Background: Hungarys’s estimated cervical cancer mortality was 6.9/100,000 in 2012, above the average of the EU27 countries  in the same year. Since 2014, the bivalent HPV vaccine has been offered to schoolgirls aged 12–13. (2) Methods: We conducted a cross-sectional study among 1022 high school seniors  in 19 randomly selected schools in Budapest. Our anonymous questionnaire contained 54 items: basic socio-demographic data, knowledge about HPV infection/cervical cancer and HPV vaccination. (3) Results: 54.9% knew that HPV caused cervical cancer, and 52.1% identified HPV as an STD. Knowledge of risk factors such as promiscuity (46.9%) and early sexual activity (15.6%) was low, but higher than that of further HPV-induced diseases: genital warts , anal cancer , penile cancer (9.4%), and vulvar cancer (7.8%). A percentage of 14.6% feared getting infected, and 35.7% supported compulsory HPV vaccination. A percentage of 51.2% would have their future children vaccinated—significantly more girls than boys. (4) Conclusion: Our results support the findings of previous studies about young adults’ HPV-related knowledge, which was poor, especially regarding pathologies in men. Despite the low level of awareness, the students’ attitude was mostly positive when asked about vaccinating their future children.(1)  In Hungary, the mortality rate of cervical cancer was 6.23/100,000 in 2012, which is nearly two times higher than the average  of the European Union (EU27) member countries in the same year [Currently, there are three different HPV vaccines available. All of them are recombinant and assembled from the virus-like particles (VLP) of the L1 capsid protein. While the bivalent vaccine immunizes against serotypes 16 and 18, the quadrivalent protects against LR HPV serotypes 6 and 11 as well. The 9-valent vaccine has only recently been approved by the U.S. Food and Drug Administration (FDA) in December 2014. This vaccine contains HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58. The US Centers for Disease Control and Prevention (CDC) recommends the use of all three vaccines from 9–26 years of age in both sexes [In Hungary, 7th grade schoolgirls (12–13 years old) are offered two doses of the bivalent HPV vaccine nationwide since September 2014 ,6. In coSince the prevalence of HPV is the highest in the late teens and early twenties ,13,14, tUsing the online database of the Educational Authority, we randomly selected 19 high schools in Budapest . First, . Our sample represented about 2% of the high school seniors studying in Budapest during the academic years 2012/13 and 2013/14 [In our questionnaire, 26 matrix questions concerned basic demographic, socio-economic, and lifestyle factors, 13 questions assessed knowledge about HPV infection and cervical cancer, 11 examined the attitude toward the HPV vaccine, and 4 focused on cervical cancer screening. The overall response rate of 1277 distributed questionnaires  was 80%  2013/14 .p < 0.05 significance level and calculated odds ratios (ORs) with 95% confidence intervals—using IBM-SPSS v.23 .Individuals refusing to answer specific questions were excluded only from the evaluation of the questions concerned. Questionnaires were not processed if items concerning HPV infection and cervical cancer were left blank, or when multiple answers were given to single choice questions. We performed frequency analyses and Pearson chi-square tests—at N = 1022) consisted of 492 female (48.1%) and 530 male (51.9%) students. They studied in vocational (65.4%) and grammar schools (34.6%). The majority (57.4%) wanted to pursue education after high school graduation. 31.5% of mothers and 25.1% of fathers had undergraduate or graduate degrees. Percentages of 11.4% of mothers and 1.6% of fathers were healthcare providers. The majority of students (82.8%) perceived their family’s economic status as up to the national average or above it. A percentage of 25% admitted to being affiliated with religious organizations. A percentage of 75% didn’t follow any specific diet, 36.9% regularly practiced some kind of physical activity, and 89.9% used the Internet for at least 1 h a day.Our sample of high school seniors (N = 1022). The majority (64.4%) knew that HPV caused cervical cancer; however, they were less aware of other related diseases. Genital warts were recognized by less than 10%  and anal cancer by less than 3% . A percentage of 21.5% falsely believed that HPV could cause female infertility, and 14.5% were not aware of HPV-induced pathologies in men. A percentage of 13.7% considered the virus as responsible for prostate cancer. “I don’t know” answers were relatively frequent, especially for questions concerning pathologies in men (59%). N = 1022).Concerning the ways of transmission , the majN = 1022).Nearly half of the sample (46.9%) knew that promiscuity was a risk factor for HPV infection, and 41.7% considered unprotected sex to be a risk factor . Early iThe majority (66.7%) knew that cervical cancer screening and HPV vaccination (58.3%) were means of prevention .Considering the information sources of HPV infection/cervical cancer , studentn = 492 vs. n = 530). More female than male students knew that HPV could cause cervical cancer  and genital warts in women . More females than males believed that HPV could cause infertility . As for spreading the infection, more females than males knew that HPV was an STD . While exploring associations and ORs , we compCompulsory vaccination was supported by 91.2%, and 58.0% believed it was important. The majority (73.2%) had heard about the HPV vaccine, 16.1% admitted to having already received it, and a further 10.7% wished to be vaccinated in the future. While exploring the motivations behind accepting or rejecting the vaccine, we found that only 14.6% realized their own risk of getting infected by the virus. When asked about the efficacy of the vaccine, 58.7% trusted it with some doubts, while 24.3% had no doubts at all. A percentage of 35.7% of the sample preferred compulsory vaccination, and the slight majority of students (51.2%) would have the vaccine administered to their future children .Gender-related attitudes are demonstrated in We also explored the difference between the attitudes of vaccinated and unvaccinated students. Those who had already been immunized or opted to be vaccinated in the future had significantly more positive attitudes than the unvaccinated subsample. Vaccinated students were more likely to have families supportive of mandatory vaccination .According to previous international studies, disease awareness has a positive impact on the attitude of young adults toward HPV vaccination; thus, it must be considered as a protective factor ,20,21. TOur sample of high school seniors in Budapest had relatively poor knowledge about HPV infection and cervical cancer. While the majority (64.4%) was familiar with the etiology of cervical cancer, they were not aware of other pathologies caused by the virus in the anogenital region: less than 10% linked the virus to genital warts, less than 3% to anal cancer, and 9.4% to penile cancer.The knowledge about risk factors for HPV infection was also low—only promiscuity and unprotected sex were recognized as risk factors by over 40% of the sample. Concerning the ways of transmission, 52.1% knew that the disease could be mediated by sexual intercourse; skin contact was only identified by 3.1%. An alarmingly low number of students (14.6%), albeit significantly more girls than boys, recognized their personal risk of infection. In terms of prevention, around two-thirds of the sample knew about cervical cancer screening (66.7%) and HPV vaccination (58.3%). Finally, it must be stressed that the number of “I don’t know” answers was relatively high for questions assessing the knowledge and attitude of the sample.The students’ source of information was diverse: 23.9% obtained their knowledge from the Internet, 23.1% from family members and friends, and 20.5% from TV/radio. Surprisingly, healthcare professionals played only a marginal role in providing information about the disease. The most frequently consulted providers were gynecologists (13.1%), while GPs and registered nurses represented nearly the same proportions (11.4% and 10.5%). This low representation may be explained by the specific age and health status of the target population. Young adults are generally healthier; therefore, they have less contact with members of the medical staff than the ageing population. It must be emphasized that the students gained information typically from online platforms, which underlines the role and responsibility of public service and social media in health education.Female students had significantly higher level of knowledge than males. Since cervical cancer is a pathology affecting only women—targeted by a national screening and vaccination program—it is understandable that females have more knowledge about the disease and its causative agent (HPV). Furthermore, school-based HPV-specific sexual education programs tend to focus on girls rather than boys. It must also be considered that girls, when entering procreative age and starting sexual life, usually visit a gynecologist who provides them with information about HPV infection and cervical cancer. Nevertheless, analyzing the deeper knowledge of young women concerning health issues should be the subject of further investigations.In Hungary, the vaccine was licensed in 2007 but was only available on private financing. Since September 2014, the bivalent HPV vaccine is administered free of charge among schoolgirls aged 12–13 (7th graders) as part of the public vaccination program. Our research was conducted prior to this date (March 2013–May 2014); thus, it provides baseline data for future comparative studies.The majority of the students in our sample had already heard about the HPV vaccine (73.2%). Significantly more male students reported that their families supported mandatory vaccination; nevertheless, significantly more females than males were informed about the HPV vaccine. Almost 80% of the sample believed in its efficacy, although around 60% had doubts, despite their positive attitudes. Significantly more females than males trusted the efficacy of the vaccine. A percentage of 16.1% reported to having already been vaccinated, and an additional 10.5% opted for it in the future. More than half of the sample (51.2%)—significantly more females than males—would have their future children vaccinated. Compulsory vaccination of HPV was supported by 35.7% of the sample ; however, 32.9% rejected it, while the rest of the students remained undecided.Students already vaccinated or wishing to be vaccinated in the future were more supportive of immunization against HPV than the rest of the sample. This finding is consistent with the results of previous studies . Female In certain countries , the vaccine is offered to both males and females; nevertheless, its cost-effectiveness among the male population is still debated ,32,33,34Due to the low level of knowledge of high school seniors, health and sexual education of young adults should be reconsidered in Hungary, especially for the male population. It should be emphasized that HPV infection is sexually transmitted; thus, both sexes are affected, and awareness of the disease is a protective factor. Despite their lack of knowledge and low self-perceived risk, the students mostly supported the HPV vaccination program; however, higher vaccine acceptance and better vaccination coverage could be achieved by the implementation of more tailored health and sex education programs. We have been unaware of the existence of any similar nationwide campaigns in Hungary since data collection began.


