofthose significantly altered lipid elements and appeared only at 60year to 70year and 70yeargroups respectively and considered as agespecific lipidelements Table LysoPC and lysoPI mainly increasedin 60year and to 70year old patients whereas lysoPEdeclined in 60year group We also compared lipidomicprofiles between patients at early and late stages of lungcancer and found and lipid elements significantlyincreased at early and late stages respectively of whichPE PG ad PS increased in both stages lysoPI in earlystage and PC in late stage Table S8 About and elements declined at early and late stages where the majority was PA Table S9 Table demonstrates stagespecificlipid elements identified by those lipid elements elevatedor declined exclusively at early and late stages of lung cancer for example some of lysoPI and PE elevated at earlystage and lysoPC and PE at late stageWe noticed about or lipid elements significantlyincreased in patients without or with metastasis of whichlysoPI mainly elevated in patients without metastasiswhereas PC and PE in patients with metastasis Table S10The declined number of lipid elements especially PA inpatients with metastasis was significantly higher than inpatients without metastasis Table S11 There were about or elevated or declined lipid elements in patients withmetastasis of which PA was majority of declined elementsin patients with metastasis Table Lipidomic panel also differed between survived andnonsurvived patients There were only eight lipids exclusively elevated in nonsurvived patients that is lysoPS140PC PC PE PE or PE PE PS330 PS372 andPS387 However far more lipids31 elevated alone in survived patients mainly elements of lysoPC lysoPG lysoPIlysoPS and PS On the contrary there were no lipidsdeclined alone in survived patients while lipids in nonsurvived patients of which were PA elements Table clinical phenomes and lipidomesTransomic profiles betweenWe also compared the difference of lipidomic profilesbetween general metabolism statuses of patients indicatedby BMI and between degrees of clinical phenomes measured by DESS scores Levels of lysoPC or lysoPI mainlyelevated in patients with BMI ‰¤ or respectivelyTable S12 whereas the number of declined PA in patientswith BMI ‰¤ was higher than that in patients withBMI Table S13 About BMIassociated lipid elements significantly elevated or declined exclusively inpatients with BMI ‰¤ and about in patients withBMI Table Levels of lysoPC and PE or PG and PSmainly increased in patients with DESS ‰¤ or TableS14 The number of declined PA n in patients withDESS ‰¤ was lower than that in patients with DESS n Figure demonstrates the variation of transomicprofiles among lung cancer subtypes indicated by transomic nodules cross significant networks of clinical phenome and lipidome layersDISCUSSIONThe present study preliminarily found the differencesof lipidomic profiles among patients of different lungcancer subtypes genders ages stages metastatic statusbody qualities and clinical phenome severities Besides itinitially demonstrated clinical phenomeassociated lipid 0c of ZHU et alFolds Pvalues LipidsPatient age Folds Pvalues LipidsPatient age TA B L E Ageassociated lipid elements significantly elevated or declined alone in each age group of patients with lung cancer morethan twofold as compared with healthy control PvaluesPatient age LipidsElevated twofoldC1P120 MeanlysoPC sn2lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPG lysoPG d181S1PlysoPC sn1lysoPE190lysoPE191PC PC PC PE 356p PE160p205PE PE PE PE orlysoPG140lysoPI sn2lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1Folds PvalueslysoPS lysoPS150lysoPS161lysoPS170lysoPS201lysoPS202lysoPS220PA PE PE orPE PE PE PE PE PE orPI 311p PI160p160PI PI PI PI PI361TAG PA PA PG393lysoPS lysoPS lysoPS PC PE PE PG PG PG PS Declined twofoldlysoPE sn1lysoPE sn1lysoPE sn1lysoPE sn2PA PA PS PS PE PE orPE PE PG351PS354PS372PA elements and lipid elementassociated clinical phenomesusing clinical transomics Studies on lipidomic profilesof lung cancer patients have experienced three phasesto detect the difference of lipidomic profiles betweenhealthy and lung cancer patients16 the association ofmultiomics among lung cancer subtypes3 and themolecular mechanism of clinical lipidomicsbased targetlipid elements17 Of those lipidomicsbased data limitedinformation could be adopted to understand the diseaseoccurrence and development phone progression and 0cZHU of Stageassociated lipid elements significantly elevated or declined alone in patients with lung cancer at the early stage or lateFolds Pvalues LipidsPatients at late stageFolds Pvalues LipidsTA B L E stage more than two folds respectively as compared with healthy control PvaluesPatient at early stageLipidsElevated twofoldlysoPE lysoPG lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPG lysoPS lysoPS lysoPS lysoPS PC PC orlysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS lysoPS lysoPS lysoPS PC PC PE PE PE PE PE 356p PE 160p205PE PE PE PE PE PE PI PI 311p PI 160p160PI PI or or PS PS PC PC PC 375e PC 160e225 or180e205PC PC PC PC PC PC PC PC PC PC PC PC PC PC orPC PC PC PE 355p PE 160p204PE 376p PE 180p205 or181p204 or 160e226PE 377p PE 160p226PE PE PE PE orPE PE PE PE PG PG PG PG PG PG PG PG Patients at late stageFoldsDeclined twofoldlysoPS PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PG PG PS PS PvaluesContinues 0c of ContinuedTA B L E Patient at early stageLipidsPatients at late stageFolds Pvalues LipidsPG PS PS PS ZHU et alPvaluesPatients at late stageFoldsFolds Pvalues Lipidsresponse to therapy due to the lack of link between omicsdata and clinical phenomes Like other omics investigations most genomic data were not tied with clinicalinformation so that with little values to be understoodand applied for clinical precision medicine18 In orderto face the major challenge that most clinical information was descriptive and unmatched with the digitalquantity of omics data clinical phenomes were scoredby DESS and integrated with genomic and proteomicdata of patients with acute respiratory distress syndromeand chronic obstructive pulmonary disease19“ Clinicalphenomes were furthermore integrated with lipidomicprofiles in patients with pulmonary embolism acutepneumonia and acute exacerbation of chronic obstructive pulmonary diseases based on clinical transomicsprinciple15Lipidomic profiles difference between health and lungcancer has been defined and it depends upon methodologies of measurement and analysis sample preparationsand sources and patient populations and status8 Forexample serum levels of lysoPC C260 and C261 and PCC424 and C344 were different between stage I NSCLCand healthy patients22 Some elements and pathwaysof serum PC and PE profiles increased in patients withlung benign disease and earlystage NSCLC as comparedwith healthy whereas few eg PC significantlyelevated in earlystage NSCLC patients alone2 It seemsthat patterns of lipid elements may be associated with thespecificity of lung cancer and stage rather than the intactlipid pathways We performed

Thyroid_Cancer

"Pharyngitis Tonsillitis Deep learning Smartphone Automated diagnosis Telemedicine Purpose Severe pharyngitis is frequently associated with inflammations caused by streptococcal pharyngitis which can cause immunemediated and postinfectious complications The recent global pandemic of coronavirus disease COVID19 encourages the use of telemedicine for patients with respiratory symptoms This study therefore purposes automated detection of severe pharyngitis using a deep learning framework with selftaken throat images Methods A dataset composed of two classes of throat images with pharyngitis and normal throat images was collected Before the training classifier we constructed a cycle consistency generative adversarial network CycleGAN to augment the training dataset The ResNet50 Inceptionv3 and MobileNetv2 architectures were trained with transfer learning and validated using a randomly selected test dataset The performance of the models was evaluated based on the accuracy and area under the receiver operating characteristic curve ROC AUC Results The CycleGANbased synthetic images reflected the pragmatic characteristic features of pharyngitis Using the synthetic throat images the deep learning model demonstrated a significant improvement in the accuracy of the pharyngitis diagnosis ResNet50 with GANbased augmentation showed the best ROCAUC of for pharyngitis detection in the test dataset In the 4fold crossvalidation using the ResNet50 the highest detection accuracy and ROCAUC achieved were and respectively Conclusion The deep learning model for smartphonebased pharyngitis screening allows fast identification of severe pharyngitis with a potential of the timely diagnosis of pharyngitis In the recent pandemic of COVID19 this framework will help patients with upper respiratory symptoms to improve convenience in diagnosis and reduce transmission Introduction Diagnostic support in remote healthcare services has shown the ability to minimize the exposure of ill patients to healthcare providers and other patients [] The recent global pandemic of coronavirus disease COVID19 has encouraged the use of telemedicine for patients with upper respiratory symptoms [] Because smartphones have become ubiquitous many researchers are interested in utilizing them in telemedicine Deep learning technology can assist with patient examination using a smartphone when clinicians deal with limited information in a remote patient monitoring setting In particular a smartphone is useful to take a picture of the throat [] Therefore home monitoring using a smartphone will help in the diagnosis and treatment of patients with upper respiratory symptoms to improve convenience and to reduce Corresponding author Department of Ophthalmology Aerospace Medical Center Republic of Korea Air Force Danjaero Namilmyeon Cheongwongun Chungcheongbukdo Cheongju South Korea Corresponding author Epilepsy Center Neurological Institute Cleveland Clinic Euclid Ave Cleveland Ohio USA Email addresses eyetaekeunyoogmailcom TK Yoo jychoi717gmailcom JY Choi Tae Keun Yoo and Joon Yul Choi contributed equally to this work 101016jcompbiomed2020103980 Received June Received in revised form August Accepted August ComputersinBiologyandMedicine1252020103980Availableonline20August2020001048252020ElsevierLtdAllrightsreserved 0cimages were posted by users asking for medical advice via the social QA The search strategy was based on the key terms œsore throat  œpharyngitis œtonsillitis œexudative tonsillitis œtonsillopharyngitis  œthroat image and œsmartphone in Korean Japanese and English languages The most recent image from the database search was achieved on April Images that were not taken with smartphones were manually picked and excluded for this study Images with the characteristics of either pharyngitis or normal throat were manually classified by two clinicians and the ambiguous images were excluded to clarify the image domains Finally we collected the initial dataset with two classes including throat images with pharyngitis and normal throat images The dataset was randomly separated into training N validation N and test sets N to apply deep learning to an independent dataset Detailed data distribution and augmentation are described in Table Only the throat and tonsils images were used for the input data without further manipulation to reduce the intraclass variance Original images were extracted from the database in the PNG Portable Network Graphics format The images were resampled to a pixel resolution of — — in the PNG formatfor CycleGAN and deep learning models All procedures were performed in accordance with the ethical standards of the institutional and national research committee and the Helsinki declaration and its later amendments or comparable ethical standards This study did not require ethics committee approval instead the researchers used webbased and deidentified data All datasets for the development of the deep learning model are available at Mendeley Data repositories 10176328ynyhnj2kz TK Yoo transmission There have been several approaches adopting deep learning for automated diagnosis of several diseases using images captured by smartphones [] Pharyngitis which is diagnosed in more than million patients in the United States annually is a common condition associated with acute upper respiratory tract infection [] Pharyngitis is an inflammation of the back of the throat and tonsils Sore throat caused by pharyngitis is one of the main causes of medical visits for young patients [] The most common cause of acute pharyngitis is a selflimiting viral infection However Streptococcus pyogenes is the major bacterial infectious cause of pharyngitis and is responsible for an estimated “ of cases of sore throat [] Frequently severe pharyngitis with fever and exudative tonsillitis is associated with streptococcal pharyngitis which can cause immunemediated and postinfectious complications such as acute rheumatic fever [] Therefore timely diagnosis of pharyngitis for treatment is important to reduce symptoms fever and complications [] However many patients with upper respiratory infection ignore their symptoms in the early stage and medical visits do not routinely take place Moreover in recent days many patients hesitate to visit clinics because of the COVID19 outbreak The importance of a mobilebased monitoring system for patients with acute upper respiratory infections has been raised because of its applicability and effectiveness [] A previous study endeavored to collect throat images using additional equipment in conjunction with the smartphone and used the knearest neighbor algorithm in color distribution space to classify images with streptococcal tonsil [] However the need for additional equipment limited their effectiveness of this method in a realworld setting Moreover color distribution was unable to represent the characteristic features of pharyngitis Throat images exhibit variation in the size illumination and shape of the oral cavity Here we present a deep learning model with smartphonebased throat images facilitating the detection of severe pharyngitis using selftaken throat images Fig We performed automated detection of severe pharyngitis using a convolutional neural network CNN framework Methods Data collection The basic concept of our framework is throat examination using a selftaken smartphone image with computeraided diagnosis system which is similar to the previous dermatology study [] This study was performed using publicly accessible selftaken throat images on the web We collected throat images from webbased social QA systems including Naver Korea kinnavercom and Yahoo Japan chiebukuroyahoocojp The additional throat image datasets were extracted using the Google image search engine Most throat Data augmentation using GAN Because of the limited number of datasets and their imbalanced distribution data augmentation is required for deep learning training Basic data augmentation techniques such as flip translation rotation and brightness change have been applied to train deep learning models Several previous studies have attempted to train deep learning models using generative adversarial network GANbased synthetic images to increase the classification performance [] Inspired by previous works using a generative adversarial network we adopted the CycleGANbased data augmentation to increase the accuracy of diagnosis The cycle consistency in Fig allows CycleGAN to capture the characteristics of two image domains and automatically learn how these characteristics should be translated to transfer to domains without any paired datasets [] CycleGAN was developed to overcome the limitations of paired data when two generators and two discriminators are used It is considered to be a powerful technique that performs image domain transfer and face transfer [] Previous studies have demonstrated that CycleGAN can improve deep learning models by generating training situations to learn better decision boundaries between classes We built the CycleGAN augmentation model to increase the Fig Workflow of building a deep learning model for pharyngitis diagnosis using a smartphone ComputersinBiologyandMedicine12520201039802 0cTK Yoo Fig Schematics of the CycleGAN model generating new normal images and pathologic throat images with pharyngitis generalizability of the dataset and to improve the classification performance in the imbalanced dataset Before training the CycleGAN the throat images were augmented using linear transformation including left and right flip width and height translation from to random rotation from —¦ to —¦ zooming from to and random brightness change from to We defined these transformations as the basic augmentation step In this step normal throat and pharyngitis images were randomly sampled for the training set and normal throat and pharyngitis images were randomly sampled for the validation set Using the data with basic augmentation we trained the CycleGAN models to transform both normal to pharyngitis throat images and pharyngitis to normal images The trained CycleGAN model augmented the training set normal throat and pharyngitis images before augmentation and a total of throat images including normal and pharyngitis images were prepared to train the diagnostic classifier model after CycleGANbased augmentation It should be noted that supervised GAN techniques including conditional GAN and Pix2px were unable to be applied in this study because of a lack of paired normal and pharyngitis images To use a verified and predesigned image generator all the input images required resizing to a pixel resolution of — — which is the basic setup of a CycleGAN Therefore we used the default parameter settings that is the ADAM optimizer with a batch size of to optimize the GAN networks Development of CNN model We trained conventional deep learning models after data augmentation Because of a small image dataset in this study developing a custom deep learning method is challenging due to difficulty and time consuming eg a small training dataset can easily result in an over fitted model and low performance [] To overcome the problem of the small dataset transfer learning was widely used to train deep learning models using pretrained architectures [] This study also applied pretrained learning models to the classification task of throat images The last fully connected layer of the CNNs was only trained and the study used the pretrained conventional model as a feature extractor [] The CNN models including ResNet50 Inceptionv3 and MobileNet v2 were adopted to build binary classifiers [] These CNN models have been used successfully in many studies demonstrating stateoftheart performance with the saliency map [] The models were trained using the training set and the validation set was used to estimate how well the model had been trained We downloaded the CNN models which were pretrained on the ImageNet database and performed finetuning of the weights of the pretrained networks This process generally maintains the weights of some bottom layers to avoid overfitting and performs delicate modification of the highlevel features To use the images generated by CycleGAN the size of the input images for the deep learning models was set to a pixel resolution of — — and the