Patent Application: US-201515509690-A

Abstract:
the disclosure describes a method for diagnosing lung cancer in a subject by detecting in a biological sample obtained from that patient a mirna signature , the presence of which provides an earlier indication of cancer than alternative art - recognized methods , including , but not limited to , low - dose computed tomography .

Description:
the disclosure provides a “ mir - test ” for the early detection of lung cancer . an exemplary embodiment of the mir - test comprises a method of diagnosing a subject with lung cancer , including asymptomatic and / or early stage lung cancer , comprising ( a ) obtaining a biological sample from the subject ; ( b ) detecting in the biological sample a decrease in the abundance of each of hsa - mir - 92a - 3p , hsa - mir - 30b - 5p , hsa - mir - 191 - 5p , hsa - mir - 484 , hsa - mir - 328 - 3p , hsa - mir - 30c - 5p , hsa - mir - 374a - 5p , hsa - mir - 7d - 5p , and hsa - mir - 331 - 3p compared to a control abundance value corresponding to each of hsa - mir - 92a - 3p , hsa - mir - 30b - 5p , hsa - mir - 191 - 5p , hsa - mir - 484 , hsa - mir - 328 - 3p , hsa - mir - 30c - 5p , hsa - mir - 374a - 5p , hsa - mir - 7d - 5p , and hsa - mir - 331 - 3p ; and ( c ) detecting in the biological sample an increase in the abundance of each of hsa - mir - 29a - 3p , hsa - mir - 148a - 3p , hsa - mir - 223 - 3p , and hsa - mir - 140 - 5p compared to a control abundance value corresponding to each of hsa - mir - 29a - 3p , hsa - mir - 148a - 3p , hsa - mir - 223 - 3p , and hsa - mir - 140 - 5p ; wherein the detection of a decreased abundance in each of the mirna in ( b ) and an increased abundance of each of the mirna in ( c ) indicates the development of lung cancer in the subject , thereby diagnosing the subject with lung cancer . in support of the mir - test of the disclosure , data are provided for the validation of this method of diagnosing lung cancer , including asymptomatic and / or early stage lung cancer , in a large cohort of high - risk individuals . in this study , the test reached a sensitivity of 86 %, when the high and intermediate risk classes were grouped together , while 53 % of individuals were positioned in the low - mirna risk category . the npv of the mir - test was & gt ; 99 %, thus , low - risk individuals can safely avoid subsequent ldct screening . the high sensitivity and npv obtained with the mir - test are comparable to those observed with ldct alone , which indicates that the mir - test could substitute the ldct as a first - line screening tool . conversely , the lower specificity of the test compared to ldct , would not affect the overall screening result , as cases with a positive mir - test result would be required to undergo ldct to confirm diagnosis and localize neoplastic lesions for subsequent surgery . with respect to the false negative ( i . e . mir - test - negative lung cancer patients ) and the false positive ( mir - test - positive individuals , but negative upon ldct ) results , only 2 out of 15 deaths ( validation and clinical sets ) were false negatives . the death rate per 1000 patients / year was 0 , 51 , and 71 , in the low , intermediate and high - risk categories , respectively . while the relatively low number of deaths in our cohort limits the statistical power of these results , it is plausible that tumors missed by the mir - test may be rather indolent or even represent overdiagnosis by ldct . notably , 5 of 5 patients with benign tumors , who had a positive ldct result , were mir - test - negative . similarly , most individuals with nmd ( 72 out of 83 ; 87 %) were mir - test - negative . this latter result is relevant since in ldct screening trials there is a high rate of false - positive findings , up to 28 %. this complicates the interpretation of ldct results and ensuing decisions about the screening time interval : a difficulty that might be alleviated by a first - line screening test , such as the mir - test , that significantly reduces unnecessary ldcts for individuals without lung cancer . with respect to mir - test false positives , these events may not necessarily represent an intrinsic limitation of the test , because it is possible that the blood test can anticipate the ldct diagnosis . the mir - test possesses the characteristics of accuracy and robustness required to be introduced as a first - line tool in lung cancer screening programs . if implemented , the mir - test would result in a reduction in the number of ldcts by more than 50 %, while retaining the diagnostic sensitivity of