The aim of this study was to assess the possible role of HPV in the development of prostate cancer (PCa) and investigate the distribution of the p53 codon 72 polymorphism in PCa in a Turkish population. A total of 96 tissues, which had been obtained using a radical surgery method, formalin-fixed and parafin-embedded, were used in this study. The study group consisted of 60 PCa tissues  and the control group contained 36 benign prostatic hyperplasia tissues (BPH) . The presence of HPV and the p53 codon 72 polymorphism was investigated in both groups using real-time PCR and pyrosequencing. The results of the real-time PCR showed no HPV DNA in any of the 36 BPH tissue samples. HPV-DNA was positive in only 1 of the 60 PCa samples (1.7%). The HPV type of this sample was identified as HPV-57. The distribution of the three genotypes, Arg/Arg, Arg/Pro and Pro/Pro was found to be 45.6, 45.6, and 8.8% in the PCa group and 57.1%, 34.3% and 8.6% in the control group, respectively. Compared with the control group, patients with PCa had a higher frequency of the Arg/Pro genotype and Proline allele (odds ratio (OR)=1.67, 95% confidence interval (CI)=0.68-4.09, p=0.044; OR=1.13, 95% CI=0.76-1.68, p=0.021, respectively). The results of the study do not support the hyphothesis that prostate cancer is associated with HPV infection but indicated that Proline allele can be a risk factor in the development of PCa in the Turkish population. Prostate cancer (PCa) is the most common non cutaneous cancer and the second leading cause of male cancer-related death in the Western countries . Risk faThe prostate can be a target for human papillomavirus (HPV) infections due to its anatomical location . Human pMany studies have been conducted to explore the association between HPV infection and PCa; however, the possible role of HPV infection in the pathogenesis of PCa remains controversial -14. ManyThe most commonly mutated p53 tumor suppressor gene is located on chromosome 17p13 and encodes the tumor supressor protein called the “guardian of the genome”. This protein has an important role in many cellular process such as cell cycle arrest, DNA repair and apoptosis , 16.The p53 gene has several single-nucleotide polymorphisms. The most common is the codon 72 polymorphism located on the exon 4 . In the 72nd codon of the p53 gene, only one nucleotide alternates the amino acid from Arginine (Arg) (CGC) to Proline (Pro) (CCC). This alternation of the amino acid affects the biochemical and functional characteristics of the p53 protein. The Pro variant strongly activates transcription; however, the Arg variant induces the apoptosis , 17.Despite the abundance of studies on the association between PCa and the p53 codon 72 polymorphism, the results are contradictory , 18-22. The aim of the current study was to explore the presence of HPV and the distribution of the p53 codon 72 polymorphism in surgical specimens of localized prostate cancer in a Turkish population using real-time PCR and pyrosequencing methods.Formalin-fixed paraffin-embedded (FFPE) tissue samples were selected from July 2011 to June 2014 archive collection of the Pathology Laboratory of the Dıskapi Training and Research Hospital, Ankara. To eliminate the possibility of urethral and anal HPV contamination, only samples that had been obtained through radical surgery method were included in the study. The study group consisted of 60 prostate adenocarcinoma tissues  and the control group was composed of 36 benign prostatic hyperplasia (BPH) tissues . All the study and control cases were of Turkish ethnic origin. None of the patients had received neoadjuvant radiotheraphy or chemotheraphy. The study was approved by the Ethics Committee of Erzincan University.Tissue sections of 3µm wide were stained using hematoxylin eosin and the slides were evaluated by two expert pathologists in terms of the presence of cancer and confirmation of the first histological diagnosis. The histological sections were than manually dissected to determine the area of the tumor for DNA extraction. From each block, 5-10µm sections were obtained for DNA extraction. Samples displaying the characteristics of adenocarcinoma cell infiltration or hyperplasia were selected. These steps were repeated as required until 90% of neoplastic cells were visible.Tumors were graded according to the Gleason scoring system, and staging was performed according to the 2009 TNM classification . FinallyTissue sections of 5-10µm thickness were cut from each tissue block and placed in 2mL sterile tubes. For deparaffinization, the samples were incubated in 1mL xylene for 10 seconds, centrifuged at 12.000rpm for 2 minutes and the supernatant was removed. To remove the remaining xylene, the samples were washed with 1mL absolute ethanol. Following a 2-minute centrifuge at 12.000rpm, the pellets were air-dried for 10 minutes to remove the ethanol. The genomic DNA isolation from the FFPE tissue samples was performed using a QIAamp DNA FFPE Tissue Kit  according to the manufacturer’s recommendations. DNA was eluted in 50µL of buffer ATE and the DNA concentration was measured using the NanoDrop ND-1000 instrument , and then stored at-20ºC until use.TM software version 1.0.5.0 .The presence of HPV in the tissue samples was investigated using mixed primers based on broad-spectrum HPV-DNA amplification and targeting the variable region of HPV L1 ORF. A real-time PCR (EVA GreenTM chemistry) and an HPV sign® Q24 complete kit  able to identify HPV types in a broad spectrum in the Rotor Gene machine were utilized. To control inhibition, the primer set included with the kit for the detection of Human Beta-globin (β-globin) was used. The HPV sign® Q24 complete kit was used according to the manufacturer’s recommendations. Each experiment contained at least one negative amplification control (water), HPV sign® positive control and h-DNA control (β-globin control). The presence or absence of HPV DNA was determined using a melting curve analysis. Pyrosequencing was performed on samples that were found to be HPV positive in the melting curve analysis using four specific sequencing primers and the Pyromark Q24 pyrosequencing instrument . Genotype-specific sequencing primers that allowed for the synthesis of 30 base sequences were chosen. These sequences were compared with other sequences in the HPV library and identified using the HPV genotype IdentiFireThe genomic DNA isolation for p53 genotyping was performed as described in the previous section. Four samples were not genotyped due to low DNA quantities. The primers were designed using the pyrosequencing assay design software 2.0 . The PCR amplification of a 155-bp fragment of the p53 gene exon 4 was performed using forward (5’-AGACCCAGGTCCAGATGAAGC) and reverse (5’-biotin CGTAGCTGCCCTGGTAGGTT) primers  with the PyroMark PCR Kit . PCR reactions were performed in a 25µL mix containing 20ng genomic DNA and 5mM of each primer using the following protocol: initial denaturation at 95ºC for 15 min., followed by 45 cycles of 95ºC for 30 sec, 62ºC for 30 sec and 72ºC for 30 sec, and a final extension for 10 min. at 72ºC.Pyrosequencing reactions were performed using the PyroMark Gold Q24 reagents  and the PyroMark Q24 instrument according to the manufacturer’s recommendations. For the pyrosequencing, single-stranded DNA templates were obtained using PyroMark Q24 Vacuum Prep Workstation  according to the manufacturer’s recommendations. Briefly, a 10µL PCR product was immobilized in Streptavidin-coated Sepharose High Performance beads , and processed to obtain single-stranded DNA. The template DNA was incubated at 80°C in a heat block for 2 min. with 25μL of 0.3μmol/L sequencing primer (5′-ATGCCAGAGGCTGCTCCCC) specific for codon 72. To assess the quality of genotyping and raw data, the PyroMark Q24 software  was used .The data was analyzed using the SPSS 17.0 . For the analysis of categorical values, the chi-square test (χ2 test) and Fisher’s exact test were used. p<0.05 was regarded as statistically significant. The chi-square (χ2) test was performed to observe the frequency of the genotypes. The expected values of the genotype frequencies were calculated using the Hardy Weinberg equilibrium (HWE). The odds ratio (OR) and its 95% confidence interval (CI) were determined to measure the correlation between p53 codon 72 Arg/Pro polymorphism in study and control groups.A total of 96 male patients, 60 with PCa and 36 with BPH, were included in the study. 13.3% of patients with PCa and 44.4% of BPH patients were aged above 70. The mean age of total cohort was 63.04±6.67 years (range 48-75) and 67.94±8.04 years (range 53-89) in the PCa and BPH patients, respectively (p=0.089). Of the PCa patients, 29 (48.3%) were in stage T2, 30 (50%) in T3 and 1 (1.7%) was in T4. Furthermore, the Gleason score was found to be ≤6 in 29 patients (48.3%), 7 in 24 patients (40.0%), and ≥8 in the remaining 7 patients (11.7%). According to ISUP’s new grading system, 29 (48.3%) patients with prostate cancer were classified as Group 1, 14 (23.3%) as Group 2, 10 (16.7%) as Group 3, 3 (5.0%) as Group 4, and 4 (6.7%) as Group 5.® Q24 complete kit. In the Rotor gene Q analysis, all 96 samples (100%) were found to be positive in terms of human β-globin. No HPV DNA was detected using the real-time PCR (EVA GreenTM chemistry) with mixed primers targeting a hypervariable region of the HPV L1 ORF in any of the 36 BPH samples. One of the sixty (1.7%) PCa samples was found to be positive for HPV DNA using real-time PCR. Pyrosequencing analysis showed this sample had one type of HPV, namely HPV-57. The HPV-positive sample was classified as pT2cN0M0 and had a low Gleason score .The presence of HPV DNA was investigated in 60 (62%) PCa tissues and 36 (38%) BPH tissues. To assess the quality of isolated DNA, the β-globin gene was amplified using the real-time PCR method using the HPV signDue to the low quantity of the isolated DNA, p53 genotyping could not be performed on three PCa samples and one BPH sample. The genotypes identified in the p53 codon 72 polymorphism of 57 PCa samples were distributed as follows; Arg/Arg genotype in 26 samples (45.6%), Pro/Pro genotype in 5 (8.8%) and Arg/Pro genotype in 26 (45.6%). Of the 35 BPH samples, 20 (57.1%) had the Arg/Arg genotype, 3 (8.6%) had the Pro/Pro genotype, and 12 (34.3%) had the Arg/Pro genotype.Comparing the study and control groups based on these results, the Arg/Pro genotype was more frequently seen in patients with PCa than those with BPH and the difference was statistically significant . However, no statistically significant difference was found between PCa and control groups in terms of the frequency of the Arg/Arg and Pro/Pro genotypes .The frequency of the Arg allele in study and control groups was 72.1% and 77.1%, respectively. Pro allele was observed in 27.9% of PCa patients and 22.9% of BPH cases. In this study, it was also found that the frequency of Pro allele was higher in the PCa patients, thus it can be postulated that Pro allele is associated with PCa .The p53 codon 72 polymorphism in patients with prostate cancer was investigated comparing the results from Gleason score, ISUP’s new grading system and pathologic staging . The higPCa is the most commonly diagnosed cancer in men. However, the etiology and molecular pathobiology of PCa