Under stressful conditions, p53 tightly regulates cell growth by promoting apoptosis and DNA repair. When p53 becomes mutated, it loses its function, resulting in abnormal cell proliferation and tumor progression. Depending on the p53 mutation, it has been shown to form aggregates leading to negative gain of function of the protein. p53 mutant associated aggregation has been observed in several cancer tissues and has been shown to promote tumor growth. Recent studies show correlation between p53 mutant aggregation, functional loss, and tumor growth. Moreover, p53 aggregation has been observed in biopsies, patient tissues, and in vivo studies. Given the fact that over fifty percent of cancers have p53 mutation and several of them are prone to aggregation, therapeutic strategies are needed for treating p53 mutant aggregation associated cancers. Recent studies using polyarginine analogues and designer peptides for inhibiting p53 aggregation and tumor growth gives further encouragement in treating cancer as a protein aggregation disease. In this review, we highlight the recent efforts in targeting p53 aggregation in cancer and propose the use of small stress molecules as potential p53-antiaggregation drugs.  Many protein aggregation diseases are the result of protein misfolding/aggregation due to genetic mutations or environmental stress conditions . ProteinNormally, p53 proteins are switched off and are activated when cells experience stress and undergo uncontrolled division and proliferation ,10. WhenWhen p53 is mutated, it loses its function and becomes inactivated ,16,17. MSeveral methods have emerged to rescue p53 mutant function ,25. TherTreating cancer as a p53 aggregation disease is still in its early stages. Only a few studies have reported targeting cancer as a p53 mutant aggregation disease. We have shown that small stress molecules, such as polyarginine and its analogues, exhibit p53 aggregation inhibition both in vitro and in p53 mutant cancer cells ,28. AnotSeveral small stress molecules have been shown to stabilize proteins under harsh conditions. In particular, compatible solutes have been shown to stabilize protein aggregation. The molecules have shown inhibitory potential in neurodegeneration caused by peptide aggregation ,30,31,32Inspired by the above, we recently studied the ability of arginine molecules to modulate p53 aggregation and cancer cell proliferation, to identify therapeutic candidates for treating cancer due to p53 aggregation. We tested the inhibitory effects of polyarginine and its analogues on the R248Q p53 mutant mimetic peptide QRPILTIITL aggregation, and p53 mutant-bearing cancer cell growth in vitro. The study revealed that polyarginine and polyornithine significantly inhibit p53 mutant peptide aggregation in vitro and inhibit the growth of p53 mutant (R248Q) lung cancer cells H719 and p53 mutant (R175H) breast cancer cells SK-BR-3. The molecules showed no effect on p53 wildtype and p53 null cancer cell growth. In addition, the molecules enhanced p21 expression, one of the p53 target genes responsible for cell cycle arrest , indicatLactobacillus plantarum [In addition to arginine and analogues, in a separate study, we have also shown that the cationic small stress molecule acetylcholine chloride had the ability to inhibit aggregation of p53 mutant peptide WRPILTIITL (mimicking the p53 hot spot mutant R248W) in vitro . Acetylclantarum , and is lantarum . The aboRecently, Soragni et al.  developeHowever, the method has its limitations as well. The authors noted that if the wildtype p53 is partially unfolded and aggregated, the designer peptide would probably target the wildtype p53 structures as well. Hence, there can be systemic toxicity effects if this happens in normal cells. In addition, the inhibitory ability is limited to certain p53 mutant aggregations and may not be applicable to all p53 aggregation-associated cancer. Further, like all small molecule-based therapies, the peptides will have short half-life in vivo and will be cleared from circulation rapidly, and to realize the full potential, further modifications and optimization may be needed. The studies discussed in this review show that small molecules are capable of modulating p53 mutant aggregation and subsequent tumor progression. However due to their small molecular weight and size, they have a higher probability of getting rapidly cleared by the body, minimizing their potential as effective therapies. Hence, it would be ideal if the small stress molecules could be formulated in a stealth drug carrier, and to have target specificity to the tumor microenvironment to realize its full translational potential. Compared to conventional therapies which suffer from poor circulation, bioavailability, and efficacy, nanotechnology approaches can remedy some of the problems due to their tunable design . NanoparFinally, to further enhance the therapeutic potential, personalized medicine approaches could be utilized. To precisely target specific p53 mutant aggregation-associated cancer, mutant specific anti-aggregation targets could be developed. A similar concept to recent studies reported on p53 mutant antibodies to inhibit p53 mutants in cancer  could be


Cervical cancer mainly occurs among women from the developing world, and women face unique challenges in terms of their disease and treatment. Most women present with advanced cervical cancer and receive the standard curative treatment with external beam radiotherapy and brachytherapy with or without chemotherapy.To describe the quality of life (QOL) of women treated for cervical cancer during treatment (M0), at 6 months after completing treatment (M6) and at 12 months after treatment (M12).n = 153) and convenience sampling were used. Data were collected through structured interviews, and the EORTC QLQ-C30 and EORTC QLQ CX24 served as data collection instruments. Descriptive statistics were used to analyse the data, and the Kruskal–Wallis H test was used to compare the mean responses across the groups (p ≤ 0.05).A cross-sectional design, calculated sample size (The mean age of the respondents was 50.6 years (standard deviation [SD] 11.9). The global health status improved significantly in contrast with the functional scores. Financial difficulties were rampant, especially during the treatment phase. Insomnia and urinary frequency were the most cumbersome problems and remained so even after treatment.Despite an improvement in the global health, cervical cancer and its treatment had a negative influence on the QOL in all domains of lives of these women. Assessing the QOL of patients during treatment and follow-up visits would allow nurses to develop interventions to address distressing problems timeously. In addition, Africa’s nurses should assess social functioning and develop programmes to prevent social dysfunction. The significance of this study derives from the fact that best practices focus on the prevention and treatment of cervical cancer and little attention has been given to the quality of life (QOL) of women living with this condition. Although it is known that cancer and its treatment influences QOL, studies focusing on this phenomenon in the African population appear to be sparse , most commonly type 16 and 18, which is found in 70% of all cervical cancers. Cervical cancer develops slowly, usually taking 10–20 years for mild dysplasia to develop into cancer , at 6 months after completing treatment (M6) and at 12 months after treatment (M12).A cross-sectional design was selected for the study as it allows researchers to investigate time-related phenomena by collecting data from different groups at one point in time and comparing the responses  was 157 and the target population (N) was 7735.The study population consisted of all women diagnosed with cervical cancer treated at the study setting. The inclusion criteria were 18 years and older and receiving the standard curative treatment for cervical cancer at the time of data collection, or 6 or 12 months after treatment. The sample size was calculated with the assistance of a statistician. A finite population correction was used where the initial sample size (The formula used was:n = 153), thus 51 (n = 51) in each of the groups. The total sample (n = 153) was determined to achieve a confidence level of 95%. Convenience sampling was used to select the sample as it allowed the researchers to select women who were readily available .n = 153) ranged from 30 to 79 years, with a mean age of 50.6 years standard deviation (SD) ± 11.9. Nearly half  were single and unemployed , and 50.3% (n = 77) suffered from stage IIIA and more advanced disease  between study groups one (M0) and two (M6), between groups one (M0) and three (M12) (p = 0.000), and between groups two and three (p = 0.024).When calculating the global health status of the three groups, it was found that the M12 group had the better health status. A Kruskal–Wallis H test showed that the global health status was statistically significant among the study groups . A post m = 82.0), while social functioning scored the lowest overall mean (m = 60.5). In addition, social functioning had the lowest mean score in M0 compared to all the other functioning scores across the different groups. Kruskal–Wallis H tests showed there was no statistically significant difference between the role, physical, cognitive, emotional and social functioning scores among the three study groups. Financial difficulties had the highest mean score in M0 (m = 81.7), and a post hoc analysis indicated a statistically significant difference between groups one and two (p = 0.000) and between groups one and three (p = 0.001)  .m = 10.5) and insomnia had the highest overall mean (m = 33.6). Kruskal–Wallis H tests showed there was a statistically significant difference in fatigue, insomnia, dyspnoea, swelling of the feet and financial difficulties, but no statistically significant differences were found in pain and tingling of the hands or feet. A post hoc analysis indicated there was a statistically significant difference in fatigue between groups one and two (p = 0.003) and between groups one and three (p = 0.000). There was a statistically significant difference in insomnia between groups one and two (p = 0.024) and between groups one and three (p = 0.002). With regard to dyspnoea, the study revealed a significant difference in groups one and three (p = 0.001). Groups one and three had significant differences (p = 0.000) in swelling of feet as well as groups two and three (p = 0.000).When investigating the general problems, consisting of fatigue, pain, insomnia, dyspnoea, swelling of the feet and tingling of the hands of feet, dyspnoea had the lowest overall mean (m = 24.8), while diarrhoea had the highest mean score (m = 22.7). A statistically significant difference was found in diarrhoea, appetite loss, nausea and vomiting. A post hoc analysis showed a statistically significant difference between groups one and three (p = 0.000) and between groups two and three (p = 0.007) in terms of diarrhoea and nausea and vomiting. There was also a significant difference in appetite loss between groups one and two (p = 0.000) and between groups one and three (p = 0.000).Gastrointestinal, urologic and gynaecologic symptoms were also investigated . With rem = 7.2) and urinary frequency the highest (m = 44.9). In addition, urinary frequency had the highest mean score of all the symptoms investigated across the groups. There was no statistically significant difference in burning sensation when urinating, urine leakage and difficulty emptying the bladder; however, there was a statistically significant difference in urine frequency. A post hoc test showed there was a significant difference between groups one and two (p = 0.008) and between groups two and three (p = 0.010).When examining the urological data, difficulty in emptying the bladder had the lowest mean score (m = 9.2), while menopausal symptoms, hot flushes and/or night sweats had the highest mean (m = 24.2). With regard to menopausal symptoms, there was no significant difference among the three groups; however, according to a post hoc analysis, there was a statistically significant difference in vaginal discharge and vaginal bleeding. Regarding vaginal discharge, there was a significant difference between groups one and two (p = 0.000) and groups one and three (p = 0.000). There was also a significant difference between groups one and two (p = 0.000) and between groups one and three (p = 0.000) in terms of vaginal bleeding.Vaginal bleeding was the gynaecological symptom with the lowest overall mean  and between groups one and three (p = 0.000). There were significant differences in sexual enjoyment between groups one and two (p = 0.000) and between groups one and three (p = 0.000). There were statistically significant differences in vaginal functioning between groups one and two (p = 0.000) and between groups one and three (p = 0.000)  .The age profile of the sample seems to be typical of women treated for cervical cancer. Snyman and Herbst  in a SouThe study provided evidence that slightly more than half of the women suffered from stage IIIA and more advanced cervical cancer. Although a similar trend was reported in a Polish study (Pasek, Suchocka & Urbański It was positive to find that the global health status of the groups improved significantly from the time they received treatment to 12 months after treatment. Du Toit and Kidd , in a SoNone of the functioning scores showed a statistical