ldct . four independent cohorts of patients and individuals were employed in the study : i ) calibration set , ii ) validation set , iii ) specificity set , iv ) clinical set ( see fig1 ): twenty - four individuals were selected from the cosmos trial ( 12 with screen - detected lung cancer and 12 lung cancer free ; fig1 , table 2 ) and used to refine the mirna signature ( i . e . the mir - test ). the 12 lung cancer patients were previously screened to derive the 34 - mirna signature ( see , vickers k c , palmisano b t , shoucri b m , et al . nat cell biol 2011 ; 13 ( 4 ): 423 - 33 ). the mir - test was validated in an independent set of 1008 individuals enrolled in the cosmos study including 36 patients with ldct detected lung cancer and 972 individuals without lung cancer , randomly selected from a consecutive cohort from march 2011 to march 2012 . a third cohort of 83 patients was used for further validation of the mir - test . these individuals were selected from cosmos study participants and were not included in any of the other sets used in this study . this cohort was composed of : i ) 38 individuals with ct - detected solitary pulmonary nodules stable in size at 5 years of follow - up ; ii ) 16 patients with chronic obstructive pulmonary disease ; iii ) 24 individuals with pneumonia ; iv ) and 5 with operated benign lung tumor . importantly , none of these individuals developed lung cancer during a & gt ; 5 - year follow - up period by ldct . a fourth independent cohort of 74 patients diagnosed with lung cancer outside of the cosmos trial was used . these patients underwent surgery at the european institute of oncology from november 2005 to january 2008 . information about the clinical and pathological characteristics for all individuals and patients screened by the mir - test is reported in table 1 and table 2 . the mean ± standard deviation ( sd ) is reported for age ( years ) and smoking status ( pack - years ), and the median , interquartile ( q1 ; q3 ) and overall range ( min - max ) is reported for age . the information on pack - years was available for nearly all individuals except for : 25 patients ( a ) and 36 patients ( h ) ( ac , lung adenocarcinoma ; scc , lung squamous cell carcinoma ; sclc , small cell lung cancer ; lcc , large cell lung cancer ; copd , chronic obstructive pulmonary disease ; nod , stable solitary pulmonary nodules ; pn , pneumonia ; benign , patients operated with benign pulmonary nodules ( surgery false positives )). tumor stage was defined based on the tnm classification of malignant tumors published by the international union against cancer ( uicc ), 7th edition . percentages could not add up to 100 due to rounding . tumor stage at the time of diagnosis was determined according to guidelines of the american joint committee on cancer ( http :// www . cancerstaging . org /). informed consent was obtained from all participants . patients and individuals of the calibration , validation and specificity sets were all enrolled in the cosmos study , a screening program for the anticipation of lung cancer diagnosis in high - risk individuals . all enrolled individuals were smokers or former smokers with a smoking exposure of more than 20 pack - years , aged over 50 . for the experiments shown in fig3 , sera were collected before and after surgery from a group of lung cancer patients with stage i disease that did not receive chemotherapy . blood samples ( 10 ml ) were collected by standard phlebotomy before any analysis or instrumental procedure . the first 3 ml of blood were not used for serum preparation to prevent contamination by skin . serum was prepared by collecting blood in tubes with clot activator ( s - monovette 7 . 5 ml , ref01 . 1601 — sarstedt ), left at room temperature for 3 hours to clot , then spun at 3000 rpm ( 1000 g , megafuge 2 . 0 heraeus ) for 10 minutes at rt . the serum was removed immediately after centrifugation , leaving a 0 . 5 cm leftover to avoid disturbing the serum - clot interface . serum was then aliquoted in barcoded cryotubes and snap frozen in dry ice . aliquots were stored in a dedicated − 80 ° c . freezer . total rna purification , including mirnas , was based on lysis with guanidinium thiocyanate - phenol - chloroform extraction ( trizol - ls , applied biosystem ) and spin column - based total rna purification ( mirneasy mini kit , qiagen ). briefly , 0 . 3 ml of serum were mixed with trizol - ls in volumetric ratios of 3 : 1 for lysis . after denaturation , a spike of synthetic mirna ( 5 × 10 8 copies of mir - 34a ) was added to the solution to monitor the extraction efficiency . a volume of 0 . 