is still not clear. The viral etiology of prostate carcinogenesis, which includes environmental, endogenous and genetic risk factors as well as HPV, is controversial .HPV DNA was first detected in PCa and BPH tissues using PCR in 1990 in a study by McNicol and Dodd; however, the authors did not find a significant difference between the two groups . The conFrom 1990 to December 2014, there are 40 papers in the literature (excluding case reports and reviews) reporting on the results of tissue-based studies investigating the association between HPV infection and PCa. Of these, thirty were case-control studies while the remaining 10 only analysed the PCa samples. The prevalence of HPV infection in PCa samples has been reported in range of %0 to 100% .Only 4 of the 40 studies suggested a potential association between the HPV infection and PCa and found a statistically significant difference between the PCa patient group and the control group. One of the early studies was conducted by Anwar et al.  who idenLeiros et al.  reportedCuzick and Strickler suggested that early studies reported a higher positivity compared with the results of recent studies which found more negativity, and this might be due to HPV contamination with nearby tissues during sampling since HPV DNA was detected in the urethral and anal tissues. Based on this information, some researchers suggested using tissues obtained from radical surgery and microdissection of the neoplastic sample , 29.In the current study, to prevent the HPV contamination of transurethral approach, all samples were obtained using open radical prostatectomy (for the PCa samples) and transvesical open prostatectomy (for the BPH samples). Furthermore, all the tumor samples were reviewed for a second time by two expert pathologists and only the sections that contained tumor cells were included in the analysis of the presence of HPV DNA.The remaining 36 studies in the literature, however, did not report any difference between the PCa patients and the control cases in terms of the presence of HPV DNA. In the current study, only 1 of 60 PCa samples was found to be HPV DNA positive whereas all 36 BPH samples were HPV negative. Using the pyrosequencing analysis, the type of HPV positive sample was identified as HPV-57, which is considered to be low-risk and associated with skin lesions. A retrospective assessment of the patient with HPV-57 showed that the patient had genital wart. It was considered that the PCa sample was contaminated during the open radical prostatectomy. Despite the difficulty of presenting conclusive evidence for negative results, our DNA samples were of sufficient quality to amplify the human control gene (β-globin), and the internal amplification control showed that the extraction protocol did not inhibit the PCR.The data obtained in the current study is in agreement with the results of two studies; one by Bergh and the other by Sfanos. Bergh analyzed 352 samples (171 PCa and 181 BPH) and Sfanos investigated 200 PCa samples. Neither detected HPV-DNA in prostate tissue samples , 12.Storey et al. (1998) showed that the p53 homozygotes pose a high risk for the patients in terms of developing HPV-associated cervical cancer . Since tIn recent years, researchers have suggested that the p53 codon 72 polymorphism has a significant role in the development of tumors and the progression of PCa; however, contradictory results have been reported -22. Wu eOn the other hand, four studies did not find any association between the p53 codon 72 polymorphism and PCa. Huang et al.  suggesteMichopoulou et al. (2014) who conducted a study with 50 samples obtained from a Greek population, found HPV positivity in 8 PCa samples (16%) and 1 control sample (3.3%) using the real-time PCR method. Furthermore, the authors explored p53 codon 72 polymorphism in the same patients and found the distribution of three genotypes namely Arg/Arg, Arg/Pro, and Pro/Pro to be 69.6, 21.7, and 8.7%, respectively in the PCa group, and 75.0, 17.86, and 7.14%, respectively in the healthy control group. The authors did not report a statistically significant difference between HPV presence and factors such as age, stage, p53 codon 72 polymorphism and PCa .Due to these contradictory results and the lack of reliable data on the genotype distribution of the p53 codon 72 polymorphism in a Turkish population, we conducted the current research with PCa patients and a control group. Comparing the genotype frequency of the two groups, it can be postulated that in the Turkish population, the Arg/Pro genotype and Pro alleles are more frequently seen in patients with prostate cancer.The apoptosis-stimulating proteins (ASPP) of the p53 family regulate the function of apoptosis of the p53 codon 72Arg / Pro polymorphism. The apoptosis function of the p53 codon 72Pro variant is selectively inhibited by apoptosis-stimulating protein inhibitors (iASPP). The strong capacity of the p53 codon 72Arg variant to induce apoptosis results from its ability to escape from iASPP inhibition and its even greater abilitiy to localize to the mitochondria. Apoptosis is less frequently seen in people with the p53 codon 72Pro/Pro genotype compared with those with the Arg/Arg genotype and therefore Pro allele is more susceptible to the development of cancer . This isAs in all research studies, this study also has certain limitations. Since the tissue samples had been obtained from patients with localized prostate cancer treated with radical prostatectomy, the number of samples was relatively smaller and thus the statistical power and potential bias of the study were limited. We did not have access to these samples because surgical treatment is not among the treatment options for advanced stage prostate cancer and metastatic cases. Furthermore, prostate biopsy is used in the follow-up of patients with high grade PIN; therefore, the tissues of these patients were not available. In addition to that this was a retrospective study, in which we did not have access to the blood samples of the patients, we could not perform an HPV serology. The last limitation of the study was that the control group consisted of samples with BPH; however, a control group with normal prostate tissue may have been more appropriate to show the association between prostate carcinogenesis and HPV infection.This is the first study that investigated the etiological role of HPV and the p53 codon 72 polymorphism in the development of PCa in a Turkish population. The results of the present study do not support the hyphothesis that prostate cancer is associated with HPV infection but indicate that Proline allele can be a risk factor for the development of PCa in the Turkish population. Further studies with larger series are needed to investigate the potential role of HPV in prostate carcinogenesis.Int Braz J Urol. 2017; 43: 36-47


Background: Over the next 20 years, oropharyngeal cancers (OPC) will represent the majority of head and neck cancers (HNCs) in the United States. It is estimated that human papillomavirus (HPV) may account for as much as 70% to 80% of OPCs in North America and in certain parts of Europe. It is hence crucial to understand the disease risk factors and natural history of oral HPV infections. We hypothesized that poor oral health  leads to a higher degree of oral HPV-16 infections within a patient cohort from a dental school clinic. This study aims to test this hypothesis and gauge possible disease associations before larger scale studies. Subjects and Methods: 223 participants were recruited in this study from the University of Queensland Dental School clinic. Clinical oral health parameters  have been examined and determined by dental professionals. We have collected oral rinse samples from these volunteers. Results: 10 (4.5%) out of 223 participants were found to have HPV-16 DNA in their oral rinse samples using NB2 endpoint PCR and Sanger sequencing. Within the HPV-16 DNA positive subjects, 7 (70%) and 3 (30%) were associated with poor oral hygiene and periodontal disease, respectively. Conclusion: Our results show a trend towards a positive correlation between oral HPV-16 infection and poor clinical oral health status. Over the next 20 years, oropharyngeal cancers (OPC) will represent the majority of head and neck cancers (HNCs) in the United States , while oHPV is a double-stranded DNA virus, infects basal cells of the epithelium lining and has been documented to cause various epithelial lesions. More than 30 genotypes of mucosotropic HPV have been identified with low-risk types  being associated with benign lesions and high-risk types  being associated with malignant conditions  ,13. HPV-The oral cavity and oropharynx are lined with various types of specialised epithelia  . Due to n = 223) with both good and bad oral hygiene and determined their periodontal disease status, as well as other oral health parameters. This is the first study to report clinical oral health parameters examined and evaluated by dental professionals and not self-reported oral health status. We found that oral HPV-16 infection within the Australian Dental study group is 4.5%. Our results demonstrated a positive correlation between oral HPV-16 infection and poor oral health status.We hypothesised that poor clinical oral health  leads to a higher degree of oral HPV-16 infections within this dental school patient population. To test this hypothesis, we recruited participants  Clinic between October 2014 and October 2015 were recruited as study participants see . All parParticipant inclusion criteria included (1) willingness to participate and provide written informed consent; (2) never received HPV vaccination; (3) older than 18 years of age  ,25,26,27Participant demographic information included in this study was collected for gender, age and race see . ParticiParticipant clinical oral health measures were recorded, including plaque level, supra-gingival calculus level, sub-gingival calculus level , periodontal screening record (PSR)  and calculus present, no periodontal pocket probing depth greater than 3.5 mm; score 1 = BOP present, no calculus present, no periodontal pocket probing depth greater than 3.5 mm; score 2 = BOP and calculus present, no periodontal pocket probing depth greater than 3.5 mm; score 3 = BOP and calculus present, periodontal pocket probing depths greater than 3.5 mm but less than 5.5 mm, score 4 = BOP and calculus present, periodontal pocket probing depths greater than 5.5 mm)  and decaParticipants were grouped based on their periodontal disease status. If any of the six PSR scores were greater than 3, the patient was classified as a periodontal-diseased patient . If noneAppropriate environmental controls and blinded blank saline samples were collected at the UQDS to ensure that the sample are not contaminated by HPV-16 virus aerosols. Prior to sample collection, participants were asked to rinse their mouths with water to remove any food debris and other irrelevant material. Participants were then instructed to use 20 mL of 0.9% saline to swish and gargle alternatively for 30 s  and spit the sample into a sterile collection tube as per our previous work . The samOral exfoliated cell pellets were resuspended in sterile phosphate-buffered saline (PBS) and DNA was extracted using the QIAmp DNA Mini Kit, Catalog number 51304  according to the manufacturer’s instructions. DNA samples were assessed for purity and quantified on a Nanodrop 1000 Spectrophotometer  see .Two rounds of end-point PCR were used to detect HPV-16 DNA extracted from oral rinse samples. HPV-16 specific primers were used to amplify a region of the E7 gene. Human specific primers were used to detect β-globin as a reference gene to normalise for DNA input and integrity. HPV-16 primer sequences; NB2 Forward primer 5′-AGTGTGACTCTACGCTTCGG-3′, NB2 Reverse primer 5′-CTGCAGGATCAGCCATGGTA-3′. β-globin primers sequences; Forward primer 5′-CAACTTCATCCACGTTCACC-3′, Reverse primer 5′-GAAGAGCCAAGGACAGGTAC-3′. The specificity of NB2 primers was ascertained by isolating DNA from the HPV-positive cell line (SCC-2) and amplifying, followed by Sanger sequenceing identification see .The PCR setup consisted of 50 ng of DNA isolated from oral rinse samples, 1 µM of each primer, 1× Emerald AMP MAX HS PCR Mastermix  in a final volume of 12.5 µL. PCR cycling consisted of an initial denaturation at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 30 s; annealing at 65 °C for 30 s for HPV-16 NB2 primers, or 60 °C for β-Globin primers; and extension at 72 °C for 30 s. A final extension at 72 °C for 5 min before cooling to 12 °C was done. A 1 µL volume was transferred to another PCR setup tube and the above reaction conditions were repeated. The entire volume from the second post-PCR samples were subjected to gel electrophoresis at 120 V for 50 min in a 3% agarose gel prepared in TAE buffer .