images were resized for each pretrained model Most conventional deep learning models adopted a pixel resolution of — — or — — [] One deep learning research showed that the best performance was achieved at an image resolution of between — and — pixels for binary classification [] Therefore the resolution of our study was appropriate to detect pharyngitis in a binary decision The model was trained with epochs and a batch size of The ADAM optimizer with a learning rate of was also used with a crossentropy loss for all CNN models The crossentropy loss function is defined as Lcross entropy cid0ˆ‘Nipi logqi Where pi represents the ground truth value and qi represents predicted probability value from a classifier for the ith image The optimizer updated the network parameters to minimize the loss function In our experiments it tuned a fully connected layer of the CNN models For example the first layers of ResNet50 were left frozen and we trained the last fully connected layer using the training dataset which is described in Table the ADAM optimizer We chose the final classifier model which maximized the accuracy in the validation dataset To visualize the clusters to see if the classes are separable by a considerable margin the tdistributed stochastic neighbor embedding t SNE algorithm was executed using sampled instances The feature vectors from the last layer of the pretrained Inceptionv3 model were extracted to train the tSNE [] As there is a growing demand for explainable artificial intelligence methods in medicine [] we adopted githubcomjacobgilpytorch the GradCAM technique ComputersinBiologyandMedicine12520201039803 0cTK Yoo Table Throat image dataset and augmentation used in this study Number of validation set Number of training set Raw data Basic augmentation GANbased image synthesis augmentation Class Normal Pharyngitis Normal Pharyngitis Normal Pharyngitis Number of test set GAN generative adversarial network gradcam to generate the attention map [] The GradCAM visualizes the decisional areas of the CNN model using the gradients of any target flowing into the final convolutional network The heatmap has a low resolution and it was upsampled via bicubic interpolation Finally it produces heatmaps that highlight the area of interest and interprets the decision of the deep learning models The performance of the CNN models was evaluated based on the accuracy and area under the curve AUC of the receiver operating characteristic curve ROC and the precisionrecall curve PRC The Youden index which is an estimate of the optimal diagnostic threshold was adopted in this study [] After obtaining the sensitivity and specificity the Youden index was calculated at each cutoff point We selected the optimal value which maximized the Youden index These performance indexes are expressed as follows Accuracy TP TNTP TN FP FN Sensitivity Recall TPTP FNSpecificity Precision TNTN FPTPTP FP Youden index Sensitivity Specificity cid0 Where TP TN FP and FN denote true positives true negatives false positives and false negatives respectively We also performed a 4fold crossvalidation using the entire dataset to evaluate a generalized performance Google Colab Pro a cloud service for disseminating the deep learning research was adopted to implement the CycleGAN and CNN models [] Google Colab Pro provides a development environment using the Tensorflowbased deep learning libraries and a robust graphic processing unit GPU This enables the rapid processing of a heavy deep learning network without the need for a personal GPU [] The available hardware for each virtual machine varied by session but typically included top products of NVIDIA GPUs K80 T4 or P100 around “ GB of RAM and “ GB of free space on the virtual machine hard drive [] We used the Colab CycleGAN tutorial page to develop and validate the CycleGAN model and all of the code is available on the Tensorflow webpage wwwtensorflowtutorialsgene rativecyclegan We only modified the input pipeline to import our dataset The code of the CycleGAN and CNN models is presented in Supplementary Material Results We developed a deep learning model using GANbased augmentation in the challenging context of pharyngitis detection To build a balanced training dataset the CycleGAN models generated normal and pathologic throat images using the initial training dataset The color intensity distributions of the pharyngitis and normal throat images were not significantly different although most throat images had exudate regions Fig A The tSNE algorithm demonstrates that both groups are clustered and they are separable by a considerable margin Fig B The final CycleGAN model for the pharyngitis throat image was trained for epochs which required h After training normal throat images were translated into pathologic images and throat images with pharyngitis were translated into normal images Finally we constructed a balanced augmented training dataset including normal and pharyngitis images using CycleGAN The CycleGANbased synthetic images were realistic and reflected the characteristic features of pharyngitis Fig A The CycleGAN model synthesized white or gray patches and increased the redness on the throat wall and tonsils from normal images Fig B Generated throat images were reviewed by three clinicians including an otolaryngologist and an anesthesiologist All pharyngitis images generated were deemed by the three clinicians to show œmore pathologic and inflammatory results when compared to the original images This feature generation based on the CycleGAN model can be effective for generating a new sample to increase the intra class variation and generalizability The CNN models were trained using the final augmented dataset via the transfer learning technique Fig demonstrates the training process of the ResNet50 CNN model using the training and validation sets The training process for CNN took approximately h for epochs with finetuning for each training step After the 200th epoch the validation accuracy was not improved and the crossentropy of the validation result increased Therefore we considered that training for epochs was optimal in the ResNet50 model and selected the trained model at the 200th epoch Fig A shows the training validation and test datasets of CNN models with and without CycleGANbased augmentation The ROCAUCs of ResNet50 Inceptionv3 and MobileNetv2 without GAN based augmentation were and respectively Fig B We obtained the best ROCAUC of pharyngitis detection using ResNet50 with GANbased augmentation corresponding to Fig C At the optimal diagnostic cutoff value the ResNet50 model predicted pharyngitis with an accuracy of sensitivity of and specificity of The ROCAUC of Inceptionv3 and MobileNetv2 corresponded to and respectively The models with GAN based augmentation demonstrated superior performance in comparison with the models with only basic augmentation We also evaluated the performance of the models using PRCs Fig The PRCAUC also demonstrated that deep learning models with GANbased augmentation had better performance than those without GANbased augmentation ResNet50 with GANbased augmentation predicted pharyngitis with the highest PRCAUC of When a custom deep learning network without the transfer learning technique was trained the validation accuracy was lower than and was not improved during the training epochs Supplementary materials Table shows the performance of CNN models via 4fold cross validation in the entire dataset A similar tendency is observed for the average accuracy and AUC values in the crossvalidation The result shows that the highest detection accuracy and ROCAUC achieved were and respectively by using the ResNet50 Other CNN models showed lower performance than the ResNet50 with GANbased augmentation but there were no significant differences between CNN models in the 4fold crossvalidation A saliency map using the Grad CAM technique was generated to visualize the characteristic pathologic features of pharyngitis for the predicted evidence Fig A In normal throat images however some regions were highlighted Fig B When synthesized images were tested using the trained model synthesized exudates were highlighted correctly in the images as shown in Fig C Furthermore external clinical cases from previous literature were analyzed to investigate to show the capability of detecting pharyngitis of the framework developed in this study [] The representative cases with severe pharyngitis are shown in Fig One case presents an ComputersinBiologyandMedicine12520201039804 0cTK Yoo Fig Visual data exploration of pharyngitis and normal throat images A The mean redgreenblue RGB distributions B The feature space visualized using the tSNE technique For interpretation of the references to color in this figure legend the reader is referred to the Web version of this article Fig Data augmentation using CycleGAN to improve the diagnostic performance of pharyngitis A CycleGANbased augmentation for imbalanced data B Examples of pathologic throat images with pharyngitis generated by the CycleGAN Fig Training process of the ResNet50 CNN model for pharyngitis detection A Accuracy learning curves of the training and validation sets B Loss learning curves of the training and validation sets ComputersinBiologyandMedicine12520201039805 0cTK Yoo Fig Performance of pharyngitis detection deep learning models A Schematics of basic augmentation and GANbased augmentation B The receiver operating characteristic curves of deep learning models using basic augmentation C The receiver operating characteristic curves of deep learning models using GANbased augmentation Fig Precisionrecall curves of pharyngitis detection deep learning models A The deep learning models using basic augmentation B The deep learning models using GANbased augmentation Table Classification performance for severe pharyngitis detection in the 4fold crossvalidation using the developmental set AUC CI Accuracy CI Sensitivity CI Specificity CI Pvalue “ “ “ Basic augmentation ResNet50 Inceptionv3 MobileNetv2 GANbased image synthesis augmentation ResNet50 Inceptionv3 MobileNetv2 AUC area under the receiver operating characteristic curve CI confidence interval GAN generative adversarial network Comparison of receiver operating characteristics curves with the single best technique ResNet50 with GANbased image synthesis augmentation according to the Delong test “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ Reference 81yearold man with odynophagia Fig A and the other case presents a 41yearold diabetic man with throat pain Fig B The ResNet50 model was able to detect pharyngitis in both cases The GradCAM technique highlighted white patches on the throat wall as markers of severe pharyngitis in the deep learning model Discussion The current proofofconcept study investigated the possibility of deep learning using a smartphone for detecting pharyngitis A recentstudy demonstrated the ability of selftaken throat images to detect pharyngitis based on the knearest neighbor algorithm in a color space [] That study used additional equipment as well as a ComputersinBiologyandMedicine12520201039806 0cTK Yoo Fig Example of deep learning classification results with a saliency map using the GradCAM technique A Pharyngitis throat images B Normal throat images C Synthesized images smartphone to obtain throat images We utilized selftaken throat images without additional equipment using deep learning techniques for the detection of pharyngitis We showed that the trained GAN models were able to generate new realistic synthetic throat images which can improve the diagnostic accuracy The final deep learning model achieves high accuracy for automated diagnosis of pharyngitis using smartphone images Therefore this framework could be targeted toward patients with a sore throat who need screening for severe pharyngitis We believe that our study could be extended to computeraided diagnosis using images from an endoscope system in otolaryngology clinics similar to what has been done with colonoscope images using a deep learning model [] To the best of our knowledge no study has been performed to detect pharyngitis based on deep learning using smartphone images However it should be noted that this study is considered as only a preliminary and proofofconcept study because of its technical limitations due to using only Google Colab The current study framework is similar to that of Chamier which showed a deep learning framework using Google Drive and training and prediction on Google Colab [] Our proposed conceptual workflow is shown in Fig It needs the Flask servers and interfaces implemented using HTML and JavaScript to be applied in a real clinical setting This work could be part of a larger project to enable smartphonebased pharyngitis detection via a cloudbased applevel mobile data analysis We believe that a future appbased model can provide a robust solution for the costeffective and convenient screening of pharyngitis in a telemedicine setting We have highlighted the feasibility of a smartphonebased approach with deep learning to detect pharyngitis Our approach does not require Fig Classification results from the deep learning model applied to clinical cases with severe pharyngitis A An 81yearold man with odynophagia [] B A 41yearold diabetic man with throat pain [] Fig Example of a proposed smartphonebased system for pharyngitis detection ComputersinBiologyandMedicine12520201039807 0cexplored clinical datasets and webbased datasets and showed that the labeling of webbased images is often uncertain [] Therefore further clinical datasets with validation will be required to confirm the effectiveness of our framework Fourth because we collected only throat images via image search engines there was no metadata including gender race season or age According to a previous epidemiologic study these factors could affect the pharyngitis detection performance [] Fifth although the datasets were reviewed by authors images could be potentially duplicated in the dataset The duplicated images would affect the independence of the validation dataset We have shown the feasibility of deep learning for the detection of tonsil swelling and exudates in throat images The limitations of our study should be overcome by the availability of a sufficient number of throat images taken by a smartphone with a wellanized study protocol To validate the effectiveness of our framework a prospective study with many patients should be performed in a clinic once the app based framework is developed This will solve the problem of possible duplicated images in the dataset and the absence of metadata Conclusion TK Yoo additional equipment to collect throat images and we collected self taken images using a smartphone The use of webbased image capture of users from various devices and GANbased image augmentation may allow robust image processing While in the current study the implementation was performed on a desktop computer and the Colab™s remote server a smartphone application will also be possible to perform this identification for detecting pharyngitis The light deep learning models such as MobileNet can be executed on a smartphone without transmitting the images to a server [] Our result demonstrates that MobileNetv2 also has a high diagnostic performance similar to ResNet50 and Inceptionv3 In future research an increased amount of training data is needed to improve the detection accuracy using light models The utility of deep learning and smartphones may facilitate the widespread adoption of telemedicine and physical examination platforms via our approach Furthermore the presented framework enabled using a smartphone camera and deep learning techniques will help the patients in selfscreening for severe pharyngitis and may accelerate diagnostic support in remote healthcare services Because of the recent pandemic of COVID19 patients with respiratory symptoms need selfmonitoring to evaluate their pathologic status [] Advances in technology will require clinicians to embrace remote healthcare services The framework presented in this paper was designed for the timely diagnosis of pharyngitis for treatment Similar smartphoneimagebased diagnostic approaches have been introduced in several other medical image domains including skin diseases [] hematologic diseases [] oral diseases [] and eye diseases [] The main concern of deep learning models in this study

Thyroid_Cancer

" formulated a traditional Chinese medicine TCM prescription Hanshiyi Formula HSYF which was approved and promoted by the Wuhan Municipal Health Commission for treating mild and moderate coronavirus disease COVID19 We aimed to evaluate the effect of HSYF on the progression to severe disease in mild and moderate COVID19 patients We conducted a retrospective cohort study of patients with mild and moderate COVID19 in a quarantine station in Wuchang District Wuhan Using the realtime Internet information collection application and Centers for Disease Control for the Wuchang District patient data were collected through patient selfreports and followups HSYF intervention was defined as the exposure The primary outcome was the proportion of patients who progressed to a severe disease status and a stratification analysis was performed Univariate and multivariate regression analyses were performed to identify influencing factors that may affect the outcome Further we used pr sity score matching PSM to assess the effect of HSYF intervention on the conversion of mild and moderate to a severe disease status Totally mild and moderate COVID19 patients were enrolled including HSYF users exposed group and nonusers control group No cases in the exposed group and P cases in the control group progressed to severe disease and the difference between the two groups exposed groupcontrol group was cid0 [ confidence interval CI cid0 cid0 ] Univariate regression analysis revealed sex male age fever cough and fatigue as risk factors for progression to severe disease After PSM none of the HSYF users and P non users transitioned to severe disease and the difference between the two groups exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Multivariate regression analysis revealed that sex male [OR CI “ P ] and age years [odds ratio OR CI “ P ] were independent risk factors for conversion to severe disease Therefore HSYF can significantly reduce the progression to severe disease in patients with mild and moderate COVID19 which may effectively prevent and treat the disease However further larger clinical studies are required to verify our results Corresponding authors Email addresses tina_yai126com J Tian yanshiyan0927sinacom S Yan wanghan4313163com H Wang novelzhangsinacom Y Zhang luoyuorz163com Y Zheng drwhrfoxmailcom H Wu leexiuyang126com X Li zhengzhigaozezheng163com Z Gao aiyanke163com Y Ai christiana55555163com X Gou tcmxpzl126com L Zhang hely3699163com L He lfm565sohucom F Lian 13601180524139com B Liu tongxiaolinvip163com X Tong Jiaxing Tian Shiyan Yan Han Wang and Ying Zhang contributed equally to this study 101016jphrs2020105127 Received April Received in revised form August Accepted August PharmacologicalResearch1612020105127Availableonline10August2020104366182020ElsevierLtdAllrightsreserved 0cJ Tian Introduction At the end of December a series of coronavirus disease COVID19 cases emerged in Wuhan Hubei Province