24 ml of chloroform was added to the solution , which was then centrifuged for 15 min at 11 , 800 g at 4 ° c . a fixed amount ( 0 . 35 ml ; around 70 % of the final volume of the sample ) of aqueous phase was recovered . this choice was preferred to limit contamination from the interphase . the subsequent steps were performed in automation through a qiacube machine ( qiagen ), according to the “ mirneasy mini ” standard protocol . rna was eluted in 30 ml and stored at − 80 ° c . until further analysis . reverse transcription ( rt ) and pre - amplification reaction ( pre - amp ) protocols were optimized by using a microlab star liquid handling . rt was performed from a fixed volume ( 3 μl ) of total rna with taqman microrna reverse transcription kit and a custom pool of human mirna - specific stem - loop primers ( applied biosystem ) according to the manufacturer &# 39 ; s instruction , in a 2720 thermo cycler ( applied biosystem ). rt product was then diluted 1 : 2 with water ; 5 μl of diluted rt product were pre - amplified ( 12 cycles of pcr ) with taqman preamp master mix ( 2 ×) and megaplex preamp primers human poola ( applied biosystem ), according to the manufacturer &# 39 ; s instruction . the pre - amplification reaction was diluted 1 : 4 with tris - edta buffer 0 . 1 ×. next , 6 μl of diluted pre - amplification reaction were combined with 46 . 5 μl of water and with 52 . 5 μl of taqman universal master mix ii ( applied biosystem ). the final solution ( 105 μl ) was loaded into one lane of a custom taqman ® low density array microrna custom panel ( applied biosystem ). qrt - pcr was carried out on an applied biosystems 7900ht thermocycler using the manufacturer &# 39 ; s recommended cycling conditions . a hamilton microlab ® star liquid handling workstation was used to automate most of the described processes . data were exported into text format for further analysis . if qrt - pcr amplification curves presented a “ tholdfail ” error flag ( i . e . the automatic thresholding algorithm failed ), data were discarded . subsequently , raw ct values were normalized . to run the mir - test , data were normalized using mean cts ( hk - mean ) of six “ housekeeping ” mirnas genes ( mir - 197 , mir - 19b , mir - 24 , mir - 146 , mir - 15b , mir - 19a ) using the following procedure : a scaling factor ( sf ) was calculated for each sample by subtracting the “ hk - mean ” to a constant value ( k = 21 . 646 ). data were then normalized by these sfs to eliminate technical fluctuations , using the formula : if ct raw was greater than 30 . 01 or ‘ undetermined ’ by qrt - pcr , data was set to 30 . 01 and normalization skipped . this normalization strategy assures an independency from the original set of samples used to train the mir - test , and allow new samples classification without the need of an internal reference . the calibration set was used to : i ) refine diagonal linear discriminant analysis ( dlda ) weight coefficients ( w i ) of the mirna signature by using dlda as class metric ( brb - arraytools version 4 . 3 . 0 — beta_2 release ); ii ) reduce the number of mirnas in the signature . the leave - one - out ( loo ) cross - validation procedure was used to cross - validate the classifier and estimate sensitivity and specificity in the calibration set . parametric t - test set at 0 . 05 significance level was used for feature selection . statistical significance of the performance of the classifier was assessed by performing 1000 permutations of class labels . samples were then classified as positive or negative if the absolute value of the inner sum of weighted expressions ( ct norm * w i ) was greater than the absolute value of the threshold . since both weights and threshold are negative , the risk score was calculated as : rs =( σ i w i x i − thres ), for i = 1 . . . 13 ( 13 - mirna signature ) ( 2 ) where thres =− 261 . 779 , w i is the weight coefficient of the i th mirna ( table 3 ), and x i is the expression value ( ct ) of the i th mirna . all analyses were automated using a custom r script developed in - house using the r statistical software version 2 . 14 . 