® Terminator v3.1 Cycle Sequencing Kit Catalog number: 4337458 . Sanger sequencing clean-up and separation was performed by the Australian Equine Genetics Research Centre, University of Queensland. Specifically, BigDye sequencing reactions were cleaned up with the Agencourt CleanSEQ System . Capillary electrophoresis and fluorescence detection of cleaned-up reactions were done on either a 3130XL or 3730 Genetic Analyzer . Sanger sequencing results from NB2 amplicons were analysed using the Standard Nucleotide—Basic Local Alignment Search Tool (BLAST) on the National Centre for Biotechnology Information (NCBI) website to confirm HPV-16 homology  according to the manufacturer’s instructions. Gel purified NB2 amplicons were used in single primer PCR’s (NB2 reverse primer) with BigDyelogy see .p-values  for variables with 2 × 2 contingency tables ; for variables with contingency tables greater than 2 × 2 , Monte Carlo method  was used to obtain p-values . p-values less than 0.05 were considered significant. If the p value for a specific variable was significant, post-hoc testing was performed to determine p-values for each group within the variable. A significant p-value indicated that a specific sub-group within each variable was significantly associated with HPV-16.In order to examine the potential association between oral health and HPV-16 infection, HPV-16 DNA positivity results were analyzed against patient oral health status and periodontal disease status, as well as other recorded individual parameters , using Fisher’s exact test. IBM SPSS v22  was used as data analysis software. Fisher’s exact method was used to obtain n = 223) were recruited from UQDS. Ten (4.5%) of these participants were confirmed to be HPV-16 DNA positive using NB2 endpoint PCR followed by Sanger sequencing confirmation.Participants (Participants had a median age of 41 years and were distributed such that 53 participants (23.8%) were 18–30 years old, 106 (47.5%) subjects were 31–50 years old, 60 (26.9%) subjects were 51–70 years old, 4 (1.8%) were 71–90 years old. While more male 137 (61.4%) than female 86 (38.6%) participants were recruited, the age and gender differences between participants were not statistically significant. Demographically, 166 (78.3%) participants were identified as Caucasians, 26 (12.3%) Asians, 10 (4.7%) South Asians, 4 (1.9%) Aborigine or Pacific Islanders, 1 (0.5%) African, 4 (1.9%) Latino and 1 (0.5%) other race.The prevalence of daily smoking amongst participants was 19.0%, which favorably compared with previous data from a similar UQDS population (22.9%) . A signip = 0.147).More than half (52.1%) of recruited participants were identified as regular consumers of alcohol, with 10.6% consuming more than one or two standard drinks per day. In total, 35 participants did not disclose their alcohol consumption patterns. A broad variation of alcohol consumption habits was noted amongst both HPV-16 DNA positive and HPV-16 negative participants. Neither frequency nor engagement in infrequent or frequent alcohol consumption were found to be associated with HPV-16 DNA prevalence (p = 0.259). While periodontal disease status was not significantly associated with the presence of HPV-16 DNA (p = 0.367), a large periodontal disease burden (40.5%) was noted within the recruited participants requiring further dental assistance.Almost all participants recruited demonstrated some level of plaque retention (98.6%). Supra (98.6%) and sub-gingival (90.0%) calculus presence was a common clinical finding amongst recruited participants. Neither the presence nor severity of these clinical parameters of gingival health was associated with HPV-16 DNA prevalence. Participants were also categorised based on their DMFT score. Of the study cohort, 35.9% (79) had a low measure of dental decay (past/present), whilst 42.7% (94) of participants experienced a moderate level of DMFT and 21.4% (47) of participants recorded a high score of DMFT. Nevertheless, DMFT score was not significantly associated with HPV-16 DNA infection  and Antonsson et al. (2.3%) . Men shoA range of potential risk factors for oral HPV-16 infections, such as close contact with HPV-positive parents during early childhood , or mateIt has been suggested by Tezal et al. that oral HPV infections may modify periodontal disease, and interact in a complex manner ,36,40. IThe major limitations in our study are the sample size. It is likely insufficient to detect a significant increase in HPV-16 infection associated with poor oral hygiene, as a minimum sample size of 150 per group would have been required for this purpose . Another important consideration regarding the participants of this study is that the majority are patients who required treatment due to active oral disease within a university clinic setting. Consequently, the prevalence of poor oral hygiene and dental and periodontal infection in the study participants was not necessarily reflective of the general population in Australia.In our pilot study population, the prevalence of HPV-16 DNA was found to be 4.5%. The results from this study highlight a positive correlation between oral HPV infection and poor hygiene, which has previously been documented to be an independent risk factor of HPV infection. Future studies with a larger study population  with active clinical oral health parameters may yield significant results. It has been advocated that general dental practitioners should advise patients about oral sexual behavior and HPV infection risk. However, for this practice to become part of routine dental consultative practices, many cultural and social hurdles need to be overcome  and clea


To find predictive markers for response to treatment, and correlations and differences in mutated oncogenes and suppressor genes between HPV+ TSCC/BOTSSCC and HPV+ HNCUP and HPV− TSCC/BOTSCC targeted next-generation sequencing was performed of frequently mutated regions in 50 cancer related genes.Human papillomavirus positive . Differences in mutation frequency of TP53 and PIK3CA were found between HPV+ TSCC/BOTSCC and HNCUP and HPV− TSCC/BOTSCC. In HPV+ TSCC/BOTSCC presence of FGFR3 mutations correlated to worse prognosis. Other correlations to survival within the groups were not disclosed.279 HPV+ TSCC/BOTSCC mutation of PIK3CA was most frequently observed, while TP53 mutations dominated in HPV− TSCC/BOTSCC. In HPV+ TSCC/BOTSCC and HNCUP, mutations/tumor were similar in frequency and fewer compared to that in HPV− TSCC/BOTSCC. Notably, FGFR3 mutations in HPV+ TSCC/BOTSCC indicated worse prognosis.In HPV Furth (HNSCC) –14. HNSC benefit .+ tumor infiltrating lymphocyte (TIL) counts and CD98 have been proposed as such markers in HPV+ TSCC and BOTSCC [+ TSCC/BOTSCC [To better individualize treatments, several efforts have been made to identify additional predictive markers –23. Age,d BOTSCC –25. One C/BOTSCC . However+ HNCUP, since its survival after treatment is similar to that of HPV+ TSCC/BOTSCC, one hypothesis put forward is that it may originate from an HPV+ TSCC or BOTSCC [+ HNCUP and there are studies that have not found the same impact of HPV on survival [In the case of HPVr BOTSCC , 26–28. survival .+ TSCC/BOTSCC, and similarities and differences of mutated genes compared to HPV+ HNCUP and HPV− TSCC/BOTSCC, as well as potential targets for new therapies, next-generation sequencing (NGS) of hotspot mutations in 50 cancer related genes, was done. Examining TP53 and PIK3CA was of special interest, since these genes have previously been reported to be differentially mutated in HPV+ and HPV− OPSCC and their impact on outcome at the genomic level has not been studied extensively in HPV+ and HPV− OPSCC [To find more predictive markers for response to therapy for HPVV− OPSCC .+ and 51 HPV− TSCC/BOTSCC and 20 HPV+ HNCUP was analyzed using the Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) covering hotspot regions from 50 oncogenes and tumor suppressor genes. After excluding 24 samples due to poor DNA quality, 279 HPV+, 46 HPV− TSCC/BOTSCC and 19 HPV+ HNCUP samples remained for further analysis. Patient and tumor characteristics for the included samples are presented in Table DNA from 368 formalin-fixed paraffin-embedded (FFPE) tumor samples including 297 HPVVariants were called using the Torrent Variant Caller (TVC) version 5.0 and after filtering for > 5% allele frequency and an allele coverage of > 100 the remaining variants were filtered against several population genome databases as specified in the materials and methods section.