significant difference between the groups. This is in contrast with the findings of Bjelic-Radisic et al.  who, in Financial difficulties, especially during the time of treatment, had a negative influence on QOL. This does not seem to be unique to Africa, as, although not to the same extent, studies conducted across Europe and in Turkey and Poland report financial difficulties in this group of women especially during the treatment phase (Bjelic-Radisic et al. It was interesting to find that insomnia was the most troublesome general symptom, followed by pain and fatigue. Goker and others  confirm What is of concern is the significant upward trend of swelling of the feet as lymphoedema is a well-known complication of pelvic radiotherapy. Heijkoop and others  report aUrinary frequency was the most troublesome cervical cancer-related symptom experienced accompanied by a burning sensation, most commonly occurring in the treatment phase. A study conducted in the Netherlands by Heijkoop and others  supportsThe study provided evidence that all the domains of sexual functioning, sexual activity and enjoyment and vaginal functioning declined after treatment. However, when the short-term and late effects of cervical cancer and its treatment are considered, this finding does not seem surprising. It was interesting to note that other studies found a similar trend except for sexual activity, for instance Rahman and others , in a stThe study has various limitations. Using a cross-sectional design did not allow for the investigation of changes in the same respondents over time. A longitudinal design may have resulted in results that were more accurate. In addition, respondents could have provided socially acceptable answers and not reflect what is true for them. However, the researchers believe the study provided base line data regarding the QOL of women treated for cervical cancer.Nurses practising in radiation oncology care settings should be cognisant of how cervical cancer and its treatment influence the QOL of women. Assessing the QOL during treatment and follow-up visits would allow nurses to develop interventions to address distressing problems timeously. In addition, nurses in Africa should assess social functioning before and after treatment and develop programmes to assist women to optimise their functioning as social beings despite having cervical cancer and prevent social dysfunction.Despite an improvement in the global health status, cervical cancer and its treatment had a negative influence on the QOL of the women and influenced all domains of their lives. Social functioning was the most affected and did not improve significantly over time. Financial difficulties were troublesome, especially during the time of treatment; urinary frequency and insomnia remained problematic; and swelling of the feet increased significantly over time.


Child mortality rate in sub-Saharan Africa is 29 times higher than that in industrialized countries. Anemia is one of the preventable causes of child morbidity. During a humanitarian medical mission in rural South-Eastern Nigeria, the prevalence and risk factors of anemia were determined in the region in order to identify strategies for reduction. A cross-sectional study was done on 96 children aged 1-7 years from 50 randomly selected families. A study questionnaire was used to collect information regarding socioeconomic status, family health practices, and nutrition. Anemia was diagnosed clinically or by point of care testing of hemoglobin (Hb) levels. 96 children were selected for the study; 90 completed surveys were analyzed . Anemia was the most prevalent clinical morbidity (69%), followed by intestinal worm infection (53%) and malnutrition (29%). Mean age (months) at which breastfeeding was stopped was 11.8 (±2.2) in children with Hb <11mg/dl (severe anemia), 10.5±2.8 in those with Hb = 11-11.9mg/dl (mild-moderate anemia), and 9.4±3.9 in children with Hb >12mg/dl (no anemia) (P=0.0445). The longer the infant was breastfed, the worse the severity of childhood anemia was. Childhood anemia was likely influenced by the low iron content of breast milk in addition to maternal anemia and poor nutrition. A family-centered preventive intervention for both maternal and infant nutrition may be more effective in reducing childhood anemia and child mortality rate in the community. Childhood malnutrition and anemia remain significant public health concerns in Southern Nigeria and globally. While sub-Saharan Africa has made notable progress in combatting food insecurity, 25% of the population remains undernourished and below national goals . In NigeOverall, exclusive breastfeeding in Nigeria is declining, but the length of time mothers breastfeed has increased to a mean of 13 months in 2014 . The benWhile many studies have linked breastfeeding with infant anemia, few, if any, have examined risk factors for persistent anemia in childhood, despite the impact of childhood anemia on morbidity and mortality. This study sought to determine the risk factors and prevalence of childhood malnutrition and anemia in a rural community in Imo State, Nigeria, in sub-Saharan Africa.An institutional review board at the Federal University of Technology, Owerri (F.U.T.O), Imo State, Nigeria, completed a review of the planned research study and approved the study prior to study commencement.A survey of 38 questions addressing sociodemographics, agricultural diversity, and diet diversity was developed. Fifty children between the age of two and five were assessed for possible malnutrition and anemia and surveys administered to their parents or accompanying guardians. A single interviewer performed all interviews. A parent or household guardian for each of the selected children was asked to complete a demographic survey, with the help of interpreters as necessary. Completion of the questionnaire was not a requirement for medical treatment, and informed consent was obtained. Demographic information was collected for all fifty families including family size, age of parents, age of other children, and living situation. Families' socioeconomic status was scored using a series of eight questions adapted to the specific living conditions of the region using knowledge from past missions and local information. Families' agricultural and diet diversity were graded using recommendations from the United Nations Food and Agriculture Organization . QuestioNutritional status of each child was determined after measuring height, weight, and median upper arm circumference (MUAC) and compared to WHO arm circumference for age standards . Anemia  p-values were two-sided and p<0.05 was used to define statistical significance. Data analysis was conducted using SAS software , version 9.4.Categorical variables were summarized in terms of frequencies and percentages while continuous variables were summarized in terms of means ± standard deviations (SD). Logistic regression analysis, chi-square, or Fisher's exact test was used to compare categorical variables between cases and control. Odds ratios (OR) were reported along with the corresponding 95% confidence intervals. Continuous variables  were compared between cases and controls using a two-sample t-test. AllOut of 90 children tested, 57% were female (n=51) and 43% were male (n=39). The prevalence of chronic systemic disease was determined in the cohort of children. Anemia was the most prevalent (69%) followed closely by worms (53%), malnutrition (29%), upper respiratory infections (23%), cardiac disease (4%), and gastrointestinal illness (2%). The hemoglobin testing on all children revealed that 27% had a low hemoglobin, 58% had borderline hemoglobin, and 16% had normal hemoglobin.p=0.35 interaction effect), presence of upper respiratory infection (p=0.9946 interaction effect), or malnutrition (p=0.4949). There were not enough cases of gastrointestinal disease to examine the interaction between hemoglobin and gastrointestinal disease on clinical anemia. There was no correlation between median upper arm circumference and worm infestation (p=.79) or anemia diagnosis (p=.86).There was a significant association of hemoglobin levels with clinical anemia (p=0.0457), with lower levels of hemoglobin being associated with a higher rate of anemia, as expected,  p=0.25). There was no difference between hemoglobin levels and type of occupation of the head of household  (p=0.752). Example of skilled labor would be a tailor or mechanic; nonskilled labor would be a household servant or laborer; while professional could be a teacher, business manager, or profession requiring more than a secondary education. Although professionals were more likely to read to their children each week (an average of 3.7±0.5 times a week) than head of households with skilled (3.1±0.9) and nonskilled labor (2.8±0.6) (p=0.0002), there was no difference in frequency of medical checkups (p=0.9411) and likelihood of child attending school, There was no association between hemoglobin levels and socioeconomic status score (SES) , There was no significant association between hemoglobin levels and people that both sell and eat farm products compared to those that do not sell or eat farm products (p=0.4257). the number of farm animals owned in a household did not correlate with hemoglobin level (p=0.25). The number of family meals a day (p=0.742) or number of meals the child has a day did not have any association with hemoglobin levels (p=0.069). There was no significant association between hemoglobin levels and food insecurity (p=0.21) or diet diversity score (p=0.39). Children with low hemoglobin levels were breastfed a mean of 2.4 months longer than those with normal hemoglobin levels. Longer breast feeding was associated with lower hemoglobin levels  without food or nutrient supplementation on anemia , 31, 34.The prevalence of anemia in our population 69%) is consistent with other studies in tropical Africa that found 63% prevalence in children under 3 years % is cons. In contOverall, the community's diet was homogenous between families, and it was difficult to accurately assess a child's dietary intake. This likely contributed to lack of association between dietary diversity and anemia, as it was difficult to stratify diet diversity in this community. Similarly, it was difficult to stratify socioeconomic status in the community—despite using a targeted questionnaire build from a previous community needs assessment in the area. While there was no significant correlation between frequency of medical care and professional level, this is likely due to the deficiency of medical resources in the area. From our study, the importance of education appears to be understood in the community. Regardless of head of household's occupation, most children were likely to be enrolled in school. However, professional households seemed to be more consistent in promoting literacy as they were more likely to read to their children compared to nonprofessional households. However, this did not affect the healthcare choices made by both professional and nonprofessional households as both had infrequent medical care preventive visits for their children and had equally poor diet diversity or meal choices. This alludes to the fact that the overall poor financial status of both professional and nonprofessional household affected their healthcare and food choices and attitudes regardless of level of literacy , 40.A major limitation identified in this study is that it was primarily designed to assess modifiable risk factors for childhood anemia, and it did not specifically examine risk factors for prolonged breastfeeding or exclusive breastfeeding or maternal anemia status. Furthermore, it relied on field-testing kits to diagnose anemia and did not include diagnosis of possible etiologies of anemia like measures of iron deficiency using ferritin levels, for example, chronic disease, inflammation, or hemoglobinopathies. Further research is necessary in two primary areas. First, while the most likely cause of anemia in this community is iron deficiency based on large-scale population studies, further research should be done to elucidate the various causes of anemia in this community , 42. A rWhile iron supplementation would likely decrease anemia prevalence in this area, it is important to evaluate other causes of anemia, notably hemoglobinopathies, to guide public health and clinical interventions. Another limitation in this study is that the risk factors or interactions between prolonged breastfeeding and anemia were not explored or evaluated. Many studies have evaluated cultural, economic, and social determinants of breastfeeding in many settings with a wide range of conclusions –47. In NIn conclusion, persistent childhood anemia was likely influenced by the low iron content of breast milk, an indication of maternal anemia and poor nutrition. Although continuous breastfeeding is a known strategy to reduce child mortality, a family-centered preventive intervention to diagnose and treat childhood anemia could lead to reduced morbidity and mortality from anemia. Early identification of iron deficiency in children is vital to the prevention of anemia and long-term psychomotor and developmental consequences. A longitudinal study examining childhood anemia pathophysiology, determinants of exclusive breastfeeding, and treatment effects would both be cost effective given the presence of the annual medical missions and provide important data on the effects of infant anemia on childhood anemia.