China and the number of diagnosed cases have rapidly increased since then [] In the early stage of the epidemic a large number of patients with mild and moderate COVID19 were diagnosed and treated in quarantine stations Identifying effective treatments for early intervention can improve the prognosis of patients with mild and moderate COVID19 which can also help control the epidemic progression and reduce the medical burden Wuchang District is located at the center of Wuhan with a high population density It was one of the most severely affected areas with many confirmed COVID19 cases For a large number of patients with mild and moderate COVID19 in the quarantine station the Wuhan Municipal Health Commission adopted a combination of Chinese herbal medicines and used Internet data to tackle the pandemic Our study group formulated a traditional Chinese medicine TCM prescription named Hanshiyi Formula HSYF to prevent the exacerbation of the epidemic This was approved and promoted by the Wuhan Municipal Health Commission for the treatment of mild and moderate COVID19 patients in quarantine stations [] Data was collected from the realtime Internet information collection application Yuge medical system which was convenient for physicians to communicate with isolated patients in real time dynamically monitor changes in the patient™s condition observe the efficacy of HSYF in mild and moderate COVID19 patients and adjust the treatment plan if necessary In guiding the treatment of mild and moderate COVID19 patients we found that after HSYF intervention patients with mild and moderate disease experienced rapid resolution of symptoms and fewer patients transitioned to a severe disease status Therefore we compared the clinical data of mild and moderate COVID19 patients treated with HSYF with patients who were diagnosed but were not treated with HSYF Data were collected from the database of Wuhan Wuchang District Disease Control Center CDC as well as from the realtime Internet information collection application to evaluate the effect of HSYF on the proportion of COVID19 patients who progressed from a mild and moderate to severe disease status Methods Study design and participants In this retrospective cohort study conducted in the Wuchang District Wuhan data were collected from mild and moderate COVID19 patients in quarantine stations in Wuchang District Wuhan before March The exposed group included patients who were administered HSYF for more than days If there is no adverse effect or disease progression HSYF can be taken continuously until recovery The control group included patients who were diagnosed with COVID19 but were not administered HSYF including decoction granules etc at the same time Data on age sex disease duration medical history initial symptoms concomitant medication and outcomes of the two groups were collected and the difference in the proportion of mild to moderate COVID19 patients who transitioned to a severe status were analyzed Fig The diagnosis of COVID19 patients was made by the designated tertiary hospital in Wuchang District Wuhan The diagnostic criteria was according to the Diagnosis and Treatment Guideline for COVID19 released by the National Health Commission of the People™s Republic of China [] The study included patients who met the criteria of mild and moderate COVID19 the mild COVID19 patients usually presented mild clinical symptoms with no radiological manifestations of pneumonia the moderate COVID19 patients had fever and respiratory symptoms with radiological manifestations of pneumonia Severe and critical patients were excluded This study was approved by the institutional ethics board of Hubei Provincial Hospital of Traditional Chinese Medicine No HBZY2020 C0101 and was registered with chictrcn ChiCTR2000029601 Data collection Data from the exposure group was first provided by the patients who scanned the QR code on the medication box and entered data on the real time Internet information collection application After downloading and sorting the data if there were mistakes or lack of relevant information a medical staff member followed up the patients by telephone and if necessary contacted the staff of the community health service station to verify the information to ensure accuracy and completeness of the data Data from the control group were provided by the Wuhan Wuchang District CDC database Data of mild and moderate COVID19 patients diagnosed at the same time were screened and downloaded and a telephone followup was conducted by a medical staff member to ensure accuracy and completeness of the data All data were collected using a standard electronic database In order to ensure the accuracy of the data and avoid bias data were verified by two researches A and B and a third researcher C resolved any dispute between the first two researchers The results of the study were analyzed and reported in accordance with the STROBE guidelines Fig Flow chart of the observational research PharmacologicalResearch16120201051272 0cJ Tian Table Demographic and Patient Characteristics All patients Exposed group Control group Age Mean ± SD 014yr 1549yr 5064yr ‰¥65yr ‰¤48yr ＞48yr Sex Female Medical history Medical history Hypertension Coronary heart disease Diabetes Bronchial asthma Chronic obstructive pulmonary disease Hyperlipidemia Fatty liver Gallbladder disease Thyroid disease Stroke Chronic glomerulonephritis Cancer Hepatitis Tuberculosis Other diseases Initial symptoms Initial symptoms Fever Cough Diarrhea Fatigue Conjunctivitis Other symptoms Concomitant medication Antiviral treatment Oseltamivir Lopinavir Aciclovir Ribavirin Arbidol Other antiviral drugs Antibiotics Antibiotics Amoxicillin Cephalosporin Levofloxacin Moxifloxacin Clarithromycin Other antibiotics Chinese patent medicine Chinese patent medicine Lianhua Qingwen capsule Xiaochaihu granule Shuanghuanglian oral solution Huoxiang Zhengqi preparation Cough syrup Banlangen preparation Ganmao Qingre granule Other Chinese patent medicines ± ± ± Statistics Z10789 X210724 X2 X20817 X21796 X23145 X21788 X21023 X23933 X2＜ X2 X20318 X20186 X24649 X22578 X20812 X22638 X2＜ X25259 X20567 X2 X2 X2 X2 X2 X21049 X2 X20091 X27352 X2 X2 X2 X20250 X2 X21648 X20218 X27552 X210734 X20257 X22752 X27191 X23935 X24895 X2 X211160 X210579 X2 X27812 Pvalue ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ Outcomes Statistical analysis The primary outcome of this study was the proportion of mild and moderate COVID19 patients who progressed to a severe disease status We also analysed factors that may affect the outcome including the patient™s age sex disease duration medical history initial symptoms concomitant medication and grouping Pr sity score matching PSM was used to further assess the effect of HSYF intervention on the conversion from mild and moderate COVID19 to a severe status Statistical analyses were conducted using SPSS software SPSS Inc Chicago IL United States Twosided tests were used and Pvalues were considered statistically significant Numerical variables are summarized as mean ±SD The data of the categorical variables are described as numbers and percentages The ttestWilcoxon ranksum test was used to compare the ages between the two groups The chisquare testFisher™s exact test was used to compare the sex medical history initial symptoms and concomitant PharmacologicalResearch16120201051273 0cJ Tian medication between the two groups to calculate the rate difference and confidence interval CI Using the proportion of patients who transitioned to a severe status as the dependent variable the effects of age sex disease duration medical history initial symptoms concomitant antiviral drugs antibiotics Chinese patent medicines and grouping were analyzed by univariate and multivariate logistic regression For factors with a statistical significance in the univariate regression analysis a logistic regression was performed to match the two groups of patients by pr sity score in a ratio and the difference in the primary outcome between the two groups was evaluated Results Demographic and patient characteristics By March we enrolled mild and moderate COVID19 patients from quarantine stations in the Wuchang District After further screening patients who refused followup patients who were not diagnosed and two patients who took other types of TCM prescriptions were excluded Finally patients were included in our study cohort including HSYF users and nonusers including decoction granules etcAmong all patients enrolled in this study were men and were women The median age of the patients was years old The median age of the exposed group was significantly lower than that of the control group P We found that of patients had a medical history which included hypertension fatty liver and diabetes A significantly higher proportion of patients in the exposed group had a history of bronchial asthma vs P and thyroid disease vs P compared with the control group while the proportion of patients with hyperlipidemia vs P was higher in the control group than the exposed group Among all patients had experienced initial COVID19 symptoms The number of patients with diarrhea in the exposed group was significantly higher than that in the control group vs respectively P and the number of patients with fever vs P cough vs P and fatigue vs P was significantly lower in the exposed group than in the control group The patients were administered multiple medications during the treatment There were no differences in the number of patients receiving antiviral drugs antibiotics and Chinese patent medicines between the two groups In terms of antiviral treatment ribavirin vs P and arbidol vs P were the most commonly administered medications in the exposed group while oseltamivir vs P and acyclovir vs P were the most commonly administered medication in the control group In terms of antibiotics amoxicillin vs P and moxifloxacin vs P were most commonly used in the exposed group while clarithromycin vs P were most commonly used in the control group In terms of Chinese patent medicines the patients in the exposed group were usually administered Xiaochaihu granules vs P Shuanghuanglian oral solution vs P cough syrup vs P Banlangen preparation vs P and Ganmao Qingre granules vs P while the patients in the control group were administered Lianhua Qingwen capsule vs P and Huoxiang Zhengqi preparation vs P as shown in Table The majority of onset times in the two groups were from January to February vs The proportion to severe status Our primary outcome was the proportion of COVID19 patients who progressed from mild and moderate COVID19 to a severe disease status There were no cases of progression to severe disease in the exposed group while cases P in the control group progressed to severe disease The difference between the two groups in terms of progression to severe disease exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Stratification analysis showed that excluding diarrhea there were significant differences between the two groups in terms of sex age years medical history initial symptoms fever cough diarrhea fatigue concomitant medication etc P Table Efficacy evaluation The proportion to severe status The proportion to severe status Sex Male Female Age Age‰¤48yr Age＞48yr Underlying disease Yes No Initial symptoms Yes No Fever Yes No Cough Yes No Diarrhea Yes No Fatigue Yes No Chinese patent medicine Yes No All patients Exposed group Control group Statistics X2 X2 X28004 X29521 X2 X29251 X2 X2 X2 X27842 X26575 X211744 X2 X21915 X2 X2 X28085 Pvalue ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ PharmacologicalResearch16120201051274 0cFactors OR95 CI Univariate analysis Pvalue Table Univariate and multivariate regression analysis J Tian therefore these factors were accounted for in the subsequent analysis Table The univariate logistic regression analysis showed that sex male age years fever cough and fatigue were risk factors for progression to a severe status We further performed a multivariate logistic regression analysis to adjust for the above risk factors The results showed that after adjusting for all factors sex male OR CI “ P and age years OR CI “ P were independent risk factors for progression to a severe disease status Table Considering the difference in sample size between the two groups and the imbalance in confounding factors the risk factors analyzed in the univariate logistic regression analysis were used as variables for PSM caliper 025σ and the number of patients in both groups were Among them no patients in the exposed group progressed to severe disease and seven patients in the control group progressed to severe disease P The difference between the two groups in terms of progression to severe disease exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Table Discussion Age The global prevalence of COVID19 has brought tremendous medical pressure worldwide According to data from Johns Hopkins University in the United States by April the number of COVID19 cases exceeded million globally Furthermore the number of diagnosed cases in the United States were over and that in Italy and Spain were over with the number of newly diagnosed cases still rising [] With a large number of people in close contact with mildmoderate COVID19 patients all countries have adopted community control and home quarantine measures to prevent the spread of the epidemic However during home quarantine problems such as crossinfection and cluster outbreaks in the community have emerged due to the lack of strict community supervision as well as the need to purchase necessities and travel for hospital consultations In addition mild and moderate COVID19 patients who were under home quarantine lacked effective intervention strategies without the guidance from physicians which may have resulted in problems such as the progression from mild and moderate to severe disease [] In such patients an effective largescale community intervention strategy could reduce this conversion to severe disease and reduce the number of severe and critical COVID19 patients thus significantly improving and controlling the COVID19 epidemic For mild and moderate COVID19 patients antiviral antibiotic and symptomatic supportive treatments are most commonly used However most of the antiviral drugs used to treat COVID19 currently are based on previous treatments for severe acute respiratory syndrome SARS Middle East respiratory syndrome and influenza A and uncertainties on the efficacy and side effects of these drugs remain problematic One case report stated that a patient was cured with remdesivir [] In another clinical study lopinavirritonavir had no clinical benefit on the treatment of patients with severe COVID19 and adverse gastrointestinal events were identified [] Furthermore Abidol and lopinavirritonavir did not improve COVID19 symptoms or shorten the conversion of the viral nucleic acid to a negative result in a clinical study of patients [] The adverse reactions and side effects of related antiviral drugs such as hypocalcemia hemolytic anemia hypomagnesemia as well as those that emerged during the treatment of SARS by ribavirin cannot be ignored [] Hydroxychloroquine and chloroquine were urgently approved by the Food and Drug Administration to treat COVID19 but chloroquine drugs hydroxychloroquine and chloroquine phosphate can cause adverse reactions such as dizziness headache vertigo loss of appetite and nausea [] Our study showed that under the conditions of home quarantine TCM is effective in reducing the progression of mild and moderate COVID19 to severe disease which can serve as a reference for the Multivariate analysis Pvalue ＜ ＜ OR95 CI continued on next page SexRef female Medical history Ref no Fever Cough Diarrhea Fatigue PharmacologicalResearch16120201051275 0cJ Tian Table continued Factors Univariate analysis Pvalue OR95 CI Multivariate analysis Pvalue Antiviral treatment Ref no Antibiotics Ref no Chinese patent medicine Ref no Grouping Ref control group OR95 CI \u3000 The model includes age and sex The model includes age gender and medical history The model includes age sex medical history and initial symptoms The model includes age gender medical history initial symptoms and antiviral drugs The model includes age gender medical history initial symptoms antiviral drugs and antibiotics The model includes age gender medical history initial symptoms antiviral drugs antibiotics and Chinese patent medicine The model includes age gender medical history initial symptoms antiviral drugs antibiotics Chinese patent medicine and grouping prevention of the disease As a key point of COVID19 prognosis there have only been a few studies assessing the proportion of mild and moderate COVID19 patients who have progressed to a severe disease status currently In attempting to prevent and control the epidemic in China TCM has been widely used Some studies revealed that TCM can reduce the clinical symptoms of fever and cough in mild and moderate patients [] and improve relevant imaging indexes [] However these studies usually had small sample sizes and did not assess the proportion of mild and moderate patients who progressed to a severe disease status In this retrospective cohort study the results showed that the main influencing factors for the progression of mild and moderate COVID19 to severe disease included sex male age fever cough and fatigue which are consistent with other studies [“] After excluding the effects of the above factors we found that the HSYF intervention can effectively reduce the conversion of mild and moderate COVID19 to severe disease providing evidence on the effectiveness of TCM Thus TCM can be an option in addition to antibiotic therapy antiviral treatment and supportive oxygen therapy for the treatment of mild and moderate COVID19 patients HSYF administered in the study mainly contains the following Chinese materia medica Ma Huang Ephedrae Herba Shi Gao Gypsum fibrosum Xing Ren Armeniacae Semen Qiang Huo Notopterygii Rhizoma seu Radix Ting Li Zi LepidiiDescurainiae Semen Guan Zhong Cyrtomii Rhizoma Di Long Pheretima Xu Chang Qing Cynanchi paniculati Radix Huo Xiang Pogostemonis Herba Pei Lan Eupatorii Herba Cang Zhu Atractylodis Rhizoma Yun Ling Poria Sheng Bai Zhu Atractylodis macrocephalae Rhizoma Jiao Shan Zha Crataegi Fructus Jiao Shen Qu Massa medicate fermentata Jiao Mai Ya Hordei Fructus germinatus Hou Po Magnoliae officinalis Cortex Jiao Bing Lang Arecae Semen Wei Cao Guo Tsaoko Fructus and Sheng Jiang Zingiberis Rhizoma recens In TCM theory these prescriptions can improve the patients™ healthy qi and dispel evil factors Ma Huang Ephedrae Herba Shi Gao Gypsum fibrosum Xing Ren Armeniacae Semen Ting Li Zi LepidiiDescurainiae Semen focus on improving asthma symptoms in the lung Yun Ling Poria Sheng Bai Zhu Atractylodis macrocephalae Rhizoma Jiao Shan Zha Crataegi Fructus Jiao Shen Qu Massa medicate fermentata Jiao Mai Ya Hordei Fructus germinatus Hou Po Magnoliae officinalis Cortex Jiao Bing Lang Arecae Semen and Wei Cao Guo Tsaoko Fructus help strengthen the healthy qi Huo Xiang Pogostemonis Herba and Pei Lan Eupatorii Herba can dispel the evil factors Studies have shown that many Chinese components of HSYF can exert antiviral effects and improve respiratory symptoms In terms of the antiviral components methyl ephedrine Lephedrine and