+ TSCC/BOTSCC, the most common variants were PIK3CA, TP53, FGFR3, FBXW7, PTEN and CDKN2A, while in HPV− TSCC/BOTSCC mutations were most frequently found in TP53, PIK3CA, IDH2, ABL1, BRAF, CDKN2A, EGFR, NOTCH1 and PTPN11. Significant differences in frequency of variants between the two groups are shown in Table − than in HPV+ TSCC/BOTSCC with 0.92 vs. 1.68 variant/tumor respectively and were detected in 48.7% (136/279) of HPV+ and 74.5% (35/46) of the HPV− cases (p = 0.0007). This difference was mainly due to the difference in TP53 variants (9.3% vs. 63.8% for HPV+vs. HPV− TSCC/BOTSCC). Non-TP53 mutations tended to be more frequent in HPV+ compared to HPV− TSCC/BOTSCC . PIK3CA was the most frequently mutated gene in HPV+ TSCC/BOTSCC and was significantly more frequently mutated in HPV+ than in HPV− TSCC/BOTSCC (20.1% vs. 6.4%). As presented in Table + and HPV− TSCC/BOTSCC, but in these cases the numbers of affected tumors were small and the data should be treated with caution. Differences between the two groups are visualized in After filtering, 337 variants remained in 325 TSCC/BOTSCC distributed in the different target genes as presented in Table + TSCC/BOTSCC. TP53 mutations had the strongest inverse correlation with HPV+ TSCC/BOTSCC, while PIK3CA and FBXW7 showed positive correlations  are mutated in highly correlated blocks, and that most of these mutations are absent in patients with HPV+ HNCUP, 25 variants (1.32 variants/tumor) were observed in TP53, PIK3CA, CDKN2A, EGFR, IDH2, FBXW7 and VHL, for details see Table + TSCC/BOTSCC (48.7%), but less than in HPV− TSCC/BOTSCC (74.5%) (p = 0.04). TP53 was more often mutated in HPV+ HNCUP than in HPV+ TSCC/BOTSCC (p = 0.0345), but less often than in HPV− TSCC/BOTSCC (p = 0.0061). The frequency of mutated PIK3CA was similar to that in HPV+ TSCC/BOTSCC.In the 19 HPV+ TSCC/BOTSCC patients treated with curative intent for genes with mutations in > 6% of the cases i.e. TP53, PIK3CA, FGFR3, FBXW7 and of which three  are potentially targetable. A significant correlation to clinical response was found for variants in FGFR3, but for none of the other tested genes.Survival analysis was performed for the 277 HPV+ TSCC/BOTSCC with mutations in FGFR3 had a worse clinical outcome, depicted as 3-year disease free survival (DFS) (p = 0.002)   .Of note, patients with HPV) Figure . Specifir cancer , 32. Thi) Figure . When HP) Figure . Presenc− TSCC/OPSCC cases and for the 19 HPV+ HNCUP, where patients were treated with curative intent, for TP53, but not for any other mutations since the other cases were so few. No significant correlations were disclosed between TP53 and clinical outcome for either HPV− TSCC/BOTSCC or HPV+ HNCUP.Survival analysis was also performed for the 43 HPV+ and 46 HPV− TSCC/BOTSCC and 19 HPV+ HNCUP. Commonly mutated variants were found in PIK3CA, TP53, FGFR3, FBXW7, PTEN and CDKN2A in HPV+ TSCC/BOTSCC, while in HPV− TSCC/BOTSCC, mutations were most frequently found in TP53, PIK3CA and IDH2. HPV+ HNCUP showed mutations most often in TP53, PIK3CA and CDKN2A. The numbers of mutations per tumor were fewer in HPV+ TSCC/BOTSCC and HNCUP as compared to HPV− tumors. PIK3CA was most frequently mutated in HPV+ TSCC/BOTSCC, while TP53 was the most commonly mutated gene in HPV− TSCC/BOTSCC. The mutation pattern in HPV+ HNCUP, e.g. with regard to frequency of PIK3CA mutations, was similar to that of HPV+ TSCC/BOTSCC, although the frequency of TP53 mutations was a bit higher than that of HPV+ TSCC/BOTSCC. Of the variants tested, only FGFR3 variants correlated to clinical outcome in HPV+ TSCC/BOTSCC, specified as 3-year DFS, and especially those with the FGFR3 S249C variant had significantly worse prognosis.In this report, hotspot mutation regions of 50 cancer related genes were analyzed by NGS in 279 HPV+ TSCC/BOTSCC than in HPV− TSCC/BOTSCC and vice versa for TP53 was in line with several earlier reports. [− TSCC/BOTSCC as compared to some reports [The distribution of the various variants, and especially that PIK3CA mutations were more common in HPVreports. , 31, 33. reports , 33–35. + TSCC/BOTSCC is lower than in HPV− TSCC/BOTSCC is in concordance with e.g. Tinhofer et al., and could be due to the fact that high risk HPV provides active oncogenes E6 and E7 [+ and HPV− TSCC/BOTSCC in other regions needs to be analyzed further, since in this study targeted sequencing was performed using a pre-made cancer panel that covered certain areas of the selected genes. This can also explain some other differences between our study and other studies using larger panels and other experimental approaches, where they indicated larger numbers of mutations and fewer differences between HPV+ and HPV− HNSCC [+ TSCC and HNSCC respectively, were reported to have fewer gains and losses than HPV− TSCC and HNSCC [The finding that the total number of mutations in HPV6 and E7 , 36. NevV− HNSCC , 31. Stind HNSCC , 38.+ HNCUP has not been studied before and here we demonstrated that TP53, PIK3CA and CDKN2A were the most frequently altered genes in this subset of HNSCC. That HPV+ HNCUP had fewer mutations than HPV− TSCC/BOTSCC, and a similar mutation frequency of PIK3CA to HPV+ TSCC/BOTSCC strengthens the hypothesis by others and us that HPV+ HNCUP originates from HPV+ OPSCC [+ HNCUP were more common than in HPV+ TSCC/BOTSCC, but less frequent than in HPV− TSCC/BOTSCC. Thus, there were both similarities and differences between HPV+ HNCUP and HPV+ TSCC/BOTSCC. Whether, this is due to that HPV+ HNCUP should be regarded as HPV+ TSCC/BOTSCC metastasis, with additional genomic alterations, needs to be explored further. Notably, the HPV+ HNCUP group contained fewer never smokers than the HPV+ TSCC/BOTSCC group, which to some extent could explain the higher rate of TP53 mutations in the former. Attempts were made to find correlations between commonly occurring variants within each group and clinical outcome. For HPV+ TSCC/BOTSCC, mutations in PIK3CA and TP53 did not correlate to clinical outcome, while notably mutations in FGFR3 and especially the S249C variant correlated to worse prognosis. Variants of FGFR3, including S249C have been reported previously in HPV+ HNSCC, but have to our knowledge not previously been correlated to clinical outcome in HPV+ TSCC/BOTSCC [+ HNSCC varies between 0 and 24% [+ HNSCC more tumors may be affected than can be found in a mutational analysis [+ TSCC/BOTSCC, the possibility still remains that they too could be targeted in the future and present a possibility for more personalized treatment.To our knowledge, the mutational profile of HPVV+ OPSCC , 26–28. C/BOTSCC , 33, 35. and 24% , 33, 35.analysis . There aanalysis , 40. Fur− cancer group, which differs from a previous report, where mutated TP53 conferred worse outcome in HPV− HNSCC [− TSCC/BOTSCC, such as PIK3CA, were not analyzed in relation to clinical outcome due to the limited numbers of cases making it a target for further analysis. However, no such correlation was previously identified despite the larger number of HPV− HNSCC cases in the work of Tinhofer et al [In this investigation, mutation in TP53 did not correlate to clinical outcome in the HPVV− HNSCC . There iV− HNSCC . FurtherV− HNSCC . Other mer et al .+ HNCUP cases were included. Nevertheless, some interesting findings were still observed.There are limitations in this study in that only hotspot mutations of 50 oncogenes and tumor suppressor genes were analyzed, and that our inquiry was retrospective. Furthermore, only 19 HPV+ TSCC/BOTSCC and HPV+ HNCUP, compared to, and differing from HPV− TSCC/BOTSCC. Furthermore, for the first time specific FGFR3 mutations (S249C) in HPV+ TSCC/BOTSCC were disclosed as a significant risk for worse clinical outcome, also opening up for novel therapeutic options for these patients.To conclude, when studying hotspot mutations in 50 oncogenes and tumor suppressor genes, the frequency of mutations per tumor were similar and fewer in HPV+ TSCC/BOTSCC in this study was defined as having an HPV DNA positive tumor combined with overexpressing p16INK4A (p16), and if not p16 positive, as in one case, instead expressing HPV16 E7, while HPV− TSCC/BOTSCC was defined as having no HPV DNA. TSCC/BOTSCC not fulfilling these criteria, i.e. with dubious HPV status, were excluded from the analysis. By this definition this investigation initially included FFPE biopsy material from 297 HPV+ TSCC/BOTSCC and 51 HPV− TSCC/BOTSCC. Data on presence or absence of HPV DNA and p16 expression and HPV16 mRNA expression in the biopsies were derived from previous studies [+ HNCUP were obtained from a previous report where all cases but two overexpressed p16 [Patients diagnosed with TSCC (ICD-10 code C09.0-9) or BOTSCC (ICD-10 code C01.9) or HNCUP/secondary and unspecified malignant neoplasm of lymph nodes of head, face and neck (ICD-10 C77.0) between 2000-2011 at Karolinska University Hospital were included in the study. Having an HPV studies , 23. HPVDNA was extracted and HPV DNA status was assayed by a PCR-based bead based multiplex assay on a MagPix instrument (Luminex Inc.) as described previously . p16 expHotspot regions in cancer related genes were analyzed by targeted amplification by PCR using the Ion AMpliSeq Cancer Hotspot Panel v2 (CHPv2 - Thermo Fisher Scientific), which covers 2800 hotspots in the following 50 genes; ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53 and VHL.Amplicon libraries were prepared according to the manufacturer's protocol. In brief, 10 ng of genomic DNA from tumor samples was measured by Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and amplified in a single multiplex PCR reaction obtaining 207 amplicons with sizes ranging from 49 to 140 bp. Next, amplicons were treated with FuPa Reagent (Thermo Fisher Scientific) to partially digest the primers and phosphorylate the amplicon ends, and the products were ligated to the sequencing adapters with 96 unique Ion Xpress Barcodes (Thermo Fisher Scientific) according to the manufacturer's instructions. After AMPure beads purification (Beckman Coulter), all barcoded libraries were quantified by the Agilent 2100 Bioanalyser and Agilent High Sensitivity DNA Kit (Agilent Technologies). The final library concentrations were standardized to 100 pM in low Tris-EDTA (TE) buffer.Emulsion PCR and enrichment were performed on an Ion One Touch System by using the Ion PI Hi-Q OT2 200 Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Lastly, 96 pooled samples were loaded on a Ion P1 chip and sequenced on the Ion Proton benchtop sequencing platform using the Ion PI Hi-Q Sequencing 200 Kit (Thermo Fisher Scientific).The Ion Torrent Variant Caller Plugin v5.0 was used to align reads to the reference genome hg19.The Ion Torrent platform-specific pipeline software Torrent Suite used data from the initial Ion Proton runs to generate sequence reads, trim adapter sequences and filter and remove poor signal profile reads. Then, Torrent Suite Software v5.0, with a plug-in “variant caller v5.0” program (TVC), was used to call variants from the initial sequencing data. In order to eliminate base calling errors, several filtering steps were employed to generate final variant calling. First, variants samples with poor DNA quality were excluded, defined as < 80% on-target reads and/or mean coverage < 400 reads. Additional filters for variant calling were fixed at an average total coverage depth > 100, single variant coverage > 20, a variant allele frequency (VAF) between 5% and 90%. Furthermore, low impact variants were not retained, as well as germline variants, defined as variants with VAF > 1% in any of the population genome databases; 1000 genomes   from the date of diagnosis. An event was defined as recurrence in the disease. Patients who were never tumor-free were counted as having relapsed on day 0, while patients who died without prior recurrence were censored at that time-point. Statistical calculations were performed using IBM SPSS Statistics software . Survival curves and 3-year DFS were calculated using the Kaplan-Meier method and differences in survival were tested using the log-rank test. Frequencies of variants in the different genes in Table