Skin cancer is the most common type of cancer worldwide. Ozone depletion and climate changes might cause a further increase in the incidence rate in the future. Although the early detection of skin cancer enables it to be treated successfully, some tumours can evolve and become more aggressive, especially in the case of melanoma. Therefore, good diagnostic and prognostic markers are needed to ensure correct detection and treatment. Transcription factor p63, a member of the p53 family of proteins, plays an essential role in the development of stratified epithelia such as skin. In this paper, we conduct a comprehensive review of p63 expression in different types of skin cancer and discuss its possible use in the diagnosis and prognosis of cutaneous tumours. Trp53, Trp63, and Trp73 knockout mice [p63, like its sibling p73 , belongsTP63 gene gives rise to several protein isoforms resulting from the use of alternative transcription start sites and alternative C-terminus splicing events. Indeed, the TP63 gene harbours two different promoters that generate two N-terminal isoforms (TAp63 and ΔNp63) (The d ΔNp63) a. The TAd ΔNp63) a. The ∆Np63 isoform is mainly expressed in ectoderm-derived tissues, such as the epidermis, skin appendages, simple epithelia, and the thymus ,17,18,19Remarkably, no direct evidence has been provided using immunohistochemistry for p63 expression in Langerhans cells and melanocytes. However, p63 was found to be absent in normal melanocytes by polymerase chain reaction (PCR) . This obIn summary, p63 appears to be expressed in stem cells and highly-proliferating transit-amplifying cells of ectodermal origin. However, it is present at low levels or absent in differentiating cells as well as cells of mesenchymal origin. CDKN1A [HES1 [bona fide p63 target genes are components of hemidesmosomes such as BPAG1 [PERP [ITGA6 and ITGB4 [CDH3, a component of adherens junctions [FRAS1 codifying for an extracellular protein [KRT14, a component of keratin intermediate filaments [As a transcription factor, p63 can activate or repress expression of its target genes through direct binding to their promoter regions c. For inCDKN1A , a gene 1A [HES1 , one of as BPAG1  and PERPG1 [PERP ; integrind ITGB4 ; the P-cunctions ; FRAS1 c protein ; and KRTilaments . Furtherilaments  by direcilaments  and cytoilaments . Differeilaments ,40 and Kilaments  cooperatSATB1 [SMARCA4 (codifying for Brg1) [Conversely, p63 is closely involved in the process of keratinocyte differentiation in both early and late stages. ZNF750 is one of the earliest transcription factors activated during differentiation by p63 . It induSATB1  and SMARor Brg1) . Their aRecent investigations have unveiled the existence of pre-established epithelia-specific enhancers in undifferentiated keratinocytes. These might interact with the promoter regions of target genes to allow their transcription during differentiation . NotablyAltogether, p63 is well the established master regulator of epidermal homeostasis and differentiation. Its transcriptional network is an example of an extremely complex spatiotemporal system of positive and negative regulation, which allows for the proper development, self-renewal, and differentiation of the skin. p63 both impedes and triggers keratinocyte differentiation depending on the epithelial compartment, proliferative state, and the network of interacting proteins. Although mechanistic aspects of p63 function in the cutaneous basal cell carcinoma (cBCC) and rare skin tumours remain poorly investigated, the role of p63 in tumourigenesis has been intensively studied in squamous cell carcinoma (SCC). A transcriptional programme of p63 in cutaneous SCC is similar to SCC from other epithelial origins such as the head and neck . In The oncogenic properties of ΔNp63 were established in in vivo experiments that showed overexpression of ΔNp63 enhances mutant Ras-driven tumourigenesis of undifferentiated cSCC . Indeed,Skin cancer is the leading type of cancer worldwide with up to 3 million new cases every year. The most common risk factor for the development of skin cancer is ultraviolet (UV) exposure to the sun. This provokes multiple cytosine-to-thymine transitions that result in DNA mutations . HoweverTP53 and PTCH1. However, cBCC genomes are considered genetically stable compared to those from aggressive types of cancer [Cutaneous basal cell carcinoma (cBCC) is the most common type of cancer worldwide with almost 750,000 cases detected every year in the USA. cBCC accounts for up to 80% of all non-melanoma skin tumours. Basal cell carcinomas are slowly growing and rarely metastasise. Among the most frequently mutated genes are f cancer . Recent f cancer . Multiplf cancer . Moreovef cancer ,66,67,68RAS and TP53 are frequently mutated in cSCC. In contrast to cBCC, cSCC can originate from hair follicle bulge stem cells as well as interfollicular epidermal stem cells [The second most frequent type of skin cancer after cBCC is cutaneous squamous cell carcinoma. With a diagnosis rate of 15–30 per 100,000 people, cSCC accounts for 20% of all skin cancer cases. Notwithstanding a lower frequency with respect to cBCC, cSCC is characterised by a higher incidence of metastases and poorer prognoses . RAS andem cells . p63 is em cells ,67,68,71em cells  with higem cells . A rare variant of cSCC is cutaneous spindle cell squamous cell carcinoma (cSCSCC). The epidemiology of cSCSCC is associated with sun- or radiation-exposure of the skin. Radiation exposure is thought to be correlated with more aggressive phenotypes of the disease. Histologically, cSCSCC is characterised by bizarre and pleomorphic tumour cells that infiltrate deep into the dermis . SeveralAlthough several cutaneous lesions are of a benign nature, they can be precursors of skin tumours such as cSCC. The most frequent benign tumour of the skin is seborrheic keratosis (SK), which primarily affects people over the age of 50 and has low malignant potential . AnotherTakeuchi and colleagues performed a comprehensive analysis of p63 expression in several skin tumours of different origins, including very rare cases. As expected, p63 was found to be positive in epidermal tumours as well as most adnexal neoplasms (as will be discussed in the next section). In contrast, in all neural tissue tumours, mesenchymal tumours, lymphomas, and histiocytosis, p63 expression was found to be negative ,86. RemaAlthough malignant melanoma (MM) is less frequent than cBCC or cSCC, it is among the deadliest forms of skin cancer, leading to 50,000 deaths every year . MM arisTP63 gene in melanoma samples (14.7% of samples) [TP63 gene are common in epidermal dysplasia [TP63 mutation of melanomagenesis remains elusive. No consensus has been reached regarding the pattern of p63 expression in melanoma. Multiple lines of evidence suggest that p63 is virtually never expressed in MM samples  . In contysplasia . Therefop63 is used as a gold standard marker for highlighting myoepithelial cells in multiple types of cancer ,96,97. ICutaneous adnexal neoplasms (CANs) are a wide group of benign and malignant skin tumours that normally exhibit differentiation towards one of the cutaneous appendages: hair follicle, sebaceous gland, eccrine, or apocrine glands. Even though the precise origin of these neoplasms remains unknown, it has been hypothesised that CANs might arise from stem cells located within normal cutaneous adnexa . CANs arAtypical fibroxanthoma (AFX) is a rare benign tumour with a favourable prognosis that mainly occurs in sun-exposed skin on the head and neck regions of elderly patients, predominantly men. AFX is believed to be of fibroblastic origin and is characterised by myofibroblastic differentiation. The diagnosis of AFX is difficult and is performed by excluding other malignancies such as cSCSCC ,108. p63 was found to be negative in 188 out of 199 samples. Notably, in several reports the expression of p63 in AFX samples was assessed in parallel with its expression in cSCSCC. Indeed, 110 out of 130 cSCSCC samples stained positively for p63 ,112. ThuMerkel cell carcinoma (MCC) displays neuroendocrine features and is a rare but aggressive type of skin cancer. It mainly affects elderly patients (>75 years old) and can be caused by chronic UV exposure or Merkel cell polyomavirus. The origin of MCC remains elusive; however, it is speculated that this kind of cancer may arise from Merkel cell precursors as well as pre-B cells or fibroblasts, rather than mature post-mitotic Merkel cells. MCC is characterised by a high risk of metastases and death . Due to Since the discovery of p63 , researcThe reason for the discrepancy between 4A4 and p40 staining patterns remains unclear. One possible explanation might be that tumour cells express not only ΔN but also TA isoforms of p63 that are recognised by 4A4 but not p40. Another, more plausible, hypothesis is that different staining results are due to the non-specific binding of 4A4 to other members of the p53 family or even other proteins. Indeed, it has been shown that the 4A4 antibody can readily recognise p73 isoforms in vitro . Several reports have also confirmed the utility of p40 in the diagnosis of skin tumours. Lee et al. demonstrated higher specificity of p40 with respect to pan-p63  in distinguishing adnexal carcinoma from metastases in skin . As descForty years ago, the master tumour suppressor p53 was discovered, heralding a new era of understanding regarding the ubiquitous molecular mechanisms of cellular defence against cancer initiation and progression . Almost Recent studies carried out with primary keratinocytes have elucidated a very complex and thorough transcriptional programme governed by p63. Via interaction with chromatin remodelers, p63 enables the establishment of a well-organised three-dimensional genomic architecture to ensure the proper development of the skin as well as its homeostasis. Not surprisingly, in skin cancer, p63 pathways can be altered, disrupting the balance between proliferation and differentiation.The paramount importance of p63 in epithelia development encouraged researchers and pathologists to explore its expression in epithelial cancer. Multiple groups in the early 2000s carried out an extremely comprehensive study of different types of skin cancer using immunohistochemistry, providing strong evidence for high expression of p63 in virtually all types of non-melanoma skin cancer, including pre-cancerous lesions as well as rare forms of squamous cell carcinoma. By contrast, several skin-related tumours of non-epithelial origin such as atypical fibroxanthoma, along with diverse metastases to skin from other organs, are in most cases p63 negative. Even though no decision has been made regarding the use of p63 as a gold standard in skin cancer, multiple reports have proposed its utility in differential diagnosis. Furthermore, p63 has emerged as a putative marker for outcomes in Merkel cell carcinoma, where high p63 expression is associated with a poorer prognosis. Based on the pre-clinical studies, summarised in this review, we can speculate that p63 might be an important and targetable molecule, although no clinical trials have been reported that address how the level of p63 expression could modify current therapies. Nevertheless, a growing body of evidence suggests that p63 could be a promising diagnostic and predictive marker for unusual cases of skin cancer.