Dpseudoephedrine are present in Ma Huang Ephedrae Herba which can significantly inhibit the in vitro proliferation of influenza A H1N1 virus [] Patchouli alcohol in Huo Xiang Pogostemonis Herba can effectively inhibit the replication of H1N1 virus [] Hou Po Magnoliae officinalis Cortex can alter the cell cycle and promote H1N1 infected cells into the S phase [] the magnolol extract can inhibit the secretion of CD44 and CD54 reduce the levels of inflammatory factors IL1β IL6 and TNFα and relieve inflammation [] The active ingredients of Wei Cao Guo Tsaoko Fructus inhibit the binding region of severe acute respiratory syndrome coronavirus SARSCoV2 Sprotein to human ACE2 and control the replication of SARSCoV2 in the human body [] In addition to its antiviral effect Ma Huang Ephedrae Herba is a widely used Chinese herbal medicine for respiratory diseases and it also stimulates β receptors of the bronchial smooth muscle expands the bronchus exerts antiinflammatory effects [] and can stimulate central nervous system excitability and regulate body temperature [] This plays an important role in improving the symptoms of COVID19 including fever cough shortness of breath and other respiratory symptoms The clinical benefits and risks need to be balanced for the treatment of mild and moderate COVID19 The lack of understanding on the efficacy side effects and other risks associated with current antiviral treatments make it difficult to be used as a conventional intervention for mild and Table Pr sity score matching PSM Variables Mean ± SD MedianQ1Q3 Age SexRef female Fever Cough Fatigue Before PSM Exposed group n ± Control group n ± Pvalue ＜ ＜ ＜ ＜ After PSM Exposed group n ± Control group n ± Pvalue PharmacologicalResearch16120201051276 0cAppendix A Supplementary data J Tian moderate COVID19 patients However the results of our study provide evidence on the effectiveness of TCM for the treatment of mild and moderate COVID19 TCM treatment has the advantage of being widely used and inexpensive making it convenient for mild and moderate COVID19 patients isolated in the largescale community to quickly recover Furthermore our study used a realtime Internet information collection application to collect data which enabled physicians to communicate with the patients in real time and dynamically monitor changes in the patient™s condition Thus selfquarantined patients did not need to go to the hospital for a consultation which prevented cross infection Although the results of our study are significant and provides insight on the use of TCM treatment for mild and moderate COVID19 there are still some limitations Firstly our study was a retrospective study rather than a prospective randomized clinical trial RCT due to the current situation In the future we aim to conduct RCTs and experiments to further verify the efficacy and mechanism of HSYF Secondly as a retrospective cohort study some baseline factors between the exposed group and the control group were not completely accounted for and the age of the exposed group was significantly younger than that of the control group which may be related to our data collection method Using a realtime Internet information collection application is a more acceptable method in the younger population and imbalances in baseline data such as sex age and medical history could have introduced bias which may have affected the results of the study Nevertheless we have corrected these possible influencing factors during our analysis In order to improve the reliability of the study results a cohort study with a larger sample size or RCTs is necessary Conclusion [] National Health Commission of the People™s Republic of China National HSYF can significantly reduce the progression of mild and moderate COVID19 to a severe disease status which has a positive effect on the prevention and treatment of the disease However future clinical studies with a larger sample size are needed to further verify our results Funding This work was funded by the Special Project for Emergency of the Ministry of Science and Technology 2020YFC0845000 and the Traditional Chinese Medicine Special Project for COVID19 Emergency of National Administration of Traditional Chinese Medicine2020ZYLCYJ041 Declaration of Competing Interest Authors declare no competing interests Jiangsu Kanion Pharmaceutical Co Ltd provided the medications for the study while they did not participate in research design data collection data analysis data interpretation nor article writing Acknowledgements We acknowledge all volunteers and healthcare workers in the Wuchang community health service station Hubei Provincial Hospital of Traditional Chinese Medicine and Jiangsu Kanion Pharmaceutical Co Ltd Supplementary material related to this article can be found in the online version at 101016jphrs2020105127 References [] H Lu CW Stratton YW Tang Outbreak of pneumonia of unknown etiology in Wuhan China the mystery and the miracle J Med Virol “ [] Medical treatment group of Wuhan COVID prevention and control headquarters Notice Concerning the Recommendation of the Prescription of Traditional Chinese Medicine in the Treatment of COVID19 February [] XL Tong XY Li LH Zhao Discussion on traditional chinese medicine prevention and treatment strategies of coronavirus disease COVID19 from the perspective of œColddampness pestilence J Tradit Chin Med “ Administration of Traditional Chinese Medicine of the People™s Republic of China Guidance for Corona Virus Disease Prevention Control Diagnosis and Management People™s Medical Publishing House March [] World health anization Coronavirus disease COVID19 Situation dashboard whosprinklrcom [] A Kimball KM Hatfield M Arons

Thyroid_Cancer

obesity and ethnicity alter gene expression in skinJeanne M Walker18 Sandra Garcet28 Jose O Aleman34 Christopher E Mason5 David Danko5 Simone Zuffa6 Jonathan R Swann67 James Krueger2 Jan L Breslow3 peter R Holt3Obesity is accompanied by dysfunction of many ans but effects on the skin have received little attention We studied differences in epithelial thickness by histology and gene expression by Affymetrix gene arrays and PCR in the skin of obese BMI “ and normal weight BMI “ postmenopausal women paired by age and ethnicity Epidermal thickness did not differ with obesity but the expression of genes encoding proteins associated with skin blood supply and wound healing were altered In the obese many gene expression pathways were broadly downregulated and subdermal fat showed pronounced inflammation There were no changes in skin microbiota or metabolites African American subjects differed from European Americans with a trend to increased epidermal thickening In obese African Americans compared to obese European Americans we observed altered gene expression that may explain known differences in water content and stress response African Americans showed markedly lower expression of the gene encoding the cystic fibrosis transmembrane regulator characteristic of the disease cystic fibrosis The results from this preliminary study may explain the functional changes found in the skin of obese subjects and African AmericansObesity defined as a body mass index BMI greater than a0kgm21 has become a major epidemic in industrial and emerging countries The prevalence of obesity has doubled since the 1980s and it is now estimated that million adults worldwide are obese2 Obesity affects many ans of the body and it is this an dysfunction that leads to excess mortality and morbidity3 Much attention has focused on the consequences of obesity in the heart liver and pancreas and other ans in which increased inflammation and oncogenesis become apparent4 Less attention has been paid to the effects of obesity on the skinObesity increases psoriasis5 which can be ameliorated with weight loss and cutaneous infections6 Since diabetes is common in obesity disorders such as fibroepithelial polyps and acanthosis nigricans also occur in the skin of obese subjects78 Moreover physiologic changes found in obese skin include increased transepidermal water loss with lower capacitance dry rough textured skin with pronounced erythema and reduced microvascular reactivity Altered collagen formation and increased delayedtype hypersensitivity have also been reported9Adipocyte depots that exist adjacent to the epidermis have distinct morphology and physiologic characteristics and are termed dermal or subdermal adipose tissue In addition to the principal role for dermal adipocytes in lipid storage and thermal insulation10 they also promote skin immunity11 wound healing and hair follicle cycling12 Obesity is accompanied by inflammatory immune changes in subcutaneous and visceral adipose tissues13 but the role of inflammatory changes within the adipose layer of the skin has received little attention Furthermore obesity is associated with increased circulating leptin levels which appear to independently affect dermal cell proliferation and hair growth14 In addition the microanisms that live on the skin surface also affect skin immunity11 so that it is important to analyse the skin microbiome comparing obese and normal individuals1The Rockefeller University Hospital New York NY USA 2Laboratory of Investigational Dermatology The Rockefeller University New York NY USA 3Laboratory of Biochemical Genetics and Metabolism The Rockefeller University New York NY USA 4Laboratory of Translational Obesity Research New York University Langone Health New York NY USA 5Weill Cornell Medical College New York NY USA 6Department of Metabolism Digestion and Reproduction Imperial College London London UK 7School of Human Development and Health Faculty of Medicine University of Southampton Southampton UK 8These authors contributed equally Jeanne M Walker and Sandra Garcet email walkerjrockefelleredu holtprockefellereduScientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cIn view of the profound clinical and physiologic changes described in the skin in obesity it would not be surprising also to find biologically important molecular changes The present study was designed to compare gene expression in skin of healthy obese and nonobese subjects and to evaluate the potential importance of parallel changes in the microbiome and metabolites found on the adjacent skin surface and in the adipose tissue immediately below the skinMaterials and methodsSubjects Participants were recruited from the surrounding community through advertisements and from the Rockefeller University subject repository Eligible were healthy obese BMI “ a0kgm2 and nonobese BMI “ a0kgm2 postmenopausal women between the ages of and a0years The two groups were matched by age ± a0years and defined by selfreported ethnicity and by skin colour Exclusions were unstable weight change within the past three months HIV infection weight loss surgery inflammatory bowel disease history of malignancy other than nonmelanoma skin cancer in the previous a0years generalizable or systemic skin diseases history of a bleeding disorder current anticoagulant therapy or regular NSAID use current weight control medication or hypoglycaemic therapy individuals taking oestrogenprogesterone hormones and current tobacco smokers Also excluded were candidates with fasting blood glucose a0mgdl liver function tests ALT AST alkaline phosphatase greater than times the upper limit of normal ULN abnormal thyroid function test or serum creatinine — ULNFourteen obese subjects were screened two refused skin biopsies one was withdrawn due to an intercurrent inflammatory illness and one was not postmenopausal by our criteria Ten obese subjects met our inclusion criteria and underwent skin swab collections and punch biopsy Twenty nonobese subjects were screened Two subjects refused to undergo punch biopsy one was withdrawn due to an intercurrent illness two with a BMI outside the required range one withdrew consent one was excluded with a history of keloid formation one with a low platelet count one with uncontrolled hypertension One nonobese subject who underwent skin punch biopsy was not included in the analysis because we were unable to find an age and ethnicitymatched obese subject These obese and agematched ethnicitymatched nonobese postmenopausal women completed all aspects of the study Fig a0 Six participants were European American and four were African Americans in each group Postmenopausal women were chosen to exclude effects of the menstrual cycle upon study end points and to exclude gender effectsBased on preliminary data from a previous study comparing skin from seven obese and six nonobese postmenopausal women there was a variation of in a set of RTPCR genes unpublished data Assuming the same variation and proportion of differentially expressed genes to be we calculated that a sample size of n subjects per group matched by age and ethnicity would provide power at a falsediscovery rate to detect the expected number of differentially expressed genes based on a threshold of twofold changesDesign and setting This was an label comparison of a group of postmenopausal obese women and postmenopausal nonobese women who were agematched ± a0years and racematched Screening comprised a complete history and physical examination and fasting blood testing for complete blood count sedimentation rate comprehensive chemistry panel lipid panel thyroid function tests hepatitis C antibody uric acid and haemoglobin A1C Observing Good Clinical Practice guidelines all participants read and signed an informed consent document approved by the Institutional Review Board and the Advisory Committee for Clinical and Translational Science at The Rockefeller University Protocol JWA0921Procedure methods Anthropometric measurements Body weight was measured daily to the nearest a0kg using a ScaleTronix scale Welch Allyn Skaneateles Falls NY with precision of ± a0kg Subjects were weighed in a hospital gown after an overnight fast and postvoiding Height was measured to the nearest a0cm at baseline with a Seca216 stadiometer Hamburg Germany in a0cm increments Body mass index BMI was calculated as kgm2 using the NIH Standard Metric BMI calculatorBlood collection and analysis Fasting blood samples were analysed in the Clinical Pathology Laboratory of the Memorial SloanKettering Cancer Center for complete blood count electrolytes glucose creatinine blood urea nitrogen liver function Creactive protein sedimentation rate and uric acid Research serum samples were drawn pre and post intervention aliquoted and stored at ˆ’ a0°C for subsequent analysisSkin swabbing Subjects were permitted to shower but did not wash the planned biopsy area over the midlower abdomen with soap for a0days before the biopsyFor microbiome analysis two areas of skin approximately — a0cm were swabbed using the eSwab collection and preservation system for aerobic anaerobic and fastidious bacteria Copan Diagnostics Marietta CA Swabs were labelled sealed separately in the provided tubes and immediately stored at ˆ’ a0°C For metabolome analysis two different areas of skin approximately — a0cm were swabbed using salinemoistened sterile cottontipped applicators The tips were cut sealed in separate sterile collection tubes and immediately stored at ˆ’ a0°CSkin microbiome The DNA extraction protocol was adapted from the Maxwell RSC Buccal Swab DNA kit Catalogue number AS1640 Promega Corporation Madison WI Briefly a0μl of lysis buffer and a0μl of Proteinase K was mixed and added to each swab tube Swab tubes were then incubated for a0min at C using a Thermo Fisher water bath removed from the tubes and fluid was transferred to well of the Maxwell RSC Cartridge The swab head was centrifuged using a ClickFit Microtube Cat V4741 and extracted fluid was added to the corresponding well of Maxwell Cartridge and eluted in a0μl of provided elution bufferScientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Consort flow chart of eligible subjectsExtracted DNA was taken forward to the Nextera Flex protocol by Illumina Briefly a0μl of extracted DNA was taken into library prep protocol and run with cycles of PCR Libraries were cleaned up with a left sided size selection using a bead ratio of 08x The right sided size selection was omitted Libraries were then quantified using a Thermo Fisher Qubit Fluorometer and an Advanced Analytical Fragment Analyzer Libraries were sequenced on an Illumina HiSeqPE — at the Weill Cornell Epigenomics CoreAll bioinformatic analysis was performed on Weill Cornell Medicine™s Athena compute cluster a highperformance grid compute system Secondary analysis was performed on a Linux and MacOS systems Unless otherwise noted programs were run with default settingsRaw sequence data were processed with AdapterRemoval v217 to remove low quality reads and reads with ambiguous bases15 Subsequently reads were aligned to the human genome hg38 including alternate contigs using Bowtie2 v230 fast preset16 Read pairs where one or both ends mapped to the human genome were separated from read pairs where neither mate mapped Read pairs where only one mate mapped were discarded Hereafter we refer to the read sets as human reads and nonhuman readsTaxonomic profiles were generated by processing nonhuman reads with KrakenUniq v032 with a database based on all draft and reference genomes in RefSeq Microbial bacteria fungi virus and archaea ca March KrakenUniq identifies kmers that are unique to taxa in a database Reads are broken into kmers and searched against this database Finally the taxonomic makeup of each sample was given by taking the proportion reads which were assigned to each clade KrakenUniq counts the number of unique marker kmers assigned to each taxon and we filtered taxa with fewer than unique markers17We performed differential abundance testing over microbial species using the ALDEx2 R package ALDEx2 performs variance stabilization read counts using a centred log ratio transformation that models samples as Scientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cDirichletMultinomial distributions over taxa then compares taxonomic abundances across groups18 Comparison of abundances across groups was done with a Wilcoxon rank sum test and Benjamini Hochberg Correction for multiple hypothesis testingDimensionality reduction of taxonomic profiles was performed with Principal Coordinates Analysis PCA based on a matrix of JensenShannon Divergences JSD between samples Analysis of intersample beta diversity was performed using the same matrix of JSD Intrasample alpha diversity was measured by finding Shannon™s Entropy of the taxonomic profile and by counting the total number of species identified in each sample richness Shannon™s entropy accounts for the relative size of each group in diversity estimation and is defined as H ˆ’ cid31 ai log2ai where ai is the relative abundance of taxa i in the sampleWe generated profiles of antimicrobial resistance genes using MegaRes v10119 To generate profiles from MegaRes we mapped nonhuman reads to the database using Bowtie2 v230 very sensitive presets Subsequently alignments were analysed using Resistome Analyzer commit 15a52dd and normalized by total reads per sample and gene length to give RPKMs MegaRes includes an ontology grouping resistance genes into gene classes AMR mechanisms and gene groupsSkin metabolome The skin was swabbed using two sterile salinemoistened culture swabs and immediately