Human Papillomavirus (HPV) infections are known to cause cervical cancer worldwide, however, limited information is currently available on prevalence, types distribution and risk factors for HPV infection in the Arab countries. We conducted a cross-sectional observational study exclusively of women of Arabic origin residing in Qatar (n = 406) who were selected from the Women’s Hospital at Hamad Medical Corporation (HMC) and Health Centers of the Primary Health Care Corporation in Doha, Qatar over the period March 2013 to August 2014. Socio-demographic, behavioral and clinical data were collected. Four hundred and six cervical smears and 292 blood samples were included in the study. HPV typing was done using HPV type-specific primers-based real-time PCR, and Sanger sequencing. HPV-IgG and IgM were quantified using ELISA assays. The prevalence of HPV infection amongst Qatari and non-Qatari Arab women were 9.8% and 6.1%, respectively and 7.6% and 16.7% in women with normal and abnormal cytology, respectively. HPV 81 was the most commonly found genotype in women with normal cytology (34.5%), whereas HPV 81, 16 and 59 in women with abnormal cytology (25.0% each). All the HPV DNA positive women were seronegative and HPV-IgG prevalence was higher in Qatari women than in non-Qatari Arab women. None of the studied factors had any significant association with HPV-DNA positivity or HPV-IgG seropositivity. The overall identified HPV DNA prevalence and HPV seroprevalence among Arab women in Qatar were on the low side compared to global levels. Human Papillomavirus (HPV) is one of the most prevalent sexually transmitted infections worldwide, and molecular studies have implicated infection with specific HPV high-risk (HR) genotypes as etiological agents of cervical cancer –3. SeverThough the incidence of cervical cancer in the Extended Middle East and North Africa (EMENA) shows lower rates compared to the rest of the world , 7, the During HPV infection, both humoral and cellular immune responses are induced, and antibody production against HPV is important for preventing the spread of infection and re-infection ,13. AddiQatar, a country located in the Arabian Peninsula has in recent years experienced rapid economic growth and globalization resulting in a large influx of foreign expatriates from Western, other Middle Eastern, African and Asian countries. The total population of women of child-bearing age was reported as 447,298; of which 382,067 represent Arabic women (Qatari and non-Qatari) according to the Ministry of Development Planning and Statistics, Qatar . Ot. Ot20]. Viral DNA was extracted from cervical samples using QIAamp MinElute virus spin kit  as previously described . To deteReal Time PCR-based kits  and Sanger method  were used to identify the genotype(s) in the samples which tested positive for HPV DNA, as described previously , 21.Human Papillomavirus IgG (HPV-IgG) and IgM (HPV-IgM) were quantified by double-antibody sandwich ELISA kits  according to the manufacturer's instructions. Briefly, 50 μl serum samples and 100 μl HRP conjugated antibody was added to pre coated 96 flat-bottomed plates and incubated for 1h at 37°C. Following incubation, unbound substances were removed by washing solution (5X) and finally, HRP substrate (50 μl of each Chromogen A and Chromogen B solution) was added and plate was incubated for 15 min at 37°C. After appearance of yellow color in negative wells, the reaction was stopped with 50 μl stop solution. The optical density (OD) was measured at 450 nm using an ELISA plate reader and results were analyzed. The cutoff value was calculated using the manufacturer’s instructions. The average OD of the negative control wells plus 0.15 was taken as the cutoff critical value. A negative HPV result was interpreted as sample OD less than the calculated cutoff value, and samples with an OD greater than the calculated cutoff value were reported as positive for HPV IgG.Statistical analyses were performed using SPSS (IBM SPSS version 23.0). Sample characteristics including age, nationality, marital status, education level, household monthly income, smoking habits, current method of contraception, knowledge about HPV, HPV vaccine and related cervical cancer, cytology results, and clinical findings were briefed using frequency distributions. Crude association between HPV positivity and each of the previously mentioned characteristics was assessed using the chi-squared test. Significance level was considered at α = 0.05 and unadjusted odds ratios (OR) were reported with their corresponding 95% confidence intervals (CI).All enrolled Arab women were categorized into 5 broad age groups , in which 34.5% of women were aged 41–50 years, 29.8% were aged 31–40 years, 17.5% were aged 51–60 years, 13.3% were aged 21–30 years, and only 4.9% of women were aged 61 years or more. The age range across the sample was 59 years with a mean age of 42.8 years (SD = 10.5 years). Additionally, 55.4% of women  who participated in this study were Qatari nationals, followed by 26.8%  from Fertile Crescent, 10.6%  from Arabian Peninsula, and only 7.1%  from North and East Africa . This isFurthermore, data on p = 0.673). Among non-Qatari Arab women, HPV prevalence was highest in women originating from Arabian Peninsula 7.0% , followed by women from Fertile Crescent countries 6.4% . The lowest prevalence of 3.4%  was found in women originating from North and East Africa  in the age group 51–60  and lowest (3.7%) in the 21–30 age group  . HoweverThe risk factors such as marital status (widowed), level of education (no schooling and intermediate), household monthly income (<5000.00 QAR) and smoking tended to have increase of HPV infection risk , however, did not observe any statistical significance. The other risk factors like reproductive factor (contraceptive methods), knowledge about HPV, cervical cancer and HPV vaccine have no influence in HPV infection risk and had no significant association.Of the total of 406 samples collected in this study population, 33 samples (8.13%) were positive for HPV DNA. These positive samples were further screened to identify the HPV genotypes and were classified based on their oncogenic potential to high-risk (HR) and low-risk (LR) as shown in Among HPV positive women, taking into account the total frequency of occurrence of each specific genotype occurring either as single/multiple infection ), 63.6%  had at least one LR HPV genotype and 18.2%  had at least one HR HPV genotype . The mosHPV type-specific was not significantly associated with any of potential risk factors, assessed in the present study, such as marital status, education level, economic status, usage of contraception, smoking habits and knowledge about HPV, HPV vaccine and cervical cancer .We assessed the levels of IgG and IgM in the sera of 292 Arab women residing in the State of Qatar. The seroprevalence of HPV according to different variables is shown in P = 0.615). Additionally, HPV DNA positivity was also correlated with seropositivity by age group, but no significant difference was observed. However, the prevlence of HPV-DNA was higher in the age group 41–60 as compared to the HPV-IgG positive women (P = 0.705). The HPV-IgG seropositivity was slightly higher among women with college/university education, monthly income 5000.00–20,000.00 QAR, knowledge about cervical cancer but not HPV vaccine, no smoking, no use of any contraceptive methods, normal cytology and underwent routine health care, however, had no significant associations with HPV infection. It is noteworthy that all samples were negative for HPV-IgM.According to age groups, the prevalence of HPV-IgG was highest  in the age group 31–40, however, did not differ significantly according to patient age  than we previously report (6.1%) in the large cohort . HoweverIn the present study, the high-risk HPV genotypes 16 and 59 were the most common among women with abnormal cytology, while HPV 35, 33, 39, 59 were found among those with normal cytology. HPV 81, 90, 11 were the most prevalent low-risk genotypes among women with both normal and abnormal cytology, a finding that is consistent with our previous study . HPV 81 The findings on HPV DNA prevalence and genotype distribution, as well as HPV seroprevalence and potential risk factors for HPV infection, have the clinical potential to improve cervical cancer screening through identifying women at high risk for cervical dysplasia/cancer. Furthermore, they inform the development of genotype-specific vaccines for trials in population-level programs. It has been shown that HPV serology may underestimate infection exposure, as many women do not develop an HPV antibody response . Though,Interestingly, none of the women were found positive for HPV IgM antibodies and there was no evidence of an association between behavioral risk factors and HPV seropositivity. Furthermore, it has been reported that once seroconversion occurs, anti-HPV antibody levels remain detectable for years ,35, but The strengths of the present study lie in the use of a standardized and sensitive molecular assay for HPV detection, rendering our findings amenable to a detailed analysis of HPV prevalence, distribution of HPV genotypes, seroprevalence and risk factors among general population of Arab women residing in the State of Qatar, and comparison to global patterns. In the present study, we aimed for such analysis to be relevant for planning of health service provision and development of appropriate interventions based on the current characteristics of the infection burden and implied future trends for squamous intraepithelial lesions and cervical cancer. The indications that HPV infection burden might be increasing in EMEMNA, adds further importance to this investigation.The limitations of this study include the fact that, because of socio-cultural context, we were unable to collect detailed data on sexual behaviour such as number of sexual partners, age at first sexual intercourse and extramarital relationships. Additionally, the present study was based on a convenient sample from women attending the Women hospital and Gynecology clinic at PHCC. Therefore, it is not known how representative is this sample of the wider Arab women population residing in the State of Qatar.The overall HPV DNA prevalence and seroprevalence among Arab women in the State of Qatar are rather low in comparison to other countries. Despite the low prevalence, there is a diverse distribution of HPV genotypes among Arab women living in Qatar, and there appears to be an increase in prevalence over the last decade. Our study suggests that these genotypes should also included in future vaccines targeting this specific population. Furthermore, the information about the molecular and sero-prevalence of HPV infection will be helpful for policy makers in making an informed decision regarding introduction and development of policies, guidelines and implementation of HPV vaccination in the State of Qatar. Contrary to our expectations, no statistically significant association was observed between HPV DNA positivity or antibody positivity and potential risk factors for HPV infection. Though, Arab women in Qatar reported knowledge of cervical cancer, there was limited knowledge of its link to HPV infection and HPV vaccination. Awareness programs of risk associated with HPV infection and HPV vaccination are warranted in Qatar. Finally, further observational studies of HPV infection levels and HPV incidence among different age groups may help elucidate several poorly understood aspects of HPV epidemiology in this part of the world.S1 Questionnaire(PDF)Click here for additional data file.