Drugging the p53 pathway has been a goal for both academics and pharmaceutical companies since the designation of p53 as the ‘guardian of the genome’. Through growing understanding of p53 biology, we can see multiple routes for activation of both wild-type p53 function and restoration of mutant p53. In this review, we focus on small molecules that activate wild-type p53 and that do so in a non-genotoxic manner. In particular, we will describe potential approaches to targeting proteins that alter p53 stability and function through posttranslational modification, affect p53’s subcellular localization, or target RNA synthesis or the synthesis of ribonucleotides. The plethora of pathways for exploitation of p53, as well as the wide-ranging response to p53 activation, makes it an attractive target for anti-cancer therapy. The tumour suppressor p53 in its role as the ‘guardian of the genome’ exerts a multitude of effects on cells in response to cellular stress . As a keTP53, making it one of the most consistently and frequently mutated genes in cancer . HDM2 acts as an E3 ubiquitin ligase, which binds to p53 and subsequently causes its ubiquitination and nuclear export, targeting p53 for proteasomal degradation . Interestivation . The HDMtivation .Escherichia coli thioredoxin  . The morp53 not only induces the expression of its E3 ligase HDM2 but also acts as a transcription factor for the expression of WIP1/PPM1D, another of its negative regulators. This p53 inducible phosphatase 1 not only destabilizes p53 by dephosphorylating serine 15 in p53  but alsoThe deacetylation of p53 at lysine residues has been implicated in enhancing its subsequent ubiquitination by HDM2 . The claIn 2002, it was reported that HDM2 is able to promote p53 deacetylation by recruiting a complex containing HDAC1, and therefore HDAC1 inhibition could promote p53 stability and function . TrichosAnother mechanism by which ubiquitination of p53 is altered is through the inhibition of deubiquitination. The deubiquitinase USP7 is responsible for the deubiquitination of both p53 and HDM2, though it displays a preferential activity towards HDM2 . A decreNEDDylation is another postranslational modification that can be modulated with small molecules. In the case of p53, NEDDylation is favoured by HDM2 and leads to inhibition of the transcription factor function of p53 . FBX011 MLN4924 (Pevonedistat)  is thougInhibitors of the proteasome, including the clinically approved bortezomib, rapidly increase p53 protein levels . Not onlFor p53 to exert its transcription factor function, it requires nuclear localization. HDM2-mediated ubiquitination of p53 causes its nuclear export and subsequent proteasomal degradation . Therefode novo pyrimidine synthesis pathway, has gained increasing attention following a seminal paper by Indirect activation of wild-type p53 by depleting cells of ribonucleotides is another possible strategy to activate p53 without causing DNA damage . In the in vivo would be to combine with a relevant therapy. Indeed, we have already shown that combination of a DHODH inhibitor, HZ00/HZ05, with an inhibitor of p53 degradation, nutlin-3, led to a synergistic tumour cell kill both in vitro and in vivo in tumour xenograft studies -L-aspartate (PALA) has been noted for its anti-tumour activity for many years having been tested in both in vivo tumour models as well as in patients  are also potent activators of the p53 response. For example R-roscovitine (Seliciclib), which targets multiple CDKs in the nanomolar range, induces the expression of functional p53 in cells . AccordiCDK4 and HDM2 amplification are frequently linked in liposarcoma and the combination of inhibitors to each of these enzymes slightly improves the in vivo antitumor effect (V600E and BRAFV600K inhibitor, to suppress melanoma growth in vivo, by inducing p53-dependent apoptosis and growth suppression.The inhibition of cell cycle progression through inhibition of CDK4 and CDK1 has been associated with the p53/HDM2 axis. r effect . Furtherr effect . FurtherThe exploitation of the p53 pathway therapeutically has been a formidable challenge, and one that has been under investigation for more than two decades. As summarized in Figure Multiple clinical trials where HDM2 inhibitors are combined with standard chemotherapeutics or radiation are ongoing Table . StudiesFinally, one must bear in mind that HDM2 inhibitors can protect normal cells from S-phase poisons, mitotic poisons, as well as other agents such as DHODH inhibitors. Therefore, the order in which these agents are administered could be postulated to be of great importance to mitigate an undesirable outcome and to be successful as treatments.


This perspective was written in celebration of Dr Arnold J. Levine’s 80th birthday. I chose to write this prospective about restoration of p53 activity in tumors for two reasons: Arnie  has become interested in restoring p53 activity in tumor cells and such a review did not exist. Arnie was also my postdoctoral advisor and writing this perspective is just a small tribute for the huge impact he has had on my research career (and that of many others), from supporting me when I left his laboratory for a faculty position before I had even published a paper, to serving as a sounding board for numerous ideas. Arnie is an amazing thinker. He is able to almost effortlessly incorporate new knowledge into current thinking and collate a big picture. It has been my pleasure to interact with him scientifically on numerous occasions. I want to thank Arnie from the bottom of my heart for sharing with me his love of science, his critical thinking skills, and his friendship.p53 gene, should be treated with drugs that disrupt binding of these inhibitors to p53 to restore p53 function. Tumors lacking p53, on the other hand, need to reintroduce p53 through a virus encoding wild-type p53 or convert mutant p53 to wild type. Genetically modified mouse models have been used to examine p53 restoration in various contexts and will be reviewed here. Tumor responses have been heterogeneous suggesting that other factors contribute to the outcome. Resistance mechanisms will also likely emerge and need to be understood in more detail.The p53 tumor suppressor is inactivated in most  cancers via various mechanisms indicating a potent role in inhibiting tumor cell growth . The idep53 alleles that were not functional but could be reactivated in a Cre-dependent manner. The Jacks laboratory generated a wild-type p53 locus with a lox-stop-lox (LSL) cassette in the first intron effectively eliminating p53 expression but allowing p53 re-expression in the presence of Cre recombinase  of these tumors regressed and 20% showed tumor stasis (1 T-cell lymphoma and 1 osteosarcoma). One tumor lost the T2Cre-ER allele and thus grew like controls. In this context, p53 restoration led to apoptosis in lymphomas but decreased proliferation, cell cycle arrest, and senescence in sarcomas  that dramatically reduces expression of p53 to  ~7% of wild type. The p53 response upon restoration in autochthonous tumors that develop due to p53 loss was similar with lymphomas inducing apoptosis and angiosarcomas inducing senescence. Tumors regressed and in some cases completely disappeared, which coincided with increased survival of the mice. In both of the above studies, it should be noted that the p53 alleles are germline such that all cells of the mouse have less p53 than normal, and Cre recombinationrestores p53 not just in tumor cells , and the CAG-CreER Tamoxifen-inducibletransgene develop lymphomas and sarcomas. p53 was restored with the addition of Tamoxifen and in contrast to p53-null tumors that regressed in the two studies described above, lymphomas and sarcomas in this case showed tumor stasis but not tumor regression  was used , which include cytokines and chemokines with the potential to fuel tumor cell growth ; genetic reconstitution of p53 is permanent and does not mimic drug pharmacology. However, even a partial response appears to be effective in slowing tumor growth. In most of these studies as well, the tumor microenvironment (TME) has p53 alterations, distorting interpretation of the data. We also need to expand these studies beyond the tumor types (lymphomas and sarcomas) that occur with germline loss of p53.The caveats of the above studies are many: the studies were performed in mice not humans; most studies involved germline p53. This was observed in the Jacks study above  have been developed to restore p53 activity .]