frozen at ˆ’ a0°C Swab heads were removed and placed in a0ml methanol water Following sonication a0min a0ml of isopropanol was added and the solution was spun at a0g for a0min The swab was removed and the samples were dried using a vacuum concentrator operating at a0°C Prior to UPLCMS analysis samples were reconstituted in a0μl of HPLCgrade water sonicated for a0min and transferred to vials for analysisA Waters 2777C sample manager Waters Corp Milford MA USA was used for sample handling This was equipped with a a0μl Hamilton syringe a a0μl loop used for fullloop injections of prepared sample and a 3drawer sample chamber maintained at a0°C with a constant flow of dry nitrogen gas to prevent the buildup of condensation The LC component was an ACQUITY UPLC Waters Corp Milford MA USA composed of a binary solvent manager and column heatercooler module Metabolic profiles were acquired using reversedphase chromatography Water and acetonitrile each supplemented with formic acid mobile phases A and B respectively were selected for the mobile phase A — a0mm HSS T3 column was used at a0°C with a mobile phase flow rate of a0mlmin This generated a maximum pressure of psi in a wateracetonitrile gradient After a a0min isocratic separation at initial conditions A a linear gradient elution A to A in a0min proceeded followed by a quicker gradient A to A in a0min to final conditions The mobile phase flow rate was simultaneously increased to a0mlmin in the latter stage to facilitate faster column washing The MS component comprised a Xevo G2S QToF MS Waters Corp Manchester UK coupled to the UPLC via a Zspray electrospray ionization ESI source The cone gas flow was set to a0lh to protect the cone from residue accumulation during operation Both positive and negative ion modes RPC and RPCˆ’ respectively were used Raw spectra were converted into mzML files using MSConvert20 and processed with XCMS in R21 Peak picking and peak grouping were performed using inhouse scripts in R and matrices were normalized using a median fold change approach Log transformation scaling and data analysis was performed in SIMCA Umetrics Umea SwedenSkin biopsy After the skin swabbing the abdominal site was cleansed with Chloraprep swabs chlorhexidine and isopropyl alcohol Becton Dickinson Canaan CT Using sterile technique local anaesthesia was induced by infiltration of the area with a0ml of lidocaine Hospira Inc Lake Forest IL mixed with a0ml sodium bicarbonate The skin biopsy was performed using a a0mm punch Miltex Instruments York PA Fat tissue was carefully removed from the skin core of the biopsy The dermis and epidermis were divided into two halves one half placed in a cryomold for OTC flash freezing Agar Scientific Essex UK and stored at ˆ’ a0°C and the other half was placed in RNAlater Stabilization Solution Thermo Fisher Scientific Fair Lawn NJ refrigerated for a0h and then frozen at ˆ’ a0°C The fat tissue was removed from the biopsy divided between RNAlater refrigerated for a0h then frozen at ˆ’ a0°C and a dry Sarstedt tube that was flash frozen in liquid nitrogen and placed in ˆ’ a0°C The biopsy site was sutured closed and a dry sterile dressing was applied Subjects were discharged and scheduled to return for suture removalGene‘array and quantitative real‘time PCR analysis RNA was extracted followed by hybridization to Affymetrix Human U133 Plus gene arrays Santa Clara CA or quantitative RTPCR as previously described2223All statistical analyses were carried out in R Limma Log 2transformed qRTPCR measurements hARP normalized and microarray expression values were assessed with a mixedeffect The fixed factors were condition obese vs nonobese race African American vs Caucasian with random intercept for each subject Quality control of microarray chips was carried out using standard QC metrics and R package microarray quality control Images were scrutinized for spatial artefacts using Harshlight24 Expression measures were obtained using the GCRMA algorithm25 A batch effect corresponding to the hybridization date was detected by PCA and adjusted using the ComBat function from the SVA package Probe sets with at least samples with expression values were kept for further analysis Fold changes for the comparisons of interest were estimated and hypothesis testing was conducted with contrasts under the general framework for linear models with the limma package P values from the moderated paired ttests were adjusted for multiple hypotheses using the Benjamini“Hochberg procedure Hierarchical clustering was performed with Euclidean distance and a McQuitty agglomeration scheme26Data was deposited into Gene Expression Omnibus GEO repository GSE151839All study methods and procedures were carried out in accordance with Good Clinical Practice Guidelines by trained practitioners The protocol and informed consent were evaluated and approved by the Institutional Scientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression between the skin of obese and nonobese subjects A Heat map of the most differentially expressed genes in the skin of obese and nonobese subjects with an FCH fold change fdr false discovery rate B PCA principal component analysis plot of differentially expressed genes in the skin of obese and nonobese subjects with an FCH fdr Review Board and the Advisory Committee for Clinical and Translational Science at Rockefeller University prior to initiation of the study and annually thereafter Protocol JWA0921ResultsThis study was performed in ten healthy obese and ten healthy nonobese postmenopausal women matched for age and ethnicity Obese subjects had a mean weight of a0kg BMI of a0kgm2 and waist circumference of a0cm Nonobese subjects had a mean weight of a0kg BMI of a0kgm2 and waist circumference of a0cm Supplemental Table a0S1 The skin thickness for subjects with obesity was not significantly different from that of nonobese subjects Supplemental Fig a0S1Gene expression analysis of the skin The most differentially expressed genes in the skin between obese and nonobese subjects are displayed in the heat map in Fig a02a Comparing gene expression in obese versus nonobese skin showed greater gene expression of S100A7A encoding a calcium binding protein involved in psoriasis and CORIN encoding a natriuretic peptide converting enzyme which is expressed in the dermis and is involved in specifying skin colour However the expression of CREB3LA encoding a cyclic AMP response element was lower in the skin of obese subjectsA PCA model constructed on of the most differentially expressed genes between obese and nonobese subjects showed partial separation of the groups This difference was seen in PC1 which accounted for of the variation in the included genes Fig a02bThe complete list of skin genes whose expression significantly differed between the two groups are shown with fold changes in Supplemental Tables a0S2 and S3 Again the gene expression of S100A7A was 344fold higher in the obese skin compared to the nonobese skin Similarly the expression of DEFB4A Defensin B4A which encodes an antimicrobial peptide part of the betadefensive system and SPRR2C which encodes a proline rich protein strongly induced during differentiation of human epidermal keratinocytes was also significantly higher in the obese skin being and 17fold higher respectively Genes with lower expression profiles in obese subjects than nonobese included AOP that encodes aquaporin involved in water channels present in the skin PROM1 prominin involved in cell differentiation and proliferation and Keratin and important for fibrogenesis in the epidermis Also of interest was the significantly higher expression 282fold of CFTR the cystic fibrosis transmembrane conductance receptor in nonobese subjects compared to the obese groupQTPCR analysis of genes selected from the total list of significantly differentially expressed genes in skin confirmed increased expression of the S100A 373fold DEFB4A defensin B4A 329fold and CORIN fold in the skin of obese subjects Fig a0 Significantly lower gene expression in the skin of obese subjects was found with CFTR 36fold PROM1 556fold and GABRP gamma aminobutyric acid receptor 29foldScientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cGene expression pathway analysis showed a broad downregulation of many pathways in obesity with only out of of the most highly differentially expressed pathways higher in the obese Supplemental Fig a0S2 The pathways most downregulated included cardiac beta adrenergic signalling which appears to function in skin cyclin dependent Kinase CDK5 a mutation which is important in melanoma formation and functions in skin healing and gonadotrophic releasing hormone GNRH signalling which has many extra pituitary functionsGene expression analysis in skin fat We next examined differences in gene expression between the groups of subjects in subdermal fat removed from immediately below the skin portion of the biopsy A heat map of gene expression shows a markedly different pattern between the two groups Fig a04a The expression of many of the genes upregulated in obese subdermal fat are involved with inflammation and immune function including platelet activating factor PLA2G7 ILIRN involved in IL1 activation SPPI a cytokine that can increase interferon gamma and IL12 activity and several serpins mediators involved in inflammation and immune functionThe PCA plot of genes whose expression differed significantly in subdermal fat of obese and nonobese subjects Fig a04b clearly shows separation between the two groups Most of the difference was seen in PC1 which includes of the genes whose expression was determinedSupplemental Tables a0S4 and S5 show a list of genes whose expression was relatively greater in the subdermal fat of obese subjects The expression of SPPI that encodes osteopontin which can act as a cytokine augmenting the action of interferon gamma and interleukin was approximately tenfold higher in the obese subjects EGFL6 expression which encodes an epidermal growth factor found to be enhanced in obesity and alters insulin action was increased by 85fold MMP9 which encodes metalloproteinase and ILTRN was increased by sevenfold in obesity Genes significantly downregulated in obese subdermal fat included SLC27A2 acetylCoAsynthase tenfold and C6complement fivefoldBy QTPCR in subdermal fat from obese subjects the increased expression of genes encoding proteins important in inflammation and immune function was confirmed Fig a0 This includes genes encoding proteins that determine accumulation of immune cells in adipose tissues such as CD52 the high affinity immunoglobulin gamma FC receptor FCGR1β CCL3 CZXCL8 interleukin and CLEC7A a pattern recognition receptor found in monocytes and other myeloid cells IL17F a member of the IL17 family also was specifically increased in subdermal fat from the obese as compared to nonobese individualsExpression pathway analysis Supplemental Fig a0S3 showed upregulation of several inflammatory immune pathways including the Thelper dendritic cell maturation and inflammatory signalling pathways further indicating profound effects of obesity on inflammation in subdermal fat Dramatically lower in the obese subdermal fat was the LXRRXR activation pathwayGene expression analysis by race Two subgroups were observed in the gene expression profiles of the skin based on the subject™s race Using selfreported data and skin colour as criteria the data from African Americans was analysed separately from the data from European Americans This analysis showed striking differences in this very small group of subjects A heatmap of the most differentially expressed genes in skin from obese subjects divided by ethincity is shown in Fig a06a No clear differences in gene expression in the skin by ethnicity were found in nonobese subjects In contrast gene expression clearly differed between obese African American and obese European American subjects Fig a06a and also is illustrated in the PCA plot Fig a06b with PC1 responsible for the greatest variation The pattern of differences between obese and non “obese skin is further illustrated in Fig a06cA list of genes whose expression significantly differed between the obese African Americans and the obese European Americans is shown in Supplemental Tables a0S6 and S7 The expression of SLC6A4 a serotonin transporter CORIN and COL8AI a collagen gene encoding a protein that is dysregulated in atopic eczema was higher in African Americans while the expression of SCCB2A2 the secretoglobin expressed in skin sweat glands and CFTR was expressed higher in European AmericansComparing the skin of obese African Americans to obese European Americans by QTPCR the former showed significantly lower expression of MYBCPI 516fold and PROM1 43fold and CFTR Fig a0 In contrast there was a small increase in the expression of CORIN 22fold a gene encoding the atrial naturalistic peptide converting enzyme and BMP2 fold also present in the skin compared to obese European AmericansPathway analysis found no racerelated differences in the nonobese samples However in the obese Supplemental Fig a0S4 there was markedly reduced expression of oestrogen mediated sphase entry pathway aryl hydrolase receptor signalling pathway and cell cycle regulation through cyclins pathways in the African Americans compared to the skin of the European American groupSubdermal fat in African Americans exhibited few differences from that found in European Americans Fig a08A Figure a08bc show the differences by weight and by ethnicity respectively illustrating the impact of obesity in the two racial groups The expression of numerous inflammatoryimmune genes was upregulated in the fat of both groups of obese subjects Fig a0 Tables a0S5 and S6 Microbiota analysis We next examined whether the microbiota collected from skin swabs around the biopsy site differed between the obese and nonobese subjects We generated taxonomic profiles for each sample using KrakenUniq and a database built from all available microbial species in RefSeq We measured the total number of AMR genes detected in each sample by aligning reads to MegaRESOverall differences between groups were minor No significant differences were noted in average taxonomic alpha diversity as measured by either Shannon™s entropy or richness between the groups A PCA plot of the taxonomic profiles showed slight separation between obese and lean samples and slightly higher beta diversity for obese samples however these differences were minor Fig a010a The number of antimicrobial resistant AMR Scientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RT PCR between the skin of obese and nonobese subjects LS means of gene expression by RTPCR showing significant differences as p p p Figure a0 Differences in gene expression between the subdermal fat of obese and nonobese subjects A Heat map of the most differentially expressed genes in subdermal fat of obese and nonobese subjects with an FCH fdr B PCA plot of differentially expressed genes in subdermal fat of obese and nonobese subjects with an FCH fdr Scientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RTPCR between the subdermal fat of obese and nonobese subjects LS means of gene expression by RTPCR showing significant differences as p p p Figure a0 Differences in gene expression between the skin of African American and European American subjects A Heat map of the most differentially expressed genes in skin of African American and European American subjects with an FCH fdr B PCA plot of differentially expressed genes in skin of obese and nonobese African American and European American subjects with an FCH Left side of plot indicates differences in gene expression by ethnicity in nonobese subjects Right side of plot indicates differences in gene expression by ethnicity in obese subjects C PCA plot of differentially expressed genes in skin of obese and nonobese subjects by ethnicity with an FCH Left side of plot indicates differences in gene expression between obese and nonobese African American subjects Right side of plot indicates differences in gene expression between obese and nonobese European American subjectsScientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RT PCR between the skin of obese and nonobese African American and European American subjects LS means of gene expression by RTPCR showing significant differences as p p p genes identified was higher on average from obese subjects but this difference did not reach significance p Wilcox test Fig a010bDifferentially abundant taxa At the given sample size n no taxa were identified as significantly differentially abundant after Benjamini“Hochberg correction Before correction five taxa were significantly differentially abundant at p Wilcox test These five taxa were Corynebacterium aurimucosum Corynebac‘terium jeikeium Corynebacterium urealyticum Streptococcus salivarius and Streptococcus sp A12 All five taxa were more abundant in samples from obese subjects on averageMetabolome analysis As highlighted by the PCA analysis no variation in the metabolites analysed by liquid chromatographymass spectrometry from the skin swabs was observed between the obese and nonobese individuals PCA Supplemental Fig a0S6 Similarly no ethnicityrelated metabolic variation was observed These results were further confirmed by the poor predictive ability of the orthogonal projection to latent structuresdiscriminant analysis OPLSDA models comparing the two different ethnic groupsDiscussionFew comprehensive reviews of skin changes occurring with obesity have been conducted despite over of the US population being obese27 A broad review of the physiologic and clinical consequences and associations was published by Hirt et a0al in The authors include a discussion of circulatory and lymphatic changes which may enhance the frequency and severity of skin ulceration and provide a comprehensive review of skin disorders that can be associated with obesity expanding on previous reviews29 and studies in rodents30In our study the thickness of

Thyroid_Cancer