Curaçao is a Dutch-Caribbean Island located in a high-risk area for cervical cancer.Prior to introduction of a prophylactic human papillomavirus (HPV) vaccine, knowledge of the prevalence of high-risk HPV vaccine genotypes  in cervical (pre)cancer is required.To investigate the prevalence of HPV genotypes in invasive cervical cancers (ICC) and cervical intraepithelial neoplasia (CIN) grade 1, 2 and 3 in Curaçao.Paraffin-embedded blocks of 104 cervical cancers , 41 CIN3, 39 CIN2 and 40 CIN1 lesions were analysed for the presence of HPV. Sections were stained by H&E for histopathological evaluation, and DNA was extracted using proteinase K. HPV genotypes were detected using Short PCR Fragment (SPF10) PCR DNA enzyme immunoassay and a Line Probe Assay (LiPA25).HPV was found in 92 (88.5%) ICC; 87 (94.6%) had a single HPV infection and 86 (93.5%) were high-risk human papillomavirus (hrHPV)-type positive.The three most common HPV types in ICC were 16 (38.5%), 18 (13.5%) and 45 (6.7%), covering 58.7%.HrHPV vaccine genotypes 16, 18, 31, 35, 45, 52 and 58 were responsible for 73.1% of ICC. For precancerous lesions, the HPV attribution was 85.4% for CIN3, 66.7% for CIN2% and 42.5% for CIN1.Our study, the largest in the Caribbean region in (pre)cancer, shows that the prevalence of HPV-type 16 and 18 in cervical cancer is lower compared with the world population but no differences in prevalence of these two HPV types are seen in precancerous lesions.When considering HPV vaccination in Curaçao, the relatively high contribution of non-HPV 16/18 genotypes in ICC should be taken into account. Curaçao is a Dutch-Caribbean island with estimated 155 000 inhabitants last registered in 2014.The lack of an organised cervical screening and prophylactic HPV vaccination programme largely explains the high incidence of cervical cancer.Presently, three HPV prophylactic vaccines are available. A bivalent HPV vaccine against HPV16 and 18; a quadrivalent vaccine directed against HPV16 and 18 with an additional coverage to low-risk HPV 6 and 11; and recently a nonavalent vaccine directed against high-risk Human papillomavirus (hrHPV)16, 18, 31, 33, 45, 52 and 58 and lrHPV6 and 11. The bivalent and quadrivalent vaccines have shown partial to full cross-protection against certain non-vaccine HPV types.According to the report about the situational analysis of cervical cancer prevention and control in the Caribbean, only three Caribbean islands have a HPV vaccination programme up and running.In July 2016, the island started with an HPV-based national cervical cancer screening programme.Before introducing a prophylactic vaccine to women on Curaçao, it is important to know the prevalence of HPV genotypes in cervical precancerous lesions and cancer. This can help in the decision by policymakers which prophylactic HPV vaccine should be used and what the potential impact of the new vaccine might be. Here we describe the prevalence of HPV genotypes in paraffin-embedded tissues of cervical cancers and cervical precancerous lesions ) derived from the only pathology laboratory of Curaçao.A retrospective study was designed and co-ordinated by Fundashon Prevenshon (FP), Curaçao, in co-operation with DDL Diagnostic Laboratory, Rijswijk, the Netherlands; the department of pathology, VU University Medical Centre (VUmc) Amsterdam, the Netherlands and the Analytic Diagnostic Centre (ADC), Curaçao, to determine the prevalence of HPV DNA genotypes in formalin-fixed paraffin-embedded (FFPE) tissues of women with invasive cervical cancer (ICC).All ICC and CIN cases diagnosed in woman from Curaçao were selected in the PALGA system, which is the nationwide network and registry of histopathology and cytopathology in the Netherlands and Curaçao. This system includes both private and government insured patients. Only available blocks of good quality, containing enough tissue, were selected from 2012 backwards, anonymised, relabelled and analysed at DDL diagnostic laboratory. In total, FFPE blocks of 104 cervical cancers, 40 CIN1, 39 CIN2 and 41 CIN3 were collected from the ADC’s department of Pathology, Curaçao. The type of tissue from which the lesion samples are obtained is given in Eight sections were cut from each block according to the sandwich method for histopathology and DNA extraction for HPV testing.In brief, the first and last sections were 4 µm and stained with H&E for histopathological evaluation of the lesion. In between the two H&E-stained sections, six sections of 8 µm were cut. The first three were combined in a single tube and used for HPV genotyping, the last three were stored in another tube for back up.After each 10 blocks that were cut, an empty paraffin block was cut as a control for contamination during the sectioning process. Sections from the control block were tested in parallel with the samples and remained negative. For each sample, we also obtained age of the woman at diagnosis, date of sample collection and histological diagnosis.Tissue sections were incubated overnight for DNA extraction with 250 µL of a 0.1% proteinase K solution at 70ºC. The standard proteinase volume of 250 µL was reduced to 100 µL for sections containing small tissue samples as observed by visual inspection. After incubation, proteinase K was inactivated at 95ºC for 10 min. HPV DNA was amplified by the SPF10 PCR in a total volume of 50 µL using 10 µL of 10 times diluted DNA. The SPF10 DNA enzyme immunoassay (DEIA)  was used for general detection of over 69 known HPV types by hybridisation to a mixture of HPV-specific probes in a 96-well format.Amplimers from positive samples by DEIA were genotyped in the SPF10LiPA25 reverse hybridisation assay for identification of 25 HPV genotypesDNA from all samples was also tested for the presence of amplifiable human genomic DNA and PCR inhibition by a duplex real-time PCR targeting the human RNaseP gene and a plasmid spiked into the PCR mix.HPV type attribution per group was assessed in different ways to deal with multiple infections  of the medical ethics committee of FP, Curaçao .Since the results of hierarchical attribution analysis were similar to the proportional attribution analysis, we only present here the hierarchical distribution. The prevalence of HPV genotypes in multiple infections in cervical cancer and CIN lesions is shown in online Most of the histotypes were squamous cell carcinoma (SCC n=85 (81.7%)), followed by adenocarcinoma ((ADC n=15 (14.4%)) and adenosquamous carcinoma ((ASC n=4 (3.8%)) .Because of the small number of ASC, for statistical analysis we added the four ASC cases to the SCC.The large majority (88.5%) of cervical carcinoma were HPV positive of which only 5.4% had a multiple infection. The majority of positive ICC cases (93.5%) contained one hrHPV type . The mos10.1136/sextrans-2017-053109.supp2Eighty per cent of the ADC (n=15) were HPV positive.Only single HPV-type infections were detected, and all samples were high-risk HPV types. HPV18 was the most prevalent HPV type (40.0%) followed by HPV16 (26.7%) and HPV45 (13.3%) .In total, 12 of the ICC samples were HPV negative: nine SCC and three ADC.The percentages of HPV prevalence increased with the grade of CIN lesion namely CIN1 77.5%, CIN2 89.7%, CIN3 95.1% . The perHPV16 was the most prevalent HPV type in all degrees of CIN and its prevalence increased substantially with greater severity of CIN. The most prevalent HPV types in CIN3 samples were HPV16 (46.3%) and HPV 31 (12.2%). HPV types 18, 52 and 58 each covered 7.3%.In CIN 2, the most common type was HPV16 (30.8%), while HPV18, 35 and 51 were the second most common, each covered 10.3%. In CIN 1, the most common HPV types were HPV16 (15.0%), HPV31 (12.5%) and HPV51 (7.5%) .This is the first study from Curaçao to show systematic data on HPV prevalence in cervical cancer and CIN lesions. Interestingly, although the prevalences of HPV16 (38.5%) and HPV18 (13.5%) in cervical cancer of women from Curaçao are still the most prevalent HPV genotypes, their prevalence is lower compared with the world prevalence.The prevalence of vaccine types HPV31, HPV52, HPV58 and HPV X (DEIA pos. and LiPA neg.) for each HPV type was 3.8%, in total 15.2%. In ADC (n=15), HPV 18 (40.0%), HPV16 (26.7%) and HPV45 (13.3%) were the most prevalent HPV types. Twelve cervical cancers were found to be HPV negative.We performed several experiments to substantiate absence of HPV in cervical cancer and exclude experimental causes. Review of the slides by two pathologists did not change the diagnosis, and repeated HPV testing and genotyping with SPF10 yielded identical results. Inadequate DNA quality could be excluded by parallel testing with a duplex real-time PCR targeting the human RNaseP gene. Potential PCR inhibition could be excluded by a plasmid spiked into the PCR mix.All 12 samples contained amplifiable human genomic DNA and stable Cq values of the plasmid spike showed absence of PCR inhibition. Another explanation for a negative HPV result in cervical carcinoma might be integration of HPV DNA in the L1 region because the SPF10 targets the L1 gene. We did not perform HPV detection assays targeting the E6/E7 region to exclude this possibility.Our HPV 16/18 prevalence data in cervical cancer are in agreement with Martinique (57% in 131 ICC)The ranking order of HPV genotypes found in cervical cancer in Curaçao matches with those from Trinidad HPV16 (66.1%), HPV18 (17.8%) and HPV45 (8.9%).Compared with HPV 16 and 18 in cervical cancer from the Caribbean region according to the Institut Català d’Oncologia (ICO) data (57.6%), our estimated HPV16 and 18 prevalence data in cervical cancer are somewhat lower (52.0%), but our HPV 16 and 18 prevalence data in high-grade lesions  are higher compared with the ICO data .However, it should be noted that most of the published HPV prevalence data from CIN lesions in the Caribbean region are obtained from studies in women with normal cytology where these lesions were discovered incidentally during the trials. In Jamaica (n=840), the prevalence of HPV16 and 18 was found to be 50% in 14 high-grade squamous intraepithelial lesion (HSIL) and 21.3% in 61 atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LSIL).Data from Guadeloupe (n=618) only described HPV 16 and 18 prevalence in LSIL: 9.7% in LSIL (n=31) and 11.3% of ASC-US (n=106). HPV 16 and 18 prevalence in HSIL were not described because of the small sample size (n=8).An explanation for the differences in HPV prevalence between the Caribbean region may be the very heterogeneous ethnical background, which consists of a mix of African and European descent and different social and cultural behaviours in this population.Our HPV genotype prevalence data argue for the use of a prophylactic HPV vaccine with broad coverage of the non-HPV 16/18 genotypes, as present in the nonavalent vaccine. The use of HPV vaccines with a smaller HPV genotype coverage such as the bivalent vaccine can also be considered when longitudinal data show that the cross-protection of the HPV 16/18 vaccines provides also long-lasting protection against non-vaccine HPV types such as 45, 31 and 33, present in relevant percentages in the population.The strengths of our study is that all samples derived from the only Pathology laboratory in Curaçao where all cases were handled according to one protocol. Three pathologists did histological review and HPV typing was carried out following a single standardised protocol in DDL, which has ample experience with HPV genotyping in very large epidemiological studies on genotyping.A limitation of the study is the limited number of CIN lesions studied because only available blocks of good quality and containing enough tissue were included. Consequently, this should be taken into account when interpreting the HPV prevalence data. However, this is the largest series of CIN from the Caribbean area in which HPV prevalence was assessed.In conclusion, our study shows that compared with the world population the prevalence of HPV types 16 and 18 in cervical cancer is lower and consequently the contribution of the HPV genotypes 31, 45, 51, 52 and 58 is higher. When considering prophylactic HPV vaccination on Curaçao, these HPV genotypes' prevalence data in cervical (pre)cancer should be taken into account.The incidence of cervical cancer in Curaçao is high (13.1) and cervical cancer prevention programmes are lacking.The prevalence of HPV16 and 18 in cervical cancer in this large study from Curaҫao is lower compared with the world population and Trinidad.The prevalence of HPV genotypes in CIN lesions in Curaҫao is comparable with the world population.In the selection of a prophylactic HPV vaccine the high proportion of non-HPV 16/18 genotypes in cervical cancer should be taken into account.10.1136/sextrans-2017-053109.supp3