The p53 family of proteins has grown substantially over the last 40 years. It started with p53, then p63, p73, isoforms and mutants of these proteins. The function of p53 as a tumour suppressor has been thoroughly investigated, but the functions of all isoforms and mutants and the interplay between them are still poorly understood. Mutant p53 proteins lose p53 function, display dominant-negative (DN) activity and display gain-of-function (GOF) to varying degrees. GOF was originally attributed to mutant p53′s inhibitory function over the p53 family members p63 and p73. It has become apparent that this is not the only way in which mutant p53 operates as a large number of transcription factors that are not related to p53 are activated on mutant p53 binding. This raises the question to what extent mutant p53 binding to p63 and p73 plays a role in mutant p53 GOF. In this review, we discuss the literature around the interaction between mutant p53 and family members, including other binding partners, the functional consequences and potential therapeutics. In 1979 the research into p53 started off with the finding of a 53kDa protein using immunoprecipitation with the SV40 T antigen in transformed cells ,3. FortyTP53 can cause loss of p53 expression or expression of mutated proteins. The mutant proteins are almost always caused by single nucleotide changes in the DNA, resulting in mutant proteins that differ in only one amino acid from the wildtype molecule. Unlike many other tumour suppressors, mutations are found throughout the amino acid sequence of p53, although more frequently in the DNA binding domain, giving rise to thousands of different mutant proteins [The importance of p53 in tumour suppression is apparent when looking at the frequency of p53 mutations in all cancers, the early onset and frequency of cancers in Li-Fraumeni patients and the many mouse models that display tumourigenesis when p53 is lost or mutated. Using data from the TCGA (The Cancer Genome Atlas), it has become apparent that more than 50% of all cancers carry p53 mutations, but in some cancers, such as small cell lung cancer and ovarian cancer, this percentage can go up to levels higher than 90% ,7. Mutatproteins . The mosproteins .To function as a transcription factor, p53 forms dimers and tetramers that interact with the DNA. The observation that mutant p53 is often impaired in wildtype function led to the observation that it can exert a dominant-negative role over any remaining wildtype p53 protein through direct binding ,12. In aTP53, the family members TP63 and TP73 were cloned and identified as homologues of p53 that share a similar DNA binding domain but differ in N- and C-termini. All family members can be transcribed from different promotors resulting in variants with long N-termini that contain transcription activation domains (TA) or shortened N-termini (ΔN) (Shortly after ini (ΔN) a. TA isoini (ΔN) a. Spliceini (ΔN) a,b.Tp63 KO mice and Tp73 KO mice that show lethality during late gestation or early after birth due to skin and craniofacial epithelial defects [Tp53 knockout embryos survive until early onset of tumourigenesis [p63 and p73 are thought to be expressed in a more tissue-specific manner than p53, playing important roles in the skin epithelium and in the central nervous system. This is reflected in the phenotypes of  defects ,32 or ne defects  Althoughigenesis , subtle igenesis , suggestTP53, mutations in TP63 and TP73 are rarely found in cancers. This raises the question to what extent p63 and p73 contribute to tumourigenesis. Although mice that are knockout for all p63 isoforms or all p73 isoforms show early lethality, loss of TAp63 or TAp73 expression specifically promoted tumourigenesis or metastasis [In comparison to tastasis ,37,38. Ftastasis ,40. Moretastasis . Loss oftastasis .Under normal conditions, ΔN isoforms dominant-negatively interact with TA forms of their family counterparts, and isoforms can transcriptionally regulate each other ,41,42. AIn addition to inhibiting wildtype p53, many mutant p53 proteins interact with most isoforms of p63 and p73  43,44,4,444,45. Interactions between mutant p53 and p63 or p73 can be observed in vitro and are strong, indicating that mutant p53-p63 or mutant p53-p73 bind directly with each other ,45,48. FMuch work on mutant p53 interactions has so far focused on delineating how mutant p53 proteins inhibit TA variants. All TA isoforms of the p53 family contain an oligomerisation domain (OD) that is used for homo-dimerisation and tetramerisation. Remarkably, heterodimerisation between TA isoforms of the different family members is seldom observed under physiological circumstances. Mutant p53 R175H can dominant negatively inhibit wildtype p53 by binding through the OD . HoweverSome of the mouse models that were generated of N-terminally truncated p53 display a similar phenotype to mutant p53 (R172H or R270H) in promoting invasion and metastasis . As Δ133To investigate how mutant p53 inhibits p63 and p73 function, several laboratories have studied potential binding partners of the mutant p53-family member complexes . These sSome of these binding partners were found to act in a similar manner in both the mutant p53-p63 and mutant p53-p73 complexes. The topoisomerase TopBP1, for instance, promotes the formation of both complexes by binding p53 and increasing the affinity for mutant p53 to bind to p63 or p73. This leads to loss of p63 or p73 mediated transcription and an increase in invasion and metastasis . ConversInterestingly, the binding partners of the complexes do not always act on p63 or p73 in the same way. MDM2 and HSP70 differentially regulate the two complexes by promoting the dissociation of the mutant p53-p63 complex but enabling the formation of the mutant p53-p73 complex. This results in a loss of p73-mediated transcription and a gain in p63-mediated transcription ,57. InteMost binding partners have been shown to regulate only p63 or p73. For instance, SMAD and Pin1 promoted the mutant p53-p63 complex formation, inhibiting p63 function ,59. The These findings show that the interacting proteins of mutant p53-family member complexes in cancer can easily modulate some aspects of mutant p53 GOF. This regulation, if appropriately targeted, could be used to reduce the GOF of mutant p53 that is related to p63 and p73 function. In particular, looking at proteins that affect both complexes in the same manner could be interesting. Inhibition of TopBP1 or restoration of ANKRD11 expression would both lead to wild type p63 and p73 signalling, which would suppress metastasis and induce apoptosis.Over the last few years, the interaction between mutant p53 and family members has gotten significant attention from researchers interested in prion-like aggregation. In vitro, the DBD domain was shown to form aggregates, and the OD and mutant forms of p53 were prone to form amyloid ,63. Xu eMost likely, aggregation cannot be fully responsible for all gain-of-function that we see on mutant p53 expression. By aggregating, the mutant p53 complex with all of its binding partners is rendered inactive. Although mutant p53 inhibits TAp63 in activating canonical p53 target genes, some reports describe that through mutant p53, TAp63 binds to a unique set of promotors to promote gene expression ,70. In akRAS mutation, tumours were found to frequently metastasise to the liver and adjacent tissues. Loss of p53 expression in combination with loss of TAp63 expression also led to metastasis, but with a lower prevalence, suggesting that loss of TAp63 does not exclusively contribute to mutant p53 GOF in metastasis in this model [TAp63 and TAp73 knockout animals have been reported to display additional phenotypic abnormalities in ageing, metabolism or spermatogenesis, respectively, that have so far not been reported to occur in any of the mutant p53 mouse models [As mutant p53 has been shown to exert gain-of-function in many ways, it will be important to determine to what extent the interaction between mutant p53 and p53 family members, as well as with other molecules, plays a role in the different aspects of tumourigenesis in vivo. Most mutant p53 mouse models have investigated hotspot mutations that are generally considered fully impaired in wildtype function, stabilised in tumours and capable of GOF ,19. Thesis model . In addie models ,78,79. TTp53 KO mice was shown to inhibit tumourigenesis in p53 knockout animals by upregulating TA isoforms [To fully elucidate how the interactions between mutant p53 and family members work together in promoting tumourigenesis and metastasis, it will be important to combine loss of TAp63 expression with mutant p53 in the same model system and determine whether the tumour burden is exacerbated. For this, it will also be important to fully characterise the expression of isoforms of the p53 family. Loss of ΔN isoforms of p63 and p73 in isoforms . It willAlthough p53 has been studied for 40 years and is the most mutated gene in all cancers, hardly any therapies or clinical decisions are based on p53 status. This is not due to a lack of effort in trying to design clever ways to combat mutant p53 GOF or to increase wildtype function. Stabilisation of wildtype p53 has been thoroughly studied, with nutlin-mediated inhibition of MDM2 (the E3 ubiquitin ligase involved in p53 degradation) being one of the most famous protein–protein interference drugs . TherapeBAX or GADD45 [Remarkably, there are quite a few drugs that prevent mutant p53 from binding to TAp73, but no therapeutics that directly and specifically interfere in the mutant p53-TAp63 interaction . This rar GADD45 . These dr GADD45 ,88,89. PTAp73′s role is certainly not limited to apoptosis as it has been shown to play a role in invasion in pancreatic cancer cells via a protein–protein interaction with NF-Y . It woulSeveral compounds have been found that restore the folding of mutant p53 to promote wildtype function, which are listed in Many of these compounds will, therefore, also abolish mutant p53 from binding to the family members, but this is not always specifically investigated. The most well-known example of a drug that restores p53 function is Prima-1, which completed a Phase 1 clinical trial ,119. ApaAn important consideration for this review is that only a handful of p53 mutants, predominantly the hotspot mutations, have been thoroughly investigated. Due to large cancer genome sequencing efforts, we now know that thousands of different mutant p53 proteins exist in cancers. Traditional classification into the DNA contact mutants and the structurally unfolded mutants has not been able to attribute specific functions or cancer characteristics to either one of these classes of mutants. Although unfolded p53 mutants were more able to interact with p63 and p73 than DNA binding mutants, both types of mutants have been shown to inhibit p63 and p73 function. In addition, RETRA and peptide aptamers were able to increase TAp73 function in cells expressing the folded mutant p53 R273H ,85. ImpoInstead of using the folding state of p53, perhaps we can classify mutants in a different way. Although the traditional six hotspots can be detected in almost all cancers with p53 mutations, certain cancers seem to have a predisposition for certain mutants. An example of this is hepatocellular carcinoma (HCC), in which there appears to be a selection for expression of one hotspot, mutant p53 R249S . Remarka