obesity and ethnicity alter gene expression in skinJeanne M Walker18 Sandra Garcet28 Jose O Aleman34 Christopher E Mason5 David Danko5 Simone Zuffa6 Jonathan R Swann67 James Krueger2 Jan L Breslow3 peter R Holt3Obesity is accompanied by dysfunction of many ans but effects on the skin have received little attention We studied differences in epithelial thickness by histology and gene expression by Affymetrix gene arrays and PCR in the skin of obese BMI “ and normal weight BMI “ postmenopausal women paired by age and ethnicity Epidermal thickness did not differ with obesity but the expression of genes encoding proteins associated with skin blood supply and wound healing were altered In the obese many gene expression pathways were broadly downregulated and subdermal fat showed pronounced inflammation There were no changes in skin microbiota or metabolites African American subjects differed from European Americans with a trend to increased epidermal thickening In obese African Americans compared to obese European Americans we observed altered gene expression that may explain known differences in water content and stress response African Americans showed markedly lower expression of the gene encoding the cystic fibrosis transmembrane regulator characteristic of the disease cystic fibrosis The results from this preliminary study may explain the functional changes found in the skin of obese subjects and African AmericansObesity defined as a body mass index BMI greater than a0kgm21 has become a major epidemic in industrial and emerging countries The prevalence of obesity has doubled since the 1980s and it is now estimated that million adults worldwide are obese2 Obesity affects many ans of the body and it is this an dysfunction that leads to excess mortality and morbidity3 Much attention has focused on the consequences of obesity in the heart liver and pancreas and other ans in which increased inflammation and oncogenesis become apparent4 Less attention has been paid to the effects of obesity on the skinObesity increases psoriasis5 which can be ameliorated with weight loss and cutaneous infections6 Since diabetes is common in obesity disorders such as fibroepithelial polyps and acanthosis nigricans also occur in the skin of obese subjects78 Moreover physiologic changes found in obese skin include increased transepidermal water loss with lower capacitance dry rough textured skin with pronounced erythema and reduced microvascular reactivity Altered collagen formation and increased delayedtype hypersensitivity have also been reported9Adipocyte depots that exist adjacent to the epidermis have distinct morphology and physiologic characteristics and are termed dermal or subdermal adipose tissue In addition to the principal role for dermal adipocytes in lipid storage and thermal insulation10 they also promote skin immunity11 wound healing and hair follicle cycling12 Obesity is accompanied by inflammatory immune changes in subcutaneous and visceral adipose tissues13 but the role of inflammatory changes within the adipose layer of the skin has received little attention Furthermore obesity is associated with increased circulating leptin levels which appear to independently affect dermal cell proliferation and hair growth14 In addition the microanisms that live on the skin surface also affect skin immunity11 so that it is important to analyse the skin microbiome comparing obese and normal individuals1The Rockefeller University Hospital New York NY USA 2Laboratory of Investigational Dermatology The Rockefeller University New York NY USA 3Laboratory of Biochemical Genetics and Metabolism The Rockefeller University New York NY USA 4Laboratory of Translational Obesity Research New York University Langone Health New York NY USA 5Weill Cornell Medical College New York NY USA 6Department of Metabolism Digestion and Reproduction Imperial College London London UK 7School of Human Development and Health Faculty of Medicine University of Southampton Southampton UK 8These authors contributed equally Jeanne M Walker and Sandra Garcet email walkerjrockefelleredu holtprockefellereduScientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cIn view of the profound clinical and physiologic changes described in the skin in obesity it would not be surprising also to find biologically important molecular changes The present study was designed to compare gene expression in skin of healthy obese and nonobese subjects and to evaluate the potential importance of parallel changes in the microbiome and metabolites found on the adjacent skin surface and in the adipose tissue immediately below the skinMaterials and methodsSubjects Participants were recruited from the surrounding community through advertisements and from the Rockefeller University subject repository Eligible were healthy obese BMI “ a0kgm2 and nonobese BMI “ a0kgm2 postmenopausal women between the ages of and a0years The two groups were matched by age ± a0years and defined by selfreported ethnicity and by skin colour Exclusions were unstable weight change within the past three months HIV infection weight loss surgery inflammatory bowel disease history of malignancy other than nonmelanoma skin cancer in the previous a0years generalizable or systemic skin diseases history of a bleeding disorder current anticoagulant therapy or regular NSAID use current weight control medication or hypoglycaemic therapy individuals taking oestrogenprogesterone hormones and current tobacco smokers Also excluded were candidates with fasting blood glucose a0mgdl liver function tests ALT AST alkaline phosphatase greater than times the upper limit of normal ULN abnormal thyroid function test or serum creatinine — ULNFourteen obese subjects were screened two refused skin biopsies one was withdrawn due to an intercurrent inflammatory illness and one was not postmenopausal by our criteria Ten obese subjects met our inclusion criteria and underwent skin swab collections and punch biopsy Twenty nonobese subjects were screened Two subjects refused to undergo punch biopsy one was withdrawn due to an intercurrent illness two with a BMI outside the required range one withdrew consent one was excluded with a history of keloid formation one with a low platelet count one with uncontrolled hypertension One nonobese subject who underwent skin punch biopsy was not included in the analysis because we were unable to find an age and ethnicitymatched obese subject These obese and agematched ethnicitymatched nonobese postmenopausal women completed all aspects of the study Fig a0 Six participants were European American and four were African Americans in each group Postmenopausal women were chosen to exclude effects of the menstrual cycle upon study end points and to exclude gender effectsBased on preliminary data from a previous study comparing skin from seven obese and six nonobese postmenopausal women there was a variation of in a set of RTPCR genes unpublished data Assuming the same variation and proportion of differentially expressed genes to be we calculated that a sample size of n subjects per group matched by age and ethnicity would provide power at a falsediscovery rate to detect the expected number of differentially expressed genes based on a threshold of twofold changesDesign and setting This was an label comparison of a group of postmenopausal obese women and postmenopausal nonobese women who were agematched ± a0years and racematched Screening comprised a complete history and physical examination and fasting blood testing for complete blood count sedimentation rate comprehensive chemistry panel lipid panel thyroid function tests hepatitis C antibody uric acid and haemoglobin A1C Observing Good Clinical Practice guidelines all participants read and signed an informed consent document approved by the Institutional Review Board and the Advisory Committee for Clinical and Translational Science at The Rockefeller University Protocol JWA0921Procedure methods Anthropometric measurements Body weight was measured daily to the nearest a0kg using a ScaleTronix scale Welch Allyn Skaneateles Falls NY with precision of ± a0kg Subjects were weighed in a hospital gown after an overnight fast and postvoiding Height was measured to the nearest a0cm at baseline with a Seca216 stadiometer Hamburg Germany in a0cm increments Body mass index BMI was calculated as kgm2 using the NIH Standard Metric BMI calculatorBlood collection and analysis Fasting blood samples were analysed in the Clinical Pathology Laboratory of the Memorial SloanKettering Cancer Center for complete blood count electrolytes glucose creatinine blood urea nitrogen liver function Creactive protein sedimentation rate and uric acid Research serum samples were drawn pre and post intervention aliquoted and stored at ˆ’ a0°C for subsequent analysisSkin swabbing Subjects were permitted to shower but did not wash the planned biopsy area over the midlower abdomen with soap for a0days before the biopsyFor microbiome analysis two areas of skin approximately — a0cm were swabbed using the eSwab collection and preservation system for aerobic anaerobic and fastidious bacteria Copan Diagnostics Marietta CA Swabs were labelled sealed separately in the provided tubes and immediately stored at ˆ’ a0°C For metabolome analysis two different areas of skin approximately — a0cm were swabbed using salinemoistened sterile cottontipped applicators The tips were cut sealed in separate sterile collection tubes and immediately stored at ˆ’ a0°CSkin microbiome The DNA extraction protocol was adapted from the Maxwell RSC Buccal Swab DNA kit Catalogue number AS1640 Promega Corporation Madison WI Briefly a0μl of lysis buffer and a0μl of Proteinase K was mixed and added to each swab tube Swab tubes were then incubated for a0min at C using a Thermo Fisher water bath removed from the tubes and fluid was transferred to well of the Maxwell RSC Cartridge The swab head was centrifuged using a ClickFit Microtube Cat V4741 and extracted fluid was added to the corresponding well of Maxwell Cartridge and eluted in a0μl of provided elution bufferScientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Consort flow chart of eligible subjectsExtracted DNA was taken forward to the Nextera Flex protocol by Illumina Briefly a0μl of extracted DNA was taken into library prep protocol and run with cycles of PCR Libraries were cleaned up with a left sided size selection using a bead ratio of 08x The right sided size selection was omitted Libraries were then quantified using a Thermo Fisher Qubit Fluorometer and an Advanced Analytical Fragment Analyzer Libraries were sequenced on an Illumina HiSeqPE — at the Weill Cornell Epigenomics CoreAll bioinformatic analysis was performed on Weill Cornell Medicine™s Athena compute cluster a highperformance grid compute system Secondary analysis was performed on a Linux and MacOS systems Unless otherwise noted programs were run with default settingsRaw sequence data were processed with AdapterRemoval v217 to remove low quality reads and reads with ambiguous bases15 Subsequently reads were aligned to the human genome hg38 including alternate contigs using Bowtie2 v230 fast preset16 Read pairs where one or both ends mapped to the human genome were separated from read pairs where neither mate mapped Read pairs where only one mate mapped were discarded Hereafter we refer to the read sets as human reads and nonhuman readsTaxonomic profiles were generated by processing nonhuman reads with KrakenUniq v032 with a database based on all draft and reference genomes in RefSeq Microbial bacteria fungi virus and archaea ca March KrakenUniq identifies kmers that are unique to taxa in a database Reads are broken into kmers and searched against this database Finally the taxonomic makeup of each sample was given by taking the proportion reads which were assigned to each clade KrakenUniq counts the number of unique marker kmers assigned to each taxon and we filtered taxa with fewer than unique markers17We performed differential abundance testing over microbial species using the ALDEx2 R package ALDEx2 performs variance stabilization read counts using a centred log ratio transformation that models samples as Scientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cDirichletMultinomial distributions over taxa then compares taxonomic abundances across groups18 Comparison of abundances across groups was done with a Wilcoxon rank sum test and Benjamini Hochberg Correction for multiple hypothesis testingDimensionality reduction of taxonomic profiles was performed with Principal Coordinates Analysis PCA based on a matrix of JensenShannon Divergences JSD between samples Analysis of intersample beta diversity was performed using the same matrix of JSD Intrasample alpha diversity was measured by finding Shannon™s Entropy of the taxonomic profile and by counting the total number of species identified in each sample richness Shannon™s entropy accounts for the relative size of each group in diversity estimation and is defined as H ˆ’ cid31 ai log2ai where ai is the relative abundance of taxa i in the sampleWe generated profiles of antimicrobial resistance genes using MegaRes v10119 To generate profiles from MegaRes we mapped nonhuman reads to the database using Bowtie2 v230 very sensitive presets Subsequently alignments were analysed using Resistome Analyzer commit 15a52dd and normalized by total reads per sample and gene length to give RPKMs MegaRes includes an ontology grouping resistance genes into gene classes AMR mechanisms and gene groupsSkin metabolome The skin was swabbed using two sterile salinemoistened culture swabs and immediately frozen at ˆ’ a0°C Swab heads were removed and placed in a0ml methanol water Following sonication a0min a0ml of isopropanol was added and the solution was spun at a0g for a0min The swab was removed and the samples were dried using a vacuum concentrator operating at a0°C Prior to UPLCMS analysis samples were reconstituted in a0μl of HPLCgrade water sonicated for a0min and transferred to vials for analysisA Waters 2777C sample manager Waters Corp Milford MA USA was used for sample handling This was equipped with a a0μl Hamilton syringe a a0μl loop used for fullloop injections of prepared sample and a 3drawer sample chamber maintained at a0°C with a constant flow of dry nitrogen gas to prevent the buildup of condensation The LC component was an ACQUITY UPLC Waters Corp Milford MA USA composed of a binary solvent manager and column heatercooler module Metabolic profiles were acquired using reversedphase chromatography Water and acetonitrile each supplemented with formic acid mobile phases A and B respectively were selected for the mobile phase A — a0mm HSS T3 column was used at a0°C with a mobile phase flow rate of a0mlmin This generated a maximum pressure of psi in a wateracetonitrile gradient After a a0min isocratic separation at initial conditions A a linear gradient elution A to A in a0min proceeded followed by a quicker gradient A to A in a0min to final conditions The mobile phase flow rate was simultaneously increased to a0mlmin in the latter stage to facilitate faster column washing The MS component comprised a Xevo G2S QToF MS Waters Corp Manchester UK coupled to the UPLC via a Zspray electrospray ionization ESI source The cone gas flow was set to a0lh to protect the cone from residue accumulation during operation Both positive and negative ion modes RPC and RPCˆ’ respectively were used Raw spectra were converted into mzML files using MSConvert20 and processed with XCMS in R21 Peak picking and peak grouping were performed using inhouse scripts in R and matrices were normalized using a median fold change approach Log transformation scaling and data analysis was performed in SIMCA Umetrics Umea SwedenSkin biopsy After the skin swabbing the abdominal site was cleansed with Chloraprep swabs chlorhexidine and isopropyl alcohol Becton Dickinson Canaan CT Using sterile technique local anaesthesia was induced by infiltration of the area with a0ml of lidocaine Hospira Inc Lake Forest IL mixed with a0ml sodium bicarbonate The skin biopsy was performed using a a0mm punch Miltex Instruments York PA Fat tissue was carefully removed from the skin core of the biopsy The dermis and epidermis were divided into two halves one half placed in a cryomold for OTC flash freezing Agar Scientific Essex UK and stored at ˆ’ a0°C and the other half was placed in RNAlater Stabilization Solution Thermo Fisher Scientific Fair Lawn NJ refrigerated for a0h and then frozen at ˆ’ a0°C The fat tissue was removed from the biopsy divided between RNAlater refrigerated for a0h then frozen at ˆ’ a0°C and a dry Sarstedt tube that was flash frozen in liquid nitrogen and placed in ˆ’ a0°C The biopsy site was sutured closed and a dry sterile dressing was applied Subjects were discharged and scheduled to return for suture removalGene‘array and quantitative real‘time PCR analysis RNA was extracted followed by hybridization to Affymetrix Human U133 Plus gene arrays Santa Clara CA or quantitative RTPCR as previously described2223All statistical analyses were carried out in R Limma Log 2transformed qRTPCR measurements hARP normalized and microarray expression values were assessed with a mixedeffect The fixed factors were condition obese vs nonobese race African American vs Caucasian with random intercept for each subject Quality control of microarray chips was carried out using standard QC metrics and R package microarray quality control Images were scrutinized for spatial artefacts using Harshlight24 Expression measures were obtained using the GCRMA algorithm25 A batch effect corresponding to the hybridization date was detected by PCA and adjusted using the ComBat function from the SVA package Probe sets with at least samples with expression values were kept for further analysis Fold changes for the comparisons of interest were estimated and hypothesis testing was conducted with contrasts under the general framework for linear models with the limma package P values from the moderated paired ttests were adjusted for multiple hypotheses using the Benjamini“Hochberg procedure Hierarchical clustering was performed with Euclidean distance and a McQuitty agglomeration scheme26Data was deposited into Gene Expression Omnibus GEO repository GSE151839All study methods and procedures were carried out in accordance with Good Clinical Practice Guidelines by trained practitioners The protocol and informed consent were evaluated and approved by the Institutional Scientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression between the skin of obese and nonobese subjects A Heat map of the most differentially expressed genes in the skin of obese and nonobese subjects with an FCH fold change fdr false discovery rate B PCA principal component analysis plot of differentially expressed genes in the skin of obese and nonobese subjects with an FCH fdr Review Board and the Advisory Committee for Clinical and Translational Science at Rockefeller University prior to initiation of the study and annually thereafter Protocol JWA0921ResultsThis study was performed in ten healthy obese and ten healthy nonobese postmenopausal women matched for age and ethnicity Obese subjects had a mean weight of a0kg BMI of a0kgm2 and waist circumference of a0cm Nonobese subjects had a mean weight of a0kg BMI of a0kgm2 and waist circumference of a0cm Supplemental Table a0S1 The skin thickness for subjects with obesity was not significantly different from that of nonobese subjects Supplemental Fig a0S1Gene expression analysis of the skin The most differentially expressed genes in the skin between obese and nonobese subjects are displayed in the heat map in Fig a02a Comparing gene expression in obese versus nonobese skin showed greater gene expression of S100A7A encoding a calcium binding protein involved in psoriasis and CORIN encoding a natriuretic peptide converting enzyme which is expressed in the dermis and is involved in specifying skin colour However the expression of CREB3LA encoding a cyclic AMP response element was lower in the skin of obese subjectsA PCA model constructed on of the most differentially expressed genes between obese and nonobese subjects showed partial separation of the groups This difference was seen in PC1 which accounted for of the variation in the included genes Fig a02bThe complete list of skin genes whose expression significantly differed between the two groups are shown with fold changes in Supplemental Tables a0S2 and S3 Again the gene expression of S100A7A was 344fold higher in the obese skin compared to the nonobese skin Similarly the expression of DEFB4A Defensin B4A which