The incidence of Papillary thyroid carcinoma (PTC), the most common type of thyroid malignancy, has risen rapidly worldwide. PTC usually has an excellent prognosis. However, the rising incidence of PTC, due at least partially to widespread use of neck imaging studies with increased detection of small cancers, has created a clinical issue of overdiagnosis, and consequential overtreatment. We investigated how molecular data can be used to develop a prognostics signature for PTC.The Cancer Genome Atlas (TCGA) recently reported on the genomic landscape of a large cohort of PTC cases. In order to decrease unnecessary morbidity associated with over diagnosing PTC patient with good prognosis, we used TCGA data to develop a gene expression signature to distinguish between patients with good and poor prognosis. We selected a set of clinical phenotypes to define an ‘extreme poor’ prognosis group and an ‘extreme good’ prognosis group and developed a gene signature that characterized these.We discovered a gene expression signature that distinguished the extreme good from extreme poor prognosis patients. Next, we applied this signature to the remaining intermediate risk patients, and show that they can be classified in clinically meaningful risk groups, characterized by established prognostic disease phenotypes. Analysis of the genes in the signature shows many known and novel genes involved in PTC prognosis.This work demonstrates that using a selection of clinical phenotypes and treatment variables, it is possible to develop a statistically useful and biologically meaningful gene signature of PTC prognosis, which may be developed as a biomarker to help prevent overdiagnosis.The online version of this article (doi:10.1186/s12885-016-2771-6) contains supplementary material, which is available to authorized users. Papillary thyroid carcinoma (PTC) is not only the most common form of thyroid cancer; its incidence has been increasing faster than any other cancer type in the US –3. The lMolecularly, PTCs have few somatic alterations. They are mainly driven by mutations in the MAPK-pathway including NRAS, HRAS, KRAS and BRAF, and mutations in the PI3K-AKT signaling pathway . Some ofThe existing prognostic factors such as age at the time of diagnosis, the size of the tumor, extension into surrounding tissues, lymph node involvement, or distant metastasis help differentiate PTC patients into low and high risk . HoweverIn this report, we take advantage of the large collection of genomic data collected in the TCGA cohort in combination with clinical data on treatment. We report that a gene expression signature exists with the potential to characterize low-risk disease. These results may lead to biomarkers that can change the management of low-risk disease leading to improvements in patient quality of life and reduced financial burdens .Limited follow-up data was collected for the TCGA cohort and we used a collection of clinical phenotypes to define an ‘extreme poor’ prognosis group and an ‘extreme good’ prognosis group, based on features described in the Revised American Thyroid Association Management Guidelines for Patients with Thyroid Nodules and Differentiated Thyroid Cancer . The remPreprocessed TCGA gene expression data (generated by RNA sequencing), DNA copy number data (generated by microarray technology), mutation data (generated by exome sequencing) and PARADIGM pathway activity data, were downloaded using the Firehose pipeline  [5]. Preprocessing for these data sets was done according to the Firehose TCGA pipelines described elsewhere [5]. Additional preprocessing of this data set was done as follows: For the gene expression data, genes and patients with more than 10 % missing values were removed. All remaining missing values were estimated using KNN impute . TCGA daWe used the gene expression data to develop a prognostic classifier for thyroid cancer. We first selected the top 30 % most varying genes using the mean absolute deviation statistic, and subsequently used the z-score transformation for all genes so they have zero mean and unit variance. We used Significance Analysis of Microarrays (SAM) as previous described , to idenThe non-parametric Wilcoxon rank sum tests were used to test for significance of differences in expression between tumor and normal tissue, of genes identified as significantly associated with prognosis by SAM analysis.We tested the robustness of the gene expression signature by removing all patients defined by one of the seven poor prognosis characteristics from the previously defined group of extreme poor prognosis patients one at a time, and rebuilding the SAM signature using all remaining extreme poor and extreme good prognosis patients, and repeated this for each of the seven variables. We investigated the stability of the genes in the signature by counting the overlap of the genes in each of the seven analyses.Functional gene set enrichment (GSE) analysis was carried out using the GSE tools MSigDB  and EnriNext, we used Prediction Analysis of Microarrays (PAM) to develop a parsimonious supervised prognostic gene signature . PAM anaWe first used PAM in combination with 10-fold cross validation to determine the ability of the gene expression data to predict prognosis within extreme prognosis patients. For each fold of cross validation, the PAM model was trained on 90 % of patients and assigned class probability for good prognosis to the each of the remaining 10 % of patients based on the distance of the patient to its closest centroid. We used the Area under the ROC curve (AUC) to evaluate the performance of the model in accurately predicting the prognostic class of patients.We applied this prognostic gene expression signature to intermediate risk patients to classify them into either good or poor prognosis groups, using gene expression data for the top 20 % most varying genes . To classify a new sample, its distance is calculated to each of the centroids by using the weights as an inner product, and the sample is classified to its closest centroid. We only used classification results when probabilities were >60 % or <40 %. Low confidence assignments for the remaining borderline individuals were excluded from further analyses.We tested the robustness of the PAM classifier to split the patients into good or poor prognosis groups by removing patients featuring one of the seven poor prognosis characteristics from the previously defined group of extreme poor prognosis patients, and rebuilding the PAM classifier using all remaining extreme poor and extreme good prognosis patients. We investigated the stability of the genes in the classifier and investigated the classification assignments of the left-out group. In addition, we classified the intermediate prognosis thyroid cancer cases into a ‘intermediate-poor’ prognosis and ‘intermediate-good’ prognosis groups, and reported the distribution of mutations, clinical stage, nodal involvement, extra-thyroid extensions and histological subtypes, between these groups.T-test was used to test difference in age between prognostic groups.Significance of differences in distribution of categorical factors such as gender and nodal involvement was tested using Pearson’s chi-squared with Yates correction for small numbers of samples within some categories. Student’s p = 4.28e-07, Additional file We focused on extreme phenotypes to develop a prognostic expression signature for PTC. Using the prognostic clinical characteristics defined in the methods, we identified 79 extreme poor and 51 extreme good prognosis cases out of 494 cases in the TCGA thyroid cancer cohort, for which RNA gene expression data were available  and adjacent normal tissue (n = 58) samples within the PTC study. Of 791 genes downregulated in the extreme poor prognosis group relative to the extreme good prognosis group , 674 (85 %) were significantly differentially regulated between tumor and adjacent normal tissue. Of these, 611 (91 %) were downregulated in tumor, whereas 63 (9 %) were upregulated in tumors. All ten genes upregulated in the extreme poor prognosis group according to SAM analysis were also significantly upregulated in tumor versus normal tissue . There were no other highly enriched gene ontology (GO) terms or overlaps with gene-sets representing specific biological mechanisms such as transcription factor binding sites or organelle functions; therefore, the prognostic genes signature is not likely related to a single tumor event or characteristic, but reflects diverse abnormalities in multiple cancer pathways. Using the Enrichr enrichment analysis tool, the gene-set with which the 109 poor prognosis genes most significantly overlapped was an independent list of genes that were deregulated in PTC, derived from an independent study , with 21 overlapping genes [LTF, RASL11A, SYNM, IQGAP2, AXIN2, SLC5A8, LIFR, IGFBPL1, ZNF366) were among our poor-prognosis genes, a significant enrichment (p = 0.04). Finally, our gene list was compared to a gene-set of 39 genes consistently deregulated in thyroid cancer, identified by meta-analysis of multiple studies [TFF3, DIO1 and ITPR1 were overlapping.The prognostic gene signature of 109 genes most significantly overlapped with a list of genes that were downregulated in PTC compared to normal tissue in a previous study , with 27ng genes . Next, tng genes . Nine li studies . Of thesNext, we estimated the classification performance of a supervised classifier to predict prognosis using the extreme poor and good prognosis patients. We used the PAM classifier in combination with 10-fold cross validation and limited the classifier to maximum 100 genes. This resulted in an AUC of 0.75  and intermediate-good prognosis groups (n = 146) , we built a PAM model on the complete data set of extreme prognosis cases and classified the remaining intermediate prognosis PTC cases in two groups: intermediate-poor prognosis and intermediate-good prognosis. We then compared the distribution of key mutations and pathological variables relevant to prognosis between these groups. Out of 378 intermediate prognosis patients, PAM analysis assigned 306 patients to either intermediate-good or intermediate-poor prognosis groups with ‘high confidence’ probabilities of >60 % or <40 %, respectively. Of the 306 intermediate prognosis patients with high-confidence assignments, 111 (36 %) were classified as intermediate-good prognosis and 195 (64 %) were classified as intermediate-poor prognosis. The intermediate-poor prognosis group had higher nodal involvement, a tendency towards extra-thyroid extension and is highly enriched for BRAF mutations compared to the good prognosis group. The intermediate-good prognosis group had a mixed RAS-BRAF mutation composition, with significantly higher incidence of HRAS and NRAS mutations compared with the poor prognosis group , and members of cancer-related pathways, such as FZD9, a member of the Wnt signaling pathway, MAP2K6, a MAP kinase, IGFBPL1, a regulator of insulin-like growth factor signaling, and LRRC4 and LRRC4B involved in cell proliferation, migration and angiogenesis.We investigated the robustness of the PAM supervised predictor, i.e. its performance in predicting prognosis when each poor prognostic clinical characteristic was excluded. Extreme poor prognosis patients assigned to each of the seven poor prognostic factors were excluded, one group at a time. For each left-out group, a PAM predictor was trained using the remaining extreme prognosis samples, and the performance of the model in accurately predicting prognosis for left-out individuals was assessed. For all but one of the left out groups, between 11 and 43 % of the left out samples were classified as good prognosis without including them for training , so that THRA transcription is impeded by NR1D1 transcription [p = 0.04, n = 501), though not within a smaller set of normal adjacent tissue samples  . This is not surprising, as epigenetic mechanisms and copy number variation likely influence disease outcome through alteration of gene expression, therefore measurement of expression itself may be more directly related to disease outcomes.There is a pressing clinical need for prognostic biomarkers to direct therapy and treatment planning for patients presenting with PTC. Given the precipitous rise in this disease, robust clinical indicators may help reduce the potential consequences of overtreatment for patients with this mostly indolent disease. Our identification of a prognostic signature for PTC provides proof of concept that gene expression patterns can be used to identify patients who may otherwise be subject to overdiagnosis. This work may provide a rational first step towards identifying a prognostic test that can help clinicians to tailor therapy in patients with good prognosis and intensify management of patients with poor prognosis. The analytical methods we adopted provided an alternative to standard survival analysis to identify genes associated with prognosis, due to the small number of deaths from PTC. Unfortunately, this analytical method does not entail adjustment for potential confounding factors that may influence patient prognosis such as age or different treatments. The key limitation of this study, however, is that we were unable to validate our prognostic gene signature in independent patient cohort, due to lack of existing data or samples with clinical annotation. Our work motivates the collection of long-term clinical follow-up data to further develop, refine and validate prognostic signatures for PTC. Such a signature may be developed as a clinically applicable biomarker using technologies such as the NanoString nCounter  to rout