1 . the script allows to read the information content of the output documents from the applied biosystems viia 7 ™ and provides mir - test risk scores automatically . the calibration set was used to refine a 34 - mirna signature ( see , bianchi f , nicassio f , marzi m , et al . embo molecular medicine 2011 ; 3 ( 8 ): 495 - 503 ) by employing an optimized serum mirna detection protocol . briefly , a pre - amplification step ( preamp ) that improved the detection of circulating mirnas [˜ 23 versus ˜ 31 cycle threshold ( ct ), on average ] was added , and all steps for purification and sample preparation ( including preamp ) were automated . this procedure minimized technical variability that resulted in a lower number of mirnas in the signature from 34 to 13 , while preserving the performance of the original model ( tables 3 and 4 ; fig6 ). the 13 - mirna signature ( mir - test ) displayed a ± 5 fluctuation of mir - test scores when repeated measurements of the same sample were performed ( fig7 ). based upon these results , three categories of risk ( i . e ., high , intermediate and low ) were defined as follows : high risk scores correspond to values ≧ 5 ; intermediate risk scores correspond to values & lt ; 5 and ≧− 5 ; low risk scores correspond to values & lt ;− 5 . table 3 reports the mirna assay , accession numbers ( acc . ), sequence , and mirbase nomenclature ( release 20 ) for the 13 mirnas that comprise the mir - test . fold - change ( fold ) and p - values ( parametric t - test ) refer to the expression of mirnas in the 12 tumor sera versus the 12 normal sera of the calibration set . w i is the weight coefficient computed by diagonal linear discriminant analysis ( dlda ) and used in the mir - test to compute risk scores . the sensitivity ( sen ) and specificity ( spe ) of the original 34 - mirna signature and the reduced 13 - mirna signature ( the mir - test ) in the calibration set is depicted in table 4 . sensitivity and specificity are based on cross - validation results of the diagonal linear discriminant analysis ( dlda ) classifier ( see example 1 ). a risk score of 0 was used to compute sensitivity and specificity . a multi - tiered study was designed on high - risk individuals ( heavy smokers , aged & gt ; 50 ) enrolled in the lung cancer screening trial cosmos , and on lung cancer patients diagnosed outside of screening ( fig1 ). in an initial step , the original 34 - mirna signature was refined ( see , bianchi et al ), taking into consideration a number of technological improvements ( see example 1 ). this refinement allowed a reduction of the signature to 13 mirnas ( henceforth , the mir - test , table 2 and 3 ), which maintained the same performance as the original 34 - mirna signature ( table 4 , and fig6 ). the reduction of the signature is advantageous in terms of translation into clinical practice , as it reduces the costs and complexity of the test . the mir - test was then validated in an independent “ validation set ” of 1008 subjects enrolled in the cosmos trial ( fig1 , table 1 ). in this set , the test displayed an auc ( area under curve ) of 0 . 85 and an accuracy ( acc ), sensitivity ( se ) and specificity ( sp ) of 75 %, 78 % and 75 %, respectively , when a risk score of 0 was used as cutoff ( fig2 a , table 5 ). next , a simulation of a “ clinical ” setting was attempted , in which the mirna - test would be used as a triage screening to identify individuals who should subsequently undergo ldct . the validation set was stratified into three risk categories : high , intermediate , and low ( see example 1 ). by grouping together the high and intermediate risk categories , the sensitivity of the test increased to 86 % ( 31 of 36 tumors , table 5 ). analysis of individuals in the low risk category ( 533 of 1008 ( 53 %) including 5 tumors ; table 5 ), who would not be required to undergo ldct , revealed a negative predictive value ( npv ) of & gt ; 99 % for the mir - test . the fact that 5 of 36 ldct - detected tumors were classified as low risk ( false negatives ) by the mir - test could reflect an intrinsic limitation of the test in its present form . however , in the case of the false negatives , we did not observe any death event out of the 15 deaths registered in all individuals screened ( validation and clinical sets ). in addition , the death rate per 1000 patients / year was 0 , 51 , and 71 , in the low , intermediate and high - risk categories , respectively . while the relatively low number of deaths in our cohort limits the statistical power of these results , it is plausible that tumors missed by the mir - test may be rather indolent or even represent overdiagnosis by ldct . notably , however , none of the individuals in the validation set who were assigned a low risk score , either in the two - or three - category stratification , died of lung cancer in the follow - up period (& gt ; 30 months ) ( table 5 ). table 5 shows the performance of the mir - test in various tests . the number of