encodes an antimicrobial peptide part of the betadefensive system and SPRR2C which encodes a proline rich protein strongly induced during differentiation of human epidermal keratinocytes was also significantly higher in the obese skin being and 17fold higher respectively Genes with lower expression profiles in obese subjects than nonobese included AOP that encodes aquaporin involved in water channels present in the skin PROM1 prominin involved in cell differentiation and proliferation and Keratin and important for fibrogenesis in the epidermis Also of interest was the significantly higher expression 282fold of CFTR the cystic fibrosis transmembrane conductance receptor in nonobese subjects compared to the obese groupQTPCR analysis of genes selected from the total list of significantly differentially expressed genes in skin confirmed increased expression of the S100A 373fold DEFB4A defensin B4A 329fold and CORIN fold in the skin of obese subjects Fig a0 Significantly lower gene expression in the skin of obese subjects was found with CFTR 36fold PROM1 556fold and GABRP gamma aminobutyric acid receptor 29foldScientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cGene expression pathway analysis showed a broad downregulation of many pathways in obesity with only out of of the most highly differentially expressed pathways higher in the obese Supplemental Fig a0S2 The pathways most downregulated included cardiac beta adrenergic signalling which appears to function in skin cyclin dependent Kinase CDK5 a mutation which is important in melanoma formation and functions in skin healing and gonadotrophic releasing hormone GNRH signalling which has many extra pituitary functionsGene expression analysis in skin fat We next examined differences in gene expression between the groups of subjects in subdermal fat removed from immediately below the skin portion of the biopsy A heat map of gene expression shows a markedly different pattern between the two groups Fig a04a The expression of many of the genes upregulated in obese subdermal fat are involved with inflammation and immune function including platelet activating factor PLA2G7 ILIRN involved in IL1 activation SPPI a cytokine that can increase interferon gamma and IL12 activity and several serpins mediators involved in inflammation and immune functionThe PCA plot of genes whose expression differed significantly in subdermal fat of obese and nonobese subjects Fig a04b clearly shows separation between the two groups Most of the difference was seen in PC1 which includes of the genes whose expression was determinedSupplemental Tables a0S4 and S5 show a list of genes whose expression was relatively greater in the subdermal fat of obese subjects The expression of SPPI that encodes osteopontin which can act as a cytokine augmenting the action of interferon gamma and interleukin was approximately tenfold higher in the obese subjects EGFL6 expression which encodes an epidermal growth factor found to be enhanced in obesity and alters insulin action was increased by 85fold MMP9 which encodes metalloproteinase and ILTRN was increased by sevenfold in obesity Genes significantly downregulated in obese subdermal fat included SLC27A2 acetylCoAsynthase tenfold and C6complement fivefoldBy QTPCR in subdermal fat from obese subjects the increased expression of genes encoding proteins important in inflammation and immune function was confirmed Fig a0 This includes genes encoding proteins that determine accumulation of immune cells in adipose tissues such as CD52 the high affinity immunoglobulin gamma FC receptor FCGR1β CCL3 CZXCL8 interleukin and CLEC7A a pattern recognition receptor found in monocytes and other myeloid cells IL17F a member of the IL17 family also was specifically increased in subdermal fat from the obese as compared to nonobese individualsExpression pathway analysis Supplemental Fig a0S3 showed upregulation of several inflammatory immune pathways including the Thelper dendritic cell maturation and inflammatory signalling pathways further indicating profound effects of obesity on inflammation in subdermal fat Dramatically lower in the obese subdermal fat was the LXRRXR activation pathwayGene expression analysis by race Two subgroups were observed in the gene expression profiles of the skin based on the subject™s race Using selfreported data and skin colour as criteria the data from African Americans was analysed separately from the data from European Americans This analysis showed striking differences in this very small group of subjects A heatmap of the most differentially expressed genes in skin from obese subjects divided by ethincity is shown in Fig a06a No clear differences in gene expression in the skin by ethnicity were found in nonobese subjects In contrast gene expression clearly differed between obese African American and obese European American subjects Fig a06a and also is illustrated in the PCA plot Fig a06b with PC1 responsible for the greatest variation The pattern of differences between obese and non “obese skin is further illustrated in Fig a06cA list of genes whose expression significantly differed between the obese African Americans and the obese European Americans is shown in Supplemental Tables a0S6 and S7 The expression of SLC6A4 a serotonin transporter CORIN and COL8AI a collagen gene encoding a protein that is dysregulated in atopic eczema was higher in African Americans while the expression of SCCB2A2 the secretoglobin expressed in skin sweat glands and CFTR was expressed higher in European AmericansComparing the skin of obese African Americans to obese European Americans by QTPCR the former showed significantly lower expression of MYBCPI 516fold and PROM1 43fold and CFTR Fig a0 In contrast there was a small increase in the expression of CORIN 22fold a gene encoding the atrial naturalistic peptide converting enzyme and BMP2 fold also present in the skin compared to obese European AmericansPathway analysis found no racerelated differences in the nonobese samples However in the obese Supplemental Fig a0S4 there was markedly reduced expression of oestrogen mediated sphase entry pathway aryl hydrolase receptor signalling pathway and cell cycle regulation through cyclins pathways in the African Americans compared to the skin of the European American groupSubdermal fat in African Americans exhibited few differences from that found in European Americans Fig a08A Figure a08bc show the differences by weight and by ethnicity respectively illustrating the impact of obesity in the two racial groups The expression of numerous inflammatoryimmune genes was upregulated in the fat of both groups of obese subjects Fig a0 Tables a0S5 and S6 Microbiota analysis We next examined whether the microbiota collected from skin swabs around the biopsy site differed between the obese and nonobese subjects We generated taxonomic profiles for each sample using KrakenUniq and a database built from all available microbial species in RefSeq We measured the total number of AMR genes detected in each sample by aligning reads to MegaRESOverall differences between groups were minor No significant differences were noted in average taxonomic alpha diversity as measured by either Shannon™s entropy or richness between the groups A PCA plot of the taxonomic profiles showed slight separation between obese and lean samples and slightly higher beta diversity for obese samples however these differences were minor Fig a010a The number of antimicrobial resistant AMR Scientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RT PCR between the skin of obese and nonobese subjects LS means of gene expression by RTPCR showing significant differences as p p p Figure a0 Differences in gene expression between the subdermal fat of obese and nonobese subjects A Heat map of the most differentially expressed genes in subdermal fat of obese and nonobese subjects with an FCH fdr B PCA plot of differentially expressed genes in subdermal fat of obese and nonobese subjects with an FCH fdr Scientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RTPCR between the subdermal fat of obese and nonobese subjects LS means of gene expression by RTPCR showing significant differences as p p p Figure a0 Differences in gene expression between the skin of African American and European American subjects A Heat map of the most differentially expressed genes in skin of African American and European American subjects with an FCH fdr B PCA plot of differentially expressed genes in skin of obese and nonobese African American and European American subjects with an FCH Left side of plot indicates differences in gene expression by ethnicity in nonobese subjects Right side of plot indicates differences in gene expression by ethnicity in obese subjects C PCA plot of differentially expressed genes in skin of obese and nonobese subjects by ethnicity with an FCH Left side of plot indicates differences in gene expression between obese and nonobese African American subjects Right side of plot indicates differences in gene expression between obese and nonobese European American subjectsScientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RT PCR between the skin of obese and nonobese African American and European American subjects LS means of gene expression by RTPCR showing significant differences as p p p genes identified was higher on average from obese subjects but this difference did not reach significance p Wilcox test Fig a010bDifferentially abundant taxa At the given sample size n no taxa were identified as significantly differentially abundant after Benjamini“Hochberg correction Before correction five taxa were significantly differentially abundant at p Wilcox test These five taxa were Corynebacterium aurimucosum Corynebac‘terium jeikeium Corynebacterium urealyticum Streptococcus salivarius and Streptococcus sp A12 All five taxa were more abundant in samples from obese subjects on averageMetabolome analysis As highlighted by the PCA analysis no variation in the metabolites analysed by liquid chromatographymass spectrometry from the skin swabs was observed between the obese and nonobese individuals PCA Supplemental Fig a0S6 Similarly no ethnicityrelated metabolic variation was observed These results were further confirmed by the poor predictive ability of the orthogonal projection to latent structuresdiscriminant analysis OPLSDA models comparing the two different ethnic groupsDiscussionFew comprehensive reviews of skin changes occurring with obesity have been conducted despite over of the US population being obese27 A broad review of the physiologic and clinical consequences and associations was published by Hirt et a0al in The authors include a discussion of circulatory and lymphatic changes which may enhance the frequency and severity of skin ulceration and provide a comprehensive review of skin disorders that can be associated with obesity expanding on previous reviews29 and studies in rodents30In our study the thickness of

Thyroid_Cancer

"patients with differentiated thyroid cancer DTC tumor burden of persistent disease PD is avariable that could affect therapy efficiency Our aim was to assess its correlation with the American ThyroidAssociation ATA riskstratification system and its impact on response to initial therapy and outcomeMethods This retrospective cohort study included consecutive DTC patients referred for postoperativeradioiodine RAI treatment Patients were riskstratified using the ATA guidelines according to postoperativedata before RAI treatment Tumor burden of PD was classified into three categories ie very small small andlargevolume PD Very smallvolume PD was defined by the presence of abnormal foci on postRAI scintigraphywith SPECTCT or 18FDG PETCT without identifiable lesions on anatomic imaging Small and largevolume PDwere defined by lesions with a largest size or ‰¥ mm respectivelyResults PD was evidenced in patients Mean followup for patients with PD was ± years Thepercentage of largevolume PD increased with the ATA risk and in low intermediate and highriskpatients respectively p There was a significant trend for a decrease in excellent response rate from thevery small small to largevolume PD groups at “ months after initial therapy and respectively p and at last followup visit and respectively p On multivariate analysis age ‰¥ yearsdistant andor thyroid bed disease smallvolume or largevolume tumor burden and 18FDGpositive PD wereindependent risk factors for indeterminate or incomplete response at last followup visitConclusions The tumor burden of PD correlates with the ATA riskstratification affects the response to initialtherapy and is an independent predictor of residual disease after a mean 7yr followup This variable might betaken into account in addition to the postoperative ATA riskstratification to refine outcome prognostication afterinitial treatmentKeywords Differentiated thyroid cancer Tumor burden Riskstratification Radioiodine 18FDG PETCT Correspondence rciappuccinibaclesseunicancerfr1Department of Nuclear Medicine and Thyroid Unit Fran§ois Baclesse CancerCentre Avenue Gnral Harris F14000 Caen France2INSERM ANTICIPE Caen University Caen FranceFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cCiappuccini BMC Cancer Page of BackgroundIn patients with differentiated thyroid cancer DTCthe riskstratification system described in the American Thyroid Association ATA guidelines is auseful tool to predict the likelihood of postoperativepersistent disease PD the response to initial therapyie surgery ± radioiodine [RAI] treatment and thelongterm outcome [] Several features related to PDare likely to influence the response to treatment andthe longterm prognosis This includes the location ofPD neck lymphnodes [LN] or distant metastases the18FFluorodeoxyglucose 18FDGRAIavidity [] oravidity [] of PD the aggressiveness of pathological variants [] and the degree of celldifferentiation [] thepresence of molecular mutations BRAF TERTp []and the tumor doublingtime [] Alone or in combination with previous characteristics notably RAIaviditythe tumor burden of PD is another variable that canaffect treatment efficiency and prognosis This has beenshown in studiessometimes old and using lowresolution imaging methods focusing on patients withdistant metastases [ ] In the daily practice it is wellknown that microscopic RAIavid lesions are morelikely cured than macroscopic ones eg lung miliary vslung macronodules However no studies have specifiedthe prognostic role of tumor burden estimated usinghighresolution imaging techniques both in the settingof distant metastases and lymphnode diseaseThe aim of the study was to assess the correlationof PD tumor burden with the ATA riskstratification system and its impact on response toinitialtherapy and outcome We hypothesized thatpatients presenting postoperatively a low tumor burden of PD would have better response to initial therapy and better clinical outcomes than patients havinghigh tumor burdenMethodsPatientsThe records of consecutive patients with DTC referred to our institution for postoperative RAI treatment between January and February werereviewed For the purpose of the study patients wereriskstratified according to the ATA guidelinesbased on pathological and surgical data available aftertotalthyroidectomy and before postoperative RAItreatment postoperative risk stratification [] Dataavailable in the preoperative period such as imagingstudies showing distant metastases were also used toinform ATA risk stratification In contrast postoperative serum thyroglobulin Tg level was not used todrive RAI treatment in these patients managed before and no diagnostic RAI scintigraphy was performed before RAI treatmentrhTSHPostoperative RAI treatmentAll patients were administered an RAI regimen ± weeks after total thyroidectomy Patients were prepared after either thyroid hormone withdrawal THWinjections of recombinant humanor after two imthyrotropinThyrogen Genzyme CorpCambridge MA USA as previously described [] TSHlevel was measured the day of RAI treatment and was mUIl in all patients The RAI activity or GBq and the preparation modalities were decided byour multidisciplinary committee All patients underwenta postRAI scintigraphy combining wholebody scanWBS and neck and thorax single photon emissioncomputed tomography with computed tomographySPECTCT A complementary SPECTCT such as abdomen andor pelvis acquisition was performed in caseof equivocal or abnormal RAI foci on WBS Patientswere scanned two or file days following or GBqrespectively Initial therapy was defined as surgery iethyroidectomy ± LN dissection plus first RAI treatmentie postoperative RAI treatmentSerum Tg and antiTg antibodies TgAb assayBlood samples for stimulated serum Tg and TgAb measurements were collected immediately before the RAItreatment Serum Tg measurements were obtained withthe Roche Cobas Tg kit Roche Diagnostics Mannheim Germany with a lower detection limit of ngml and a functional sensitivity of ngml until October and with the Roche Elecsys Tg II kit Roche Diagnostics Mannheim Germany with a lower detectionlimit of ngml and a functional sensitivity of ngml thereafter TgAb was measured using quantitativeimmunoassay methods Roche Diagnostics MannheimGermany TgAb positivity was defined by the cutoffsprovided by the manufacturerPathologyPathological variants were defined according to the WorldHealth anization classification [] Poorly differentiated carcinoma widely invasive follicular carcinomaH¼rthle cell carcinoma and among PTC variants tall cellcolumnar cell diffuse sclerosing and solid variants wereconsidered as aggressive pathological subtypes [] Tumorextent was specified according to the TNM []Tumor burden of persistent diseaseAs previously described [] PD was defined as evidenceof tumor in the thyroid bed LN or distant metastasesafter completion ofinitial therapy Confirmation wasachieved either by pathology or by complementary imaging modalities neck ultrasound examination [US]postRAI scintigraphy 18FDG positron emission tomography [PETCT] CT scan or MRI and followup 0cCiappuccini BMC Cancer Page of The tumor burden of PD was classified into three categoriesie very small small and largevolume PDVery smallvolume PD was defined by the presence ofabnormal foci on posttherapeutic RAI scintigraphy withSPECTCT or 18FDG PETCT without identifiable lesions on anatomic imaging neck ultrasound CT scan orMRI Small or largevolume PD were defined by thepresence of metastatic lesions with a largest size or ‰¥ mm respectively regardless of RAI or 18FDGuptake Examples of patients with very small small orlargevolume PD are presented in Fig RAI and 18FDG uptake in persistent diseaseThe RAI or 18FDG uptake profile was defined at time ofPD diagnosis PD was considered RAIpositive RAI ifat least one metastatic lesion showed RAI uptake andRAInegative RAI otherwise Similarly PD was defined18FDGpositive 18FDG if at least one metastatic lesionpresented significant 18FDG uptake and 18FDGnegative18FDG otherwiseClinical outcome assessmentAs previously described [] clinical assessment ofpatients with a negative postRAI scintigraphy wasscheduled at three months with serum TSH Tg andTgAb measurements while on levothyroxine LT4treatment When the Tg level at three months was ngml in the absence of TgAb the disease status wasassessed at “ months by serum rhTSHstimulated