CDKN1B plays a key role in many cellular events.3 p27 is a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, which functions to negatively regulate cell cycle progression at the G1/S boundary in response to antiproliferative stimuli. In addition, numerous p27 functions, not related to CDK inhibition, have been described. For instance, cytosolic p27 plays a role in the regulation of cytoskeleton assembly/disassembly, therefore, regulates the cell morphology and movement. In addition, p27 is involved in apoptosis and autophagy modulation.6The cell cycle regulation and tumor suppressor p27 encoded by CDKN1B) was cloned in 19947 and mapped to chromosome 12p13. Later on, p27 mutations were discovered in several types of human cancers including breast cancer, sporadic parathyroid adenomas, endocrine neoplasia, small intestine neuroendocrine tumors.14Mutations, abnormal expression and mislocalization of p27 have been found in many diseases suggesting the important role of p27 in the pathogenesis of diseases. Human p27 gene . Treatment with an AKT inhibitor rescues the mislocalization of p27 to the cytoplasm in endometrial carcinoma cells.18 Similar findings are observed in other types of human cancers.23 p27 is reduced in premalignant and non-invasive cancerous lesions, including ductal carcinoma in situ of the breast. The reduced p27 expression is prognostic for subsequent development of oral squamous carcinoma. In addition, in benign prostatic hypertrophy and low malignant potential of ovarian tumors, the p27 expression levels are decreased compared to normal tissues.Reduced expression and mislocalization of p27 have been identified as an early event in some types of diseases. A study by Mc-Campbell et al demonstrates that loss of p27 expression is an early event in the progression of endometrial carcinoma in the setting of obesity. p27 expression is severely reduced and/or mislocalized to the cytoplasm in histologically “normal” endometrial glands and endometrial complex hyperplasia with atypia from obese women (CAH) as compared to normal weight women. In luteal phase endometrium, p27 expression is high and primarily nuclear. In contrast, in the majority of endometrial CAH, p27 expression is severely reduced or absent in >70% of these early lesions, and is harshly reduced or absent in 89% of primary endometrial carcinoma. These data indicate that loss of p27 is retained as a feature of early (CAH) and neoplastic endometrial lesions arising in the setting of obesity.Tsc2 tumor suppressor gene are a genetically-defined model for endometrial hyperplasia that processes to endometrial carcinoma by 16 months of age.18 At the early stage of this model, appearing “pre-hyperplastic” glands with activated mTORC1 signaling correlate with loss of the wild-type Tsc2 allele. Early life exposure to xenoestrogen accelerates the development of endometrial hyperplasia in adult female rats.24 Similar to human disease, loss of p27 occurs early in association with the development of obesity-associated endometrial hyperplasia. The energy balance intervention study by McCampbell et al demonstrates that caloric restriction is capable of reducing weight, providing a favorable to leptin/adiponectin ratio, and decreasing the circulating insulin levels in response to early life exposure to genistein. Importantly, caloric restriction also significantly decreases hyperplasia incidence with increased p27 expression levels and relocalization of p27 to the nucleus.18For animal study, Eker rats carrying a defect in the 25 and ovarian cancers,26 decreased expression of p27 correlates with reduced survival in response to platinum-based chemotherapy. In breast cancer,27 decreased expression of p27 is associated with poor outcome after adjuvant chemotherapy. In head and neck squamous cell carcinomas,28 p27 expression serves as a significant predictor of chemotherapy response in multivariate analysis.In human, the effect of chemotherapy can also be predicated according to the expression levels of p27 in some types of cancers. For instance, in non-small cell lung cancer30 Does this dietary intervention also work for uterine fibroids through the same mechanism? Further understanding the mechanism and role of p27 may lead to the development of novel treatment options against many challenging diseases.Although progresses have been made to understand the role of p27 in the pathogenesis of diseases, there remains a gap in our knowledge regarding the abnormal expression and subcellular localization of p27, which contribute to the pathogenies of varied diseases. How these events link to the processes of abnormal cell cycle and development of diseases related to the network of signaling pathways and epigenome? What is the role of p27 in favorable and unfavorable effects of chemotherapy? Also, more pre-clinical studies are needed to determine the effect of treatments in varied types of cancers and diseases. For instance, the energy balance intervention study shows a potent inhibitory effect on hyperplasia incidence in Eker rat model. In addition to endometrial hyperplasia, Eker rats are also a genetically-defined model for the development of uterine fibroids.


M2 gangliosides are a group of lysosomal lipid storage disorders that are due to mutations in HEXA, HEXB and GM2A. In our study, 10 patients with these diseases were enrolled, and Sanger sequencing was performed for the HEXA and HEXB genes. The results revealed one known splice site mutation  and three novel mutations  in HEXA. In HEXB, one known mutation (c.1597C>T [p.R533C]) and one variant of uncertain significance (c.619A>G [p.I207V]) were identified. Five patients had c.1597C>T in HEXB, indicating a common mutation in south Iran. In this study, a unique large deletion in HEXA was identified as a homozygous state. To predict the cause of the large deletion in HEXA, RepeatMasker was used to investigate the Alu elements. In addition, to identify the breakpoint of this deletion, PCR was performed around these elements. Using Repeat masker, different Alu elements were identified across HEXA, mainly in intron 5 and intron 10 adjacent to the deleted exons. PCR around the Alu elements and Sanger sequencing revealed the start point of a large deletion in AluSz6 in the intron 6 and the end of its breakpoint 73 nucleotides downstream of AluJo in intron 10. Our study showed that HEXA is an Alu-rich gene that predisposes individuals to disease-associated large deletions due to these elements.G HEXA), hexosaminidase subunit beta (HEXB) and GM2 ganglioside activator (GM2A). The HEXB gene produces a subunit of two related enzymes that include beta-hexosaminidase A and beta-hexosaminidase B. Both enzymes have two subunits. Beta-hexosaminidase A is made from one alpha subunit produced from the HEXA gene and one beta subunit (produced from the HEXB gene). However, beta-hexosaminidase B is only produced from two beta subunits encoded from the HEXB gene. Beta-hexosaminidase A and B, which are mainly found in lysosomes, break down sphingolipids, oligosaccharides and glycoproteins, and are critical in the central nervous system as they have very essential roles in breaking down the GM2 ganglioside in this system, preventing the accumulation of this product and its damage to the nervous system.1–3GM2 gangliosides are a group of lysosomal lipid storage disorders that resulted from disease-causing mutations in three autosomal recessive genes, including hexosaminidase A (http://www.hgmd.cf.ac.uk/ac/index.php), more than 182 disease-causing mutations of the HEXA gene have been identified in patients affected by Tay-Sachs disease (MIM #272800), including missense/nonsense, 96; splicing, 33; small deletions, 28; small insertions, 8; small indels, 3; and gross deletions, 1 (7.6 kb incl. promoter+ex. 1). Regarding the HEXB gene, to date, more than 107 pathogenic mutations in this gene have been reported in patents affected by Sandhoff disease (MIM #268800), including missense/nonsense, 39; splicing, 18; small deletion, 18; small insertion, 3; and gross deletion, 6. These mutations result in the accumulation of GM2 ganglioside in the central nervous system, damaging this systems and presenting the signs and symptoms identified in patients with the corresponding disorders. On the basis of the fully nonfunctional HEXA and HEXB proteins or reduced activity of these proteins resulting from mutations in their genes, these disorders are presented as a severe form found in infancy or a less severe type with a later onset. Tay-Sachs and Sandhoff diseases  are autosomal recessive, progressive neurodegenerative disorders. Their classical infantile forms develop in infancy with developmental retardation, paralysis, dementia, blindness and death usually by 2 or 3 years of age.4–6 A study conducted by Smith et al.3 showed that the clinical findings in infantile-onset disease were generally as follow: hyperacusis with excessive startle response in 85% of individuals; increased myotatic reflexes in 85% of individuals ; a pattern of combined axial hypotonia with limb spasticity in 86% of individuals; visual deterioration in 89% of individuals; and seizures in 84% of individuals. Considering the importance of identifying genetic causes in our patients that are suggestive of Tay-Sachs and Sandhoff diseases, the aim of our study was to identify disease-causing mutations in these patients from the south of Iran and report their clinical findings.According to the Human Gene Mutation Database  according to the manufacturer’s instructions. The genomic DNA concentration was measured by a NanoDrop  and stored at –20 °C until use.Ten unrelated patients suspected of GM2 ganglioside diseases from the south of Iran were recruited in the current study. All patients gave informed consent before undergoing genetic analysis of the HEXA and HEXB genes were amplified by the PCR primers given in https://www.ensembl.org) and the use of NCBI BLAST(https://blast.ncbi.nlm.nih.gov), ENSEMBLE BLAST/BLAT (https://www.ensembl.org), UCSC  and Allele ID 7.5 (a bioinformatics software that helps design primers and probes). The total volume of the PCR reaction was 50 μl, which contained 1 μl of the forward and reverse primers (20 pmol/μl), 3 μl of DNA template (50–200 ng), 25 μl of TEMPase Hot Start 2x Master Mix Blue  and 20 μl of dH2O. PCR was carried out according to the Amplicon TEMPase Hot Start program. Ten microliters of PCR amplicons were visualized on a 2% agarose gel containing SYBR Safe . Then, the PCR products were sequenced using Sanger sequencing, and their results were analyzed using NCBI BLAST and Codon Code Aligner software .All of the exon and exon–intron boundaries of the HEXA gene due to Alu elements, RepeatMasker (http://www.repeatmasker.org) was used with the RMBlast search engine (a version that was compatible with the NCBI Blast tool suite). The Repeat masker program screens DNA sequences of interspersed repeats and low complexity DNA sequences. Sequence comparisons in RepeatMasker are carried out using one of the different search engines, including nhmmer, cross_match, ABBlast/WUBlast, RMBlast and Decypher. RepeatMasker uses information from curated libraries of repeats, Dfam and Repbase. We investigated any Alu repeats  in the whole genomic structure of HEXA, covering a total of 34,695 bp, starting 1.4 kb upstream of exon 1 to 1.4 kb downstream of exon 14. Moreover, to identify the breakpoint of the identified large deletion around exons 6 and 10 in our patient, we performed a second PCR around the predicted Alu elements using the primers given in AluJo and AluSx in intron 5, and the reverse primers downstream of AluJo in intron 10). The PCR products were then used for Sanger sequencing. In addition, a coding sequence translator (http://www.fr33.net/translator.php) was used to predict the possible effect of this deletion on the amino-acid sequence of the HEXA protein.In addition, to predict the cause of a new large deletion in the http://www.ebi.ac.uk/Tools/msa/tcoffee/) was applied to compare the amino-acid sequence of HEXA proteins across different species to obtain information about the conservation of amino acids at the site of mutation.To predict the functional effects and conservation status of a newly identified missense mutation in another patient, we also used different bioinformatics tools, including MutationTaster, MutationAssesor, Polyphen, Grantham, PhastCons, GERP, CADD, Fathmm and SIFT. The T-coffee multiple sequence alignment program : c.919G>A [p.E307K], the deletion of exons 6–10 in a homozygous state and NM_000520 (HEXA-201): c.622delG [p.D208Ifsx15], which are suspected as being homozygous, were identified. One variant of uncertain significance (NM_000521 (HEXB-201): c.619A>G [p.I207V])8 and one known mutation (NM_000521 (HEXB-201):c.1597C>T [p.R533C])9 in HEXB were found in a homozygous state  in HEXA was identified in our patient. Using the repeat masker tool, different Alu elements were found across the HEXA gene , one in intron 7, one in intron 10 flanking exon 10 (the last exon which was deleted in the deleted block) and three Alus at the 3ʹ untranslated region. The exact breakpoint of the deletion around exons 6 and 10 were determined using different PCR products flanking the Alu elements in their corresponding intron-flanked deleted exons. In addition, the consequence of this deletion was predicted to be an in-frame deletion of 192 amino acids of the HEXA protein, leading to its abnormal function , revealing the homozygous