individuals assigned to difference mir - test categories is reported . in brackets , percentage out of total . tumor stage was defined based on the tnm classification of malignant tumors published by the international union against cancer ( uicc ), 7 th edition . § mir - test performance using two - category stratification pos : ≧ 0 ; neg : & lt ; 0 . in validation set , sensitivity ( se ) was 78 % and specificity ( sp ) was 75 %. in specificity set , sp was 87 %. in clinical set , se was 70 %. * mir - test performance using three - category stratification : high : ≧ 5 ; intermediate : & lt ; 5 and ≧− 5 ; low : & lt ;− 5 . example 3 : analysis of the performance of the mir - test in various clinically relevant settings a series of experiments to analyze the performance of the mir - test in various clinically relevant settings was performed next . to assess the ability of the mir - test to distinguish between non - malignant lung disease ( nmd ) and lung cancer , a “ specificity set ” was assembled selecting individuals from the cosmos study ( fig1 , table 1 ) suffering from chronic obstructive pulmonary disease ( copd ), benign pulmonary nodules or pneumonia ( see methods ). in this set , 11 out of 83 ( 13 %) individuals were assigned a positive score by the mir - test when the two - category stratification was applied ( fig2 b , left panel ; table 5 ). of these , only three ( 4 %) were classified as high - risk , when the three - category stratification was applied ( table 5 ). importantly , 5 of 5 patients operated with a benign tumor ( surgery false positives ), were assigned a negative score by the mir - test ( fig2 b ). thus , the mir - test displayed a high specificity ( 87 %) in a set of individuals with nmd , further corroborating its reliability . the mir - test was then applied to a third independent set , the “ clinical set ”, composed of patients who were diagnosed with lung tumors outside of the cosmos trial ( fig1 , see example 1 ). this analysis allowed to evaluate the performance of the test in an unselected population harboring more advanced lung cancers ( stage ii - iii ), which are normally under - represented among screen - detected tumors ( table 1 ). the performance of the mir - test in the clinical set was comparable to that in the validation set ( se , 70 %; table 5 ). moreover , no major differences in performance were observed among the different tumor stages ( stage i , se 69 %; stage ii - iii , se 72 %; table 5 ) and subtypes ( fig2 b ) in the clinical set . in addition , in adenocarcinoma patients from the clinical set , no significant differences were observed among the risk scores of smokers , ex - smokers (& gt ; 5 years ) and never smokers ( p = 0 . 78 ; fig2 c ). finally , serum samples of a group of stage i , non - small cell lung cancer ( nsclc ) patients before and after surgery were analyzed , to evaluate mir - test tumor specificity ( see example 1 ). at one month post - surgery , there was no significant overall decrease in the mir - test risk scores ( p = 0 . 39 ; fig3 ). in some patients the risk increased in the absence of any residual disease , possible to long - term stability of serum mirnas and / or a release of mirnas during surgery . notably , at 5 months post - surgery there was a significant decrease in most patients ( p = 0 . 017 , fig3 ). in two patients , whose sera were available at 12 months post - surgery , the mir - test risk score continued to decrease with respect to the 5 months risk score and yielded a negative test result in both cases ( fig3 ). example 4 : origin of circulating mir - test mirnas in lung cancer patients an interesting biological question pertains to the origin of circulating mirnas and of their fluctuations in cancer patients . mirna might be present in the serum due to their passive release from apoptotic cells , or active release from in microvesicles or in complex with mirna - binding proteins , which protect them from degradation . the expression of circulating mirnas in the nci - 60 tumor cells dataset was analyzed . while such an approach has obvious caveats ( e . g ., expressed mirnas are not necessarily secreted , and even if they are , their levels of expression might not reflect their levels of secretion ), it was a feasible initial step which could direct further analyses . initially , an unsupervised clustering analysis of all mirnas that can be reliably detected in serum ( 147 - mirna , see bianchi et al .) was performed . this yielded three main groups : i ) mirnas preferentially expressed in lines of hematopoietic origin , which we