Tgassay and neck US and in recent years by Tg II assayon LT4 and neck US If there was an excellent responselevel ngmlat “ months according to the ATA criteria iestimulatedTgor nonstimulatedTglevel ngml without TgAb and negative neck USpatients were followed up on an annual basis For anything other than an excellent response imaging modalities such as CT scan of the neck and thorax 18FDGPETCT or MRI were performed In case of a secondRAI regimen given “ months after the first RAI therapy for RAIavid PD postRAI scintigraphy with SPECTCT was also used to assess initial treatment responseResponses to initial therapy as assessed at “ monthsand status at lastvisit were categorized as excellent response indeterminate response biochemical incompleteresponse or structural incomplete response according tothe ATA guidelines []Data analysisQuantitative data are presented in mean ± standard deviation SD except for Tg levels which are presented inmedian range Patients™ characteristics were comparedusing Chisquare or Fisher™s exact test the Wilcoxontest or the KruskalWallis test as appropriate TheCochranArmitage trend test was used to examineproportions of excellent response over the differentsubgroups in the following order verysmall small andlargevolume PD The analysis of diseasespecific survival and progressionfree survival was performed usingthe Cox regression model The analysis of prognosticfactors was performed using logistic regression Statistical significance was defined as p All tests wereFig Examples of very small small and large tumor burden in patients with persistent disease PD On the left side a 43yearold female patientwith a 40mm PTC at lowrisk after initial surgery T2NxMx and very smallvolume PD ac posttherapeutic 131I WBS showed a solitary bonyfocus on the right hip a arrow Fused transaxial image of 131I SPECTCT b arrow confirmed the bony uptake and hybrid CT c arrow did notdisplay any bone abnormality On the middle part a 74yearold female patient with a 40mm PTC at lowrisk after initial surgery T2N0Mx andsmallvolume PD df posttherapeutic 131I WBS showed pulmonary metastases d red and black arrows Fused transaxial image e red arrowand hybrid CT scan f red arrow depicted RAIavid lung micronodules ef mm On the right side an 88yearold female patient with a 40mmPTC tall cell variant at highrisk after initial surgery T2N1bM1 and largevolume PD gi no abnormal RAI uptake on posttherapeutic 131I WBSwith SPECTCT whereas 18FDG PETCT showed pulmonary and mediastinal metastases g Maximum intensity image arrows Fused transaxialimage h arrow and hybrid CT scan i arrow showed high 18FDG uptake SUVmax by an 18mm lung nodule 0cCiappuccini BMC Cancer Page of twosided SAS statistical software SAS InstituteInc Cary NC USA was used for data analysisstratification Patients™ characteristics are reported inTable ResultsCharacteristics of patientsThe study group included papillary thyroid cancers PTC follicular thyroid cancers FTC and poorlydifferentiated thyroid cancers PDTCThere were women and men The mean agewas ± years Three hundred and seventytwo patients were prepared with rhTSH stimulation Eightytwopatients presented positive TgAb at the time of postoperative RAI treatment In the postoperative setting priorto RAI administration patients were at lowriskLR at intermediaterisk IR and athighrisk HRaccording to the ATA riskPersistent disease and tumor burdenOverall PD was detected in patientsTheir characteristics in terms of ATA risk RAI preparation modality PD sites and RAI or 18FDG uptake arepresented in Table Of patients had very smallvolumelargevolume smallvolume and PDFigure shows two points First the rate of PDincreased from in LR patients and in IR to in HR patients p Second the percentage of patients with largevolume PD increased with risk stratification from LRIR to HR patients and respectively p Table Characteristics of patients according to the ATA riskstratification system in the postoperative settingMean age ± SD yrsSex ratio FemaleMean tumor size ± SD mmHistologyPTCFTCPDTCAggressive pathological subtypesNoYesExtrathyroidal extensionMinimalGrossT status TNM T1a T1bT2T3a T3bT4a T4bN status TNM NxN0N1a N1bM status TNM M0M1Positive TgAb levelStimulated Tg level at RAI treatment rangeaaIn patients without positive TgAb levelLRn ± ± IRn ± ± “ “HRn ± ± “p 0cCiappuccini BMC Cancer Page of Table Characteristics of patients with persistent disease according to the tumor burdenVery smallvolumePD n SmallvolumePD n LargevolumePD n Postoperative ATA riskLRIRHRPreparation modalityTHWrhTSHPD siteLNLN DMDMTB diseaseTB disease DMRAI and 18FDG statusRAI18FDG or NPRAI18FDGRAIˆ’18FDGRAIˆ’18FDGRAIˆ’18FDG NPa21 RAI18FDG NP and one RAI18FDGb15 RAI18FDG NP and two RAI18FDGc10 RAI18FDG NP and six RAI18FDG 22a 17b 16c pFig Tumor burden in patients with persistent disease correlation to the ATA riskstratification system The figure first shows that the rateof PD increased from in LR patients in IR to in HR patients p Second the percentage of patients with largevolume PDincreased with risk stratification from LR IR to HR patients and respectively p 0cCiappuccini BMC Cancer Page of Table Characteristics of patients with persistent diseaseaccording to the ATA riskstratification systemLRn IRn HRn pPD tumor burdenVery smallvolume SmallvolumeLargevolume The distribution of very small small andlargevolume PD in LR IR and HR patients is presented in Table Outcome of patients with persistent diseaseTreatment modalities within the first year of management and during the remaining followup are detailed inTable Mean followup for patients with PD was ± years and was similar between the three groups of tumorburden p Of the patients with PD at “months after initial therapy had excellent response indeterminate response biochemical incomplete response and structuralincomplete response At last followup visit the figureswere and respectively The outcome in each of the tumor burden groupsis presented in Table There was a significant trend fora decrease in excellent response rate from the verysmall small to the largevolume PD groups at “months after initial therapy and respectivelyp and at last followup visit and respectively p Fig Among the patients died related to DTCduring followup Seven were in the largevolume PDgroup and one in the smallvolume PD group All hadstructural incomplete response at “ months after initial therapy with 18FDGpositive diseaseFigures and show diseasespecific survival DSSand progressionfree survivalPFS according to theATA riskstratification 18FDG status and tumor burdenSignificant differences in DSS were observed for bothATA riskstratification and 18FDG status but not fortumor burden Patients with 18FDGpositive disease hadshorter PFS Hazard Ratio 95CI “ thanthose with 18FDGnegative disease Also IR HazardRatio 95CI “ and HR patients HazardRatio 95CI “ had shorter PFS than LRpatients Finally patients with small Hazard Ratio 95CI “ and largevolume PD Hazard Ratio 95CI “ had shorter PFS than those withverysmall volume PDPrognostic factor analysis in patients with persistentdiseaseMultivariate analysis controlling for age sex postoperative ATA riskstratificationaggressive pathologicalTable Treatment modalities and outcome of patients with PD at “ months after initial therapy and at last followup visitaccording to tumor burdenVery smallvolumePD n “ months after initial therapySmallvolume PDn Largevolume PDn pVery smallvolumePD n At last followup visitSmallLargevolume PDvolume PDn n p a Treatment modalities at “ months after initial therapy treatments given within the first year of followup treatment modalities at last followup visittreatments given after the first year during followupb Local treatment of DM external radiation beam therapy surgery or radiofrequencyAbbreviations PD Persistent disease RAI Radioiodine DM Distant metastasesTreatment modalitiesaRAINeck surgeryNeck external radiationbeam therapyLocal treatment of DMbTyrosinekinase inhibitorsChemotherapyOutcomeExcellent response Indeterminate responseBiochemical incompleteresponseStructural incompleteresponse 0cCiappuccini BMC Cancer Page of Fig Excellent response rate according to tumor burden “ months after initial therapy a and at last followup visit b in patients withpersistent disease There is a significant trend for a decrease in excellent response rate from the very small small to the largevolume PD groupsat “ months after initial therapy and respectively p and at last followup visit and respectively p subtypes site of PD tumor burden of PD and RAI or18FDG uptake showed age ‰¥ years Odds ratio [OR] p distant andor thyroid bed disease OR p smallvolume OR p andlargevolume tumor burden OR p and18FDGpositive disease OR p to be independent risk factors for indeterminate biochemical orstructuralincomplete response at last followup visitTable DiscussionThis study confirms that the incidence of PD aftertotal thyroidectomy and postoperative RAI treatmentis limited in LR patients as compared to IR or HR patients Moreover it demonstrates thatthe tumor burden of PD is correlated to postoperativeriskstratification with very smallvolume lesions preferentially observed in LR patients and small and largevolume in IR or HR patients Most importantly tumorburden of PD is shown as an independent predictor ofresponse to initial therapy and to outcome These findings confirm that tumor burden of PD is a variablewhich might be taken into account to refine outcomeprognosticationTumor burden covers a large range of locoregionalandor distant metastases from a unique microscopic lesion to multiple macroscopic ones sometimes clinicallyevident Also tumor burden encompasses structural egvisible on conventionalfunctionalradiology andorlesions eg visible on RAI scintigraphy or 18FDG PETCT The diagnostic performances of imaging methodsand consequently the concept of tumor burden havedramatically evolved in the last decades The detectionof small LN disease has been improved by the combination of highresolution neck US postRAI SPECTCTand 18FDG PETCT imaging Regarding distant metastases although postRAI WBS still remains the referencefor detecting lung miliary disease the routine use ofdiagnostic CT scan and MRI now enables the detectionof infracentimetric lung bone or brain lesionsIn the past tumor burden of PD as a potential indicator of successful treatment and prognosis was assessedusing different approaches In a study on DTC patients with lung metastases diagnosed from to multivariate analysis showed that lung nodules visible on XRay vs those not visible RAIrefractory lunglesions and multiple metastatic sites were associatedwith poor survival [] In Gustave Roussy™s experienceoverall survival was reported in DTC patients withdistant metastases lung bone or other sites diagnosedbetween and [] Tumor extent was classifiedinto three categories according to both postRAI planarscintigraphy and Xrays Category consisted in lesionsvisible on postRAIscan but with normal Xraycategory in metastatic lesions cm on Xrays andcategory in lesions cm regardless of RAI avidityOverall metastases were RAIavid in of patientsmore frequently in patients years than 0cCiappuccini BMC Cancer Page of Fig Diseasespecific survival in the patients with PD according to ATA riskstratification a 18FDG status b and tumor burden cyears Multivariate analysis demonstrated that female sex young age years well differentiatedtumor RAI avidity and limited extent category wereindependent predictors ofrecentlyRobenshtok reported the outcome of patientssurvival Morewith RAIavid bone metastasis without structural correlate on CT scan or MRI among DTC patients withbone metastases between and [] After afollowup period of years all patients were alive nonehad evidence of structural bone metastases and none 0cCiappuccini BMC Cancer Page of Fig Progressionfree survival in the patients with PD according to ATA riskstratification a 18FDG status b and tumor burden chad experienced skeletalrelated events confirming theexcellent prognosis after RAI treatmentIn DTC patients with persistent nodal disease there isalso indirect evidence supporting that tumor burden affects treatment response and outcome In a recent retrospective study Lamartina reported the outcome of patients without distant metastases who underwenta first neck reoperation for nodal persistentrecurrentdisease [] Male sex aggressive histology and the presence of more than LN metastases at reoperation wereshown to be independent risk factors of secondary relapse following complete response achieved with first 0cCiappuccini BMC Cancer Page of Table Risk factors for indeterminate biochemical or structural incomplete response at last followup visitVariableAge years ‰¥ SexFemaleMaleInitial ATA riskstratificationLRIRHRAggressive histological subtypesNoYesSite of PDLN onlyDM andor TB disease with or without LNTumor burden of PDVery smallvolumeSmallvolume mmLargevolume ‰¥ mmRAI and 18FDG status of PDRAI18FDG or NPRAIˆ’18FDG or NPRAI or RAI18FDGPatients at risk nInitial modelOR CIp valueFinal modelOR CIp value““““““““““““““““reoperation Conversely the excellent outcome of microscopic nodal involvement detected on SPECTCT at RAIablation was demonstrated by a study from Schmidt [] Of patients with RAIavid LN metastasesat ablation only three still showed nodes with significantuptake on a diagnostic RAI scintigraphy at monthsThe LN successfully treated by RAI were less than cmexcept in one patient whereas those still visible at months were above cm confirming that RAI is highlymore efficientin microscopic than in macroscopiclesionsIn the present study multivariate analysis showed thatage over years distant andor thyroid bed diseasesmall or largevolume tumor burden and 18FDGpositive disease were independent risk factors for indeterminate or incomplete response at last followup visit Incontrast ATA risk stratification and aggressive pathological subtypes did not emerge from multivariate analysis possibly because of the number of patients thenumber of variables tested and confounding variablesHoweverand progressionfreethe diseasespecificsurvival curves confirmed the high prognostic value ofthe ATA riskstratification In practice data supportsthat LR patients have a better outcome than the IR andHR groups not only because PD is uncommon in thosepatients but also because the excellent response rate ishigher in very smallvolume than in small or largevolume lesions We suggest that tumor burden usingthis threeclass discrimination could be implemented inthe assessment of patients with structural incomplete response to help refining the risk prediction This variablecould also be incorporated with the other risk predictorssuch as RAI or 18FDG uptake molecular profile tumorhistology degree of cell differentiation and Tg level andtumor volume doubling time to further improve riskestimatesAlthough retrospective the present study presents several strengths including the large cohort of consecutivepatients and the significant followup Patients diagnosedbetween and were uniformly evaluated usingmodern imaging studiesincluding postRAI scintigraphy with neck and thorax SPECTCT [] and 18FDG 0cCiappuccini BMC Cancer Page of PETCT with a dedicated headandneck acquisition [] Tumor burden was assessed combining functionaland anatomic imaging as adapted from previous papersof our group [ ] One can argue that it would havebeen even more pertinent to assess tumor burden withquantitative values rather than with a threeclass discrimination ie very small small and largevolumeActually a quantitative volumetric assessment is notfeasible because of the RAIavid nodal or metastatic lesions without structural correlate Also a quantitativeassessment based on RAI or 18FDG uptake is notpossible either because of RAIrefractory or nonhypermetabolic lesions Nevertheless we believe that ourdefinition is simple to use in routine practice and easilyreproducibleConclusionsThe tumor burden of PD correlates with the postoperative ATA riskstratification affects the response to initialtherapy and is an independent predictor of residual disease after a mean 7yr followup This variable might betaken into account in addition to the postoperative ATAriskstratification to refine outcome prognostication afterinitial treatmentAbbreviationsATA American thyroid association DM Distant metastasesDTC Differentiated thyroid cancer 18FDG 18FfluorodeoxyglucoseFTC Follicular thyroid cancers HR Highrisk IR Intermediaterisk LN Lymphnodes LR Lowrisk MRI Magnetic resonance imaging NP Not performedOR Odds ratio PD Persistent disease PDTC Poorlydifferentiated thyroidcancers PETCT Positron emission tomography with computed tomographyPTC Papillary thyroid cancers RAI Radioiodine rhTSH Recombinant humanthyrotropin SPECTCT Single photon emission computed tomography withcomputed tomography Tg Thyroglobulin TgAb AntiTg antibodiesTHW Thyroid hormone withdrawal TB Thyroid bed US Ultrasoundexamination WBS Wholebody scanAcknowledgmentsWe are indebted to Gee Knight for the reviewing of the manuscriptAuthors™ contributionsRC and SB conceived the study and its design RC ALC VSR CL VLH DV EBand SB performed data acquisition and analysis NH performed the statisticalanalysis RC and SB drafted the manuscript All authors read and approvedthe final manuscriptFundingNot applicableAvailability of data and materialsThe datasets used and analysed during the current study are available fromthe corresponding author on reasonable requestEthics approval and consent to participateAll procedures were in accordance with the ethical standards of theinstitutional committee and with the Helsinki declaration and its lateramendments Baclesse Cancer Centre has licensed from the FrenchCommission for Data Protection and Liberties CNIL MR004 ref v0This study was approved by the institutional review board of Baclesse hospital and all subjects gave written informed consentConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nuclear Medicine and Thyroid Unit Fran§ois Baclesse CancerCentre Avenue Gnral Harris F14000 Caen France 2INSERM ANTICIPE Caen University Caen France 3CETAPS EA Rouen UniversityRouen France 4Department of Head and Neck Surgery Fran§ois BaclesseCancer Centre Caen France 5Department of Pathology Fran§ois BaclesseCancer Centre Caen France 6Department of Oncology Fran§ois BaclesseCancer Centre Caen France 7Department of Cancer Biology and GeneticsFran§ois Baclesse Cancer Centre Caen France 8Department of Head andNeck Surgery University Hospital Caen FranceReceived February Accepted August ReferencesHaugen BR Alexander EK Bible KC Doherty GM Mandel SJ Nikiforov YEPacini F Randolph GW Sawka AM Schlumberger M Schuff KG Sherman SISosa JA Steward DL Tuttle RM Wartofsky L American ThyroidAssociation management guidelines for adult patients with thyroid nodulesand differentiated thyroid Cancer the American Thyroid Associationguidelines task force on thyroid nodules and differentiated thyroid CancerThyroid “Durante C Haddy N Baudin E Leboulleux S Hartl D Travagli JP Caillou BRicard M Lumbroso JD De Vathaire F Schlumberger M Longtermoutcome of patients with distant metastases from papillary andfollicular thyroid carcinoma benefits and limits of radioiodine therapy J ClinEndocrinol Metab “Robbins RJ Wan Q Grewal RK Reibke R Gonen M Strauss HW Tuttle RMDrucker W Larson SM Realtime prognosis for metastatic 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