status. In her parents, all of the exons produced PCR bands and bands around Alu elements, indicating its heterozygous status  followed by Sanger sequencing, the results revealed that the breakpoints of the large deletion occurred in AluSz6  in the intron 6 and 73 nucleotides downstream of AluJo in intron 10 : c.346+1G>A, IVS2+1G>A)us state . From 10EXA gene , includifunction  and 3. Is status . Using PHEXA was also identified as homozygous in one patient and was confirmed as being heterozygous using Sanger sequencing in her parents . Alu elements are considered to be one of the most successful mobile elements.11 They are ~300 nucleotides in length and have a dimeric structure. The 3ʹ-end of an Alu element has a poly A-like structure, which plays a key role in its amplification mechanism.12–15 There are different subfamilies of Alu sequences that share similar nucleotides in each group, and the most common Alu subfamily in the human genome is the Alu S class (the most common Alu is AluSx).16Alu sequences are responsible for the instability of the genome, which resulted from intrachromosomal and interchromosomal recombination events between these sequences.18 Deletions due to Alu repeats have been identified in different previously reported genes, such as the low-density lipoprotein receptor,19 NACHT, leucine-rich repeat, PYD-containing 720 and HEXB genes.21Alu elements, one of the deletion breakpoints in our study was observed 73 bp downstream of the AluJo element in intron 10 . As shown in AluSz6 and AluJo as the breakpoint occurred in the last nucleotides of AluSz6 and 73 bp downstream of AluJo. Although there is an AluJo in intron 6, its direction is opposite of AluJo located in intron 10 (Alu/Alu occurs). In addition, the similarity between AluSz6 and AluJo is 76% . In addition, in most of the observed events, Alu has a mismatch (homeologous) at the start of recombination.22–24It is worth noting that while most breakpoints of deletions occur in o is 76% , which iAluJo is that that some microhomology was observed at this breakpoint, such as with nucleotide adenine  to lysine, a positively charged, polar amino acid (p.E307K). This amino acid is predicted to be essential for the proper functions of this protein , which lacks all of the amino acids after position 373, caused nonsense-mediated decay. Therefore, all of these amino acids are vital for the normal function of the HEXA protein. Since the only identified damaging variation across HEXA and HEXB in both parents of the patient was this deletion and all of the phenotypes of the patient overlapped with the clinical abnormalities found in Tay-Sachs disease, this frameshift mutation can be reported to be the genetic cause of Tay-Sachs disease in this family.Regarding c.622delG and p.D208Ifsx15, it is obvious that this mutation causes a frameshift deletion that led to a premature translation termination. It is worth noting that one of the reported transcripts of http://exac.broadinstitute.org), was identified as homozygous in one of our patients  that has a high allele frequency (0.15148 for G) in the main database, ExAC (HEXA is an Alu-rich gene and is predisposed to disease-associated large deletions. We also found different mutations in the HEXA and HEXB genes and that the most common mutation in HEXB was c.1597C>T, which suggests that screening the HEXB gene for this prevalent mutation is a cost-effective test in individuals with suspicion of having Sandhoff disease. Such studies can help understand the different possible mechanisms of genetic changes in different diseases and help to investigate large deletions in HEXA and HEXB in individuals affected by Sandhoff and Tay-Sachs diseases who do not have any point mutations or small deletions and insertions. In addition, with identification of all of the Alu elements across HEXA in this study, other researchers can look for any possible deletions more easily in special cases of Sandhoff and Tay-Sachs diseases. Until now, no drugs have been used to treat Sandhoff and Tay-Sachs diseases, and a better understanding of their exact pathomechanism in the future can shed light on therapeutic approaches for these diseases using components involved in their biological processes.Current studies have shown that Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


To evaluate the frequency of NIH category IV prostatitis, and the use of expressed prostatic secretions tests in an effort to improve the reliability of prostate specific antigen as an indicator, to avoid unnecessary prostate biopsy.178 expressed prostatic secretion positive patients with serum prostate specific antigen levels of ≥ 2.5 ng / mL were included in present prospective study. The diagnostic evaluation included detailed history and physical examination, digital rectal examination, urine analysis, urine culture, and expressed prostatic secretions tests. Transrectal ultrasonography was used both to measure prostate volume and conduct 12 core prostate biopsy.The prevalence of NIH category IV prostatitis was 36.9% (178 / 482) in our population of men. In our study patients (n: 178) prostate biopsy results were classified as; 66 prostatitis, 81 BPH, and 31 Pca. In asymptomatic prostatitis group, expressed prostatic secretion mean leucocyte ratio was higher compared to other two groups (p < 0.0001). The relation between number of expressed prostatic secretion leucocytes and prostatitis, benign prostate hyperplasia, and prostate cancer is analyzed. If 16 is taken as the cut of number for leucocyte presence, its sensitivity is 0.92 (AUC = 0.78 p = 0.01).The number of leucocytes in expressed prostatic secretion is higher in the chronic prostatitis group. If the leukocyte presence of 16 and above is taken as the cut off point, the sensitivity becomes 0.92 (AUC = 0.78). We firmly believe that our new cut off value may be used as to aid prostate specific antigen and derivates while giving biopsy decision. In 1979 Wang et al. discovered Prostate Specific Antigen (PSA) . Pca is National Institute of Health (NIH) classified prostatitis into 4 categories in 1995. NIH defined Category IV as asymptomatic chronic prostatitis due to the presence of inflammatory cells either in expressed prostate secretions (EPS) or during histopathological examination of prostatic biopsies from asymptomatic men , tissue  and urine based  prostate cancer biomarker candidates have been presented during the last ten years -15. ThesThe aim of this prospective study was to further investigate the predictive role of the number of leukocytes in expressed prostate secretions in differentiating histological inflammation from PCa and other non neoplastic lesions. Also, to evaluate the use of EPS tests in an effort to improve the reliability of PSA as an indicator, to avoid unnecessary prostate biopsy.After institutional review board  approval, all study participants provided informed written consent before enrolment and 482 men were recruited in the present prospective study. Patients underwent a four-specimen study according to Meares-Stamey method . After pThe prevalence of NIH category IV prostatitis was 36.9% (178 / 482) in our population of men. In our study patients (n: 178) prostate biopsy results were classified as; 66 prostatitis, 81 BPH, and 31 Pca . None ofWhen prostatitis, BPH, and Pca groups were evaluated according to tPSA levels, there was a statistically significant difference between BPH and Pca (p = 0.022). In terms of tPSA, there was no statistically significant difference between the BPH-prostatitis (p = 0.21) groups and Pca-prostatitis groups (p = 0.45) (p > 0.5). In terms of free PSA (fPSA), no statistically significant difference was seen between in the BPH-Prostatitis group (p = 0.99), and in BPH-Pca (p = 0.72), or Prostatitis-Pca (p = 0.84) groups.In the prostatitis group the number of leukocytes in the EPS was higher than in the other two groups (p < 0.0001). The relation between the number of leukocytes in the EPS and prostatitis, BPH, and Pca was also evaluated. The most appropriate cut off value for the amount of leucocytes in the prostate massage fluid was determined as ≥ 395 (in a high enhancement area with a mean of ≥ 16) as shown in The incidence of Pca patients who diagnosed at low stage, increased because of widespread using PSA testing. The rate of detecting Pca clinically in elevated levels of serum PSA ranges between 17.5% and 38% , 19. DetChronic prostatitis is a disease still without a clear etiology. Polymorphonuclear leukocyte invasion is determined in the intra-prostatic channels and peri-prostatic tissue during histopathological analyses , 21. ChrVarious studies about the prevalence of prostatitis subtypes determined by NIH have been conducted. In studies made with needle biopsy analyses, prostatitis incidence ranging on a large scale between 17.2% and 42% have been reported , 23. HowPSA, an important tumor marker, is organ but not cancer specific, studies about new methods verifying PSA results are in progress. To reduce unnecessary biopsies and to improve on PSA specificity, past research primarily investigated PSA derivatives. These have included PSA velocity, PSA density, age-specific PSA, fractionated PSA and percentage of free prostate-specific antigen (% fPSA) . ForexamUrologists often manage asymptomatic men with a high serum PSA level by observation after antibiotic treatment. Because inflammation can often result in elevated PSA at documented chronic bacterial prostatitis patients. But studies examining this subject in the asymptomatic population have yielded conflicting results. While some studies state that serum PSA can be reduced with a course of antibiotics , 6, otheProstatitis should be diagnosed if there are 10 and more leukocytes present in a high enhancement area (40 X). According to the findings of the present study, number of polymorphous nuclear leucocytes in prostatitis group is higher than in BPH and Pca groups (p < 0.0001). Normally, 10 or more leucocyte counts are sufficient for prostatitis diagnosis. However, the number of leukocytes at the prostate massage fluid in the prostatitis group was higher than the other two groups, so that have us an idea.We thought that, if a new cut off value for leucocyte presence in prostatic fluid is determined, the predictability of prostatitis will be increased and thus unnecessary biopsies may be avoided. The outcome of the Roc curve analysis revealed that average leucocyte presence of ≥ 16 or total leucocyte presence of ≥ 395 is most appropriate and should be taken as the new cut off point in a large enhancement area. The analyses revealed AUC = 0.78 and sensitivity for prostatitis as 0.92.In summary, if the number of leucocytes is 16 and above in the prostatic massage fluid, this may most probably an indicator of prostatitis driven PSA increase. Previous to a biopsy decision, this new cut off value may be applied in clinical practice when encountering a asymptomatic patient presenting for the first time with an elevated PSA and without clinical evidence of prostatitis. Also, we recommend in these patients, it may be more beneficial to utilize other tools such as PSA velocity, PSA density, complexed PSA or newer clinical tools such as the prostate health index. Our new cut off value may be used to lend assistance these tools before biopsy decision.Our study has some methodologic factors that might affect accuracy of our estimates. We didn't correlate the EPS results with histology results of our biopsy. Because, we classified chronic inflammation histologically as; available or unavailable. We didn't make detailed classification. Chronic inflammation of the prostate was defined as infiltration of prostate biopsy specimens by inflammatory cells, lymphocytes, plasma cells and / or histiocytes. Irani et al. classified inflammation with regard to the histological grade and aggressiveness of the inflammatory process: histological grades 0 and 1 were regarded as the “non-inflammation group”, and histological grades 2 and 3 as the “inflammation group” . This clThe number of leucocytes in EPS is higher in the chronic prostatitis group. If the leukocyte presence of 16 and above is taken as the cut off point, the sensitivity becomes 0.92 (AUC = 0.78). We firmly believe that our new cut off value may be used as to aid PSA and derivates while giving biopsy decision. Further investigations will be necessary to evaluate our new cut-off value accuracy and determine exact cut-off points.