named the “ inflammatory - like ” cluster ; ii ) mirnas preferentially expressed in lines of epithelial origin , which we named the “ epithelial - like ” cluster ; iii ) mirnas without a clear differential pattern of expression ( fig4 a ). when the analysis was restricted to lung cancer and leukemic cell lines , the three - cluster structure was retained and more evident , regardless of whether the dataset was interrogated with the 147 - mirna signature ( fig4 a , right panel ) or with the 34 - and 13 - mirna signatures ( fig4 b ). statistical analyses confirmed a significant regulation of several mirnas belonging to the 34 - and 13 - mirna signatures in lung cancer or leukemic cell lines ( 16 and 8 of the 34 - and 13 - mirna signatures , respectively ; table 6 ). the above findings suggest a dual origin for the mirnas of our signatures . thus , the impact of the two components on the performance of the signature was investigated . first , it was established that both components are needed to maintain a good compromise between sensitivity and specificity ( table 7 ). next , the levels of the epithelial - and inflammatory - like mirnas , present in our signatures , in sera from individuals in the validation set were analyzed . the “ inflammatory - like ” mirnas displayed a quasi - stereotypical behavior , with 5 of 6 mirnas showing an increase in cancer patients vs . healthy individuals ( fig4 c ). the “ epithelial - like ” cluster , conversely , displayed a more heterogeneous behavior ( fig4 c ). finally , the expression of the epithelial - and inflammatory - like mirnas in mir - test - positive individuals in the validation and specificity sets ( fig4 d ) was analyzed . this analysis unveiled two interesting features : i ) a substantial fraction of the “ inflammatory - like ” mirnas were increased in the serum of nmd patients ( false positives ), similarly to lung cancer patients , which is consistent with the presence of chronic or severe lung inflammation ( fig4 d ); ii ) in the “ epithelial - like ” cluster , we found that mir - 29a was significantly increased in patients with lung cancer ( true positives ), but not in patients with nmd , relative to healthy individuals ( p = 0 . 008 ; fig4 d ), thereby representing a true tumor - specific mirna . accordingly , increased serum quantities of mir - 29a correlated with an adverse prognosis ( p = 0 . 025 ; fig5 a ). in addition , patients with benign tumors ( surgery false positives ) had lower amounts of mir - 29a ( p = 0 . 003 ) in the serum compared to lung cancer patients ( fig5 b ). significantly regulated mirnas of the 34 - mirna ( or 13 - mirna ) signature in the nci - 60 cell line panel , restricted to lung cancer ( lc ) and leukemic cell lines ( le ) are shown in table 6 . gene expression data were centered on their means ( mirna id , microrna osu v3 chip relative pre - mirna id ; accession , mirbase accession number ; mirna , mature mirna name ; in bold , mature mirnas ( n = 16 ) belonging to the original 34 - mirna signature ; mir - 32 was excluded from the analysis because of its presence in both clusters ; cluster type , identification of mirna based on clustering analysis as described in the main text ). p - values were calculated by the two - tailed t - test . the sensitivity ( sen ) and specificity ( spe ) of the complete 13 - mirna mir - test , and the mir - test without the inflammatory - like ( epithelial - like ) or epithelial - like ( inflam - like ) components , in the validation set ( n = 1008 ) is shown in table 7 . a risk score of 0 was used as cutoff to assign positive and negative test results . the details of one or more embodiments of the disclosure are set forth in the accompanying description above . any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present methods and materials now described . other features , objects , and advantages of the invention will be apparent from the description and from the claims . in the specification and the appended claims , the singular forms include plural referents unless the context clearly dictates otherwise . unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . all patents and publications cited in this specification are incorporated by reference . the foregoing description has been presented only for the purposes of illustration and is not intended to limit the invention to the precise form disclosed , but by the claims appended hereto . 1 . siegel r , naishadham d , jemal a . cancer statistics , 2013 . ca : a cancer journal for clinicians 2013 ; 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