SDHB and SDHD) mutations represent the most frequent cause of hereditary pheochromocytoma and paraganglioma (PPGL). Although truncation of the succinate dehydrogenase complex is thought to be the disease causing mechanism in both disorders, SDHB and SDHD patients exihibit different phenotypes. These phenotypic differences are currently unexplained by molecular genetics. The aim of this study is to compare disease dynamics in these two conditions via a Markov chain model based on 4 clinically-defined steady states. Our model corroborates at the population level phenotypic observations in SDHB and SDHD carriers and suggests potential explanations associated with the probabilities of disease maintenance and regression. In SDHB-related syndrome, PPGL maintenance seems to be reduced compared to SDHD (p = 0.04 vs 0.95) due to higher probability of tumor cell regression in SDHB vs SDHD (p = 0.87 vs 0.00). However, when SDHB-tumors give rise to metastases, metastatic cells are able to thrive with decreased probability of regression compared with SDHD counterparts (p = 0.17 vs 0.89). By constrast, almost all SDHD patients develop PGL (mainly head and neck) that persist throughout their lifetime. However, compared to SDHB, maintenance of metastatic lesions seems to be less effective for SDHD (p = 0.83 vs 0.11). These findings align with data suggesting that SDHD-related PPGL require less genetic events for tumor initiation and maintenance compared to those related to SDHB, but fail to initiate biology that promotes metastatic spread and metastatic cell survival in host tissues. By contrast, the higher number of genetic abnormalities required for tumor initiation and maintenance in SDHB PPGL result in a lower penetrance of PGL, but when cells give rise to metastases they are assumed to be better adapted to sustain survival. These proposed differences in disease progression dynamics between SDHB and SDHD diseases provide new cues for future exploration of SDHx PPGL behavior, offering considerations for future specific therapeutic and prevention strategies.Succinate dehydrogenase subunit B and D ( Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that arise either in head and neck parasympathetic paraganglia or paraaortic chromaffin tissue, which comprise sympathetic adrenal medulla and extra-adrenal paraganglia. Tumors that derive from either parasympathetic or sympathetic paraganglia are collectively named paragangliomas (PGLs) with the term pheochromocytoma (P) being restricted to adrenal PGL .et al. described the first PGL syndrome related to deficiency in succinate dehydrogenase (SDH) enzyme activity due to mutations in SDH subunit D (SDHD), part of mitochondrial complex II and the tricarboxylic acid (TCA) cycle  (where the superscript “b” stands for SDHB); i.e: 70% will remain without disease corresponding to 30% penetrance, and among patients with active disease: 6% HNPGL and 14% sympathetic PPGL, 10% metastatic disease). For the particular value p11 = 0.75, a transition matrix Tsdhb was estimated and is shown in ijp). This choice is p11 is discussed in the Sensitivity Analysis section.For modeling disease dynamics in sdhb  was: P*sdhb = , describing exactly the clinical picture of the disease Pbobs. Our principal findings show that for SDHB sub-group, the stabilization into a clinical state with the development of PPGL is a process driven by high rate of regression to a state without a  tumor (low p21 and p31). However, when PPGL develops, it easily gives rise to metastasis and metastatic PPGL sustains survival (high p44).The model allowed us to simulate PPGL initiation and spontaneous regression of tumors and demonstrate how these two processes can contribute to the disease phenotype. In this case, the asymptotic probability distribution P*SDHD disease dynamics with the following set of Pdobsv = , where the superscript “d” stands for SDHD . The Tsdhd matrix is shown in sdhd = . These results accurately describe the observed probabilities.A first model was parameterized to simulate SDHD PPGL steady-state values were characterized by high values of p12, p11, p22 and p43 and low values of p21 and p31. Therefore, it seems that SDHD PPGL (mainly those from parasympathetic paraganglia) develop more easily than SDHB ones and persist throughout the life (high p12) of a patient. Furthermore, although SDHD primary PPGL can transition into metastatic disease, it mostly fails to develop a stable advanced disease state and are therefore not diagnosed as metastatic (low p44).The observed SDHB and SDHD carriers have different dynamics in PPGL occurrence, regression, and progression thus, providing new insights into the understanding of these tumors. Tables SDHB and SDHD regarding the probabilities for tumor maintenance or regression. First, tumor maintenance is reduced in SDHB patients (p22 and p33 = 0.04) compared to SDHD patients (p22 and p33 = 0.95). Second, the probability of staying in metastatic state is, however, higher for SDHB than SDHD patients (p44 = 0.83 vs 0.11).Our model corroborates clinical observations by showing that 11) had to be fixed prior to the estimation procedure, which was unavoidable to ensure that the number of free-parameters to estimate was equal to 4, the number of observations, as described in the Sensitivity Analysis section.The sensitivity analysis shows robustness of the model with respect to reasonable variations in the 4 percentages of disease, which supports the validity of the modeling method. One parameter  with a more complex pattern and a greater genomic instability compared to SDH with gains and losses confined to other chromosomes. It is, therefore, possible that chromosome 11p loss is necessary and sufficient to trigger SDHD and SDHAF2 tumorigenesis, whereas SDHB tumors require more complex changes with amplification or deletion of multiple driver genes located on different chromosomes, especially 1p. Assuming a constant rate of genetic instability, this could be in agreement with differences in penetrance of the SDHD and SDHB disease. A second assumption is that additional genetic events contribute to the transformation of PPGL cells into a biology that favours metastatic spread , to be in state i throughout life, which is closest to observed value Pobvs[i]. In others words, mathematically, these transition probabilities were estimated by using the method of the mean least squares, which select the pij, which minimizes the quantity: (P*[1]—Pobvs[1])2 +(P*[2]—Pobvs[2])2 +(P*[3]—Pobvs[3])2 +(P*[4]—Pobvs[4])2.Transition probabilities, which are “invisible” for clinicians, were estimated in order to fit with the limits P* corresponding to the phenotype observed in The following assuptions were made in calculating transition probabilities:p11 was fixed; this choice being discussed below;the transition probability 21 = p31 and p22 = p33;to match the number of free parameters with the number of steady-states, it was assumed that transition probabilities do not depend on tumor location (i.e. head and neck PGL vs. sympathetic PGL): p23 and p32 were fixed at zero;since the coexistence of head and neck PGL and sympathetic PPGL is very rare condition, the transition probabilities p21, p22, p24, p44), the other parameters being either fixed (p11 = 0.75 for SDHB and p11 = 0.10 for SDHD), or set at zero , or constrained by the two assumed relationships (p21 = p31), (p22 = p33), and, obviously, by the fact that the transition probabilities from one state must sum to one. Then the 4 free-parameters were estimated to fit the 4 clinically-defined steady states.These assumptions have allowed reduction of free-parameters to 4: . Therefore, the stability of the model has been assessed by running many computations against different probabilities of stable states drawn at random: the 95% confidence interval for SDHB at state 1 was set to 60–80%  and to 0–10% for SDHD . The uncertainty on the proportion of patients in states  has been described with two independant Gaussian laws of distribution with a relative standard deviation of 20% . The proportion of patients in a metastatic state was logically chosen so that the sum of the four probabilities would be one. For each random observation generated by this procedure, a check was done to verify that the four probabilities were in , then the model was solved with the least-square method already described; the quality of the fit was verified by checking residuals and the output of the solving algorithm and the probabilities of transition were stored. Eventually (after 500 runs), the 95% confidence intervals on the transition probabilities were extracted.Since PPGL penetrance and the proportion of patients with either HNPGL, sympathetic PGL, or metastatic disease are 11, since this parameter has been fixed in the model. Simulations showed a good fit between clinical observations and model predictions in a wide range of p11 values:  for SDHB and  for SDHD. In SDHB-related PPGL, the quality of fit decreased rapidly for values of p11 below 0.70. However, the results remain qualitatively unchanged and lead to the same biological interpretations regarding transition probabilities. For simplicity, it was decided to provide the estimated results of transition probabilities for p11 = 0.75 for SDHB and p11 = 0.10 for SDHD, since these values are linked to the overall disease penetrance of both SDHB and SDHD diseases.The robustness of the estimation procedure has been evaluated for various values of p