Search

// Attila T. Lorincz 1 , Mayura Nathan 2 , Caroline Reuter 1 , Rhian Warman 1 , Mohamed A. Thaha 4, 5 , Michael Sheaff 3 , Natasa Vasiljevic 1 , Amar Ahmad 1 , Jack Cuzick 1 and Peter Sasieni 1 1 Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, Queen Mary University of London, EC1M 6BQ, UK 2 Homerton Anal Neoplasia Service, Homerton University Hospital NHS Foundation Trust, London E9 6SR, UK 3 Cellular Pathology, Barts Health NHS Trust, London E1 2ES, UK 4 National Bowel Research Centre, Blizard Institute, Queen Mary University of London, London E1 2AT, UK 5 Barts Anal Neoplasia Centre, Department of Colorectal Surgery, Surgery and Cancer CAG, The Royal London Hospital, Barts Health NHS Trust, Whitechapel, London E1 1BB, UK Correspondence to: Attila T. Lorincz, email: a.lorincz@qmul.ac.uk Keywords: anal cancer, intraepithelial neoplasia, DNA methylation, high-risk human papillomavirus, HPV genotyping Received: February 10, 2017 Accepted: May 06, 2017 Published: May 18, 2017 ABSTRACT We studied DNA methylation patterns of human papillomavirus (HPV) and tumor suppressor gene EPB41L3 in 148 anal and perianal biopsies to determine whether high levels of methylation would be associated with anal intraepithelial neoplasia (AIN). The most prevalent HPV type was HPV16, detected in 54% of the 30 benign biopsies, 33% of the 43 low-grade AIN (lgAIN), 82% of the 59 high grade AIN (hgAIN) and 4 of the 5 anal cancers. A methylation score was developed (0.561*HPV16me+0.439* EPB41L3 ) which had increasing values with severity of disease: the mean was 8.1% in benign, 13.2% in lgAIN, 22.3% in hgAIN and 49.3% in cancers ( p < 0.0001). The methylation score as a triage classifier at a cut-off of 8.8 gave a sensitivity of 90.6% (95% CI: 82.8, 96.9), specificity of 50.7% (95% CI: 39.7, 61.6) and area under the curve of 0.82 (95% CI: 0.75–0.89) for separating hgAIN and cancer from benign and lgAIN biopsies. We conclude that methylation of HPV16 and EPB41L3 show highly significant association with increasing severity of AIN and cancer and may be useful as biomarkers in anal disease.

// Xueying Mao 1, * , Fei Luo 2, 3, * , Lara K. Boyd 1, * , Bowei Zhou 2, 3 , Yanling Zhang 4, 5 , Elzbieta Stankiewicz 1 , Jacek Marzec 1 , Natasa Vasiljevic 6 , Yongwei Yu 7 , Ninghan Feng 8 , Jia Xu 3 , Attila Lorincz 6 , Yong Jiang 3 , Claude Chelala 1 , Guoping Ren 4 , Daniel M Berney 1 , Shan-Chao Zhao 2 , Yong-Jie Lu 1 1 Centre for Molecular Oncology, Barts Cancer Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, EC1M 6BQ, UK 2 Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China 3 Key Laboratory of Proteomics of Guangdong Province and Key Laboratory of Transcriptomics and Proteomics of Human Diseases Supported by The Ministry of Education of China, Southern Medical University, Guangzhou, 510515, China 4 Department of Pathology, The First Affiliated Hospital, Zhejiang University Medical College, Hangzhou, 310009, China 5 Department of Gynecology and Obstetrics, Sir Run Run Shaw Hospital, Zhejiang University Medical College, Hangzhou, 310009, China 6 Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, EC1M 6BQ, UK 7 Department of Pathology, Changhai Hospital, The Second Military Medical University, Shanghai, 200433, China 8 Department of Urology, Wuxi Second People’s Hospital, Nanjing Medical University, Wuxi, 214002, China * These authors contributed equally to this work Correspondence to: Shan-Chao Zhao, email: zhaoshanchao@263.net Yong-Jie Lu, email: y.j.lu@qmul.ac.uk Keywords: NKAIN2, tumor suppressor, prostate cancer, chromosomal deletion and truncation, population difference Received: December 16, 2015 Accepted: August 22, 2016 Published: August 30, 2016 ABSTRACT Recurrent chromosome breakpoints at 6q22.31, leading to truncation and potential loss-of-function of the NKAIN2 gene, in Chinese prostate cancer patients were previously identified. In this study we investigated genomic, methylation and expression changes of NKAIN2 in a large number of prostate cancer samples and determined its functional role in prostate cancer cells. Fluorescence in situ hybridization analysis confirmed that NKAIN2 truncation is specific to Chinese while deletion of the gene is frequent in both Chinese and UK prostate cancers. Significantly reduced expression of NKAIN2 was also detected at both RNA and protein levels. Somatic mutations of NKAIN2 in prostate cancer samples exist but at very low frequency, suggesting that it is a putative tumor suppressor gene (TSG) with haploid insufficiency. Our functional studies showed that overexpression of NKAIN2 in prostate cancer cells inhibits cellular growth by promoting cell apoptosis, and decreasing cell migration and invasion. Conversely, knockdown of NKAIN2 promotes prostate cancer cell growth by inhibiting cell apoptosis, and increasing cell migration and invasion. These data imply that NKAIN2 is a novel TSG whose activity is commonly reduced in prostate cancer. It may restrain the disease development and progression by inducing apoptosis and suppressing cancer cell growth, migration and invasion. This study provides new insights into prostate carcinogenesis and opportunities for development of novel therapies for prostate cancer.

OBJECTIVES: To evaluate associations of DNA methylation of the human tumour suppressor gene EPB41L3 with high-grade cervical intraepithelial neoplasia (CIN2+) and HIV-related factors among women living with HIV-1 (WLHIV) in Burkina Faso and South Africa. DESIGN: Case-control study of WLHIV aged 25-50 with histology-determined CIN2+ (cases, N = 152) and ≤CIN1 (controls, N = 210). METHODS: EPB41L3 methylation was measured by pyrosequencing of bisulphite converted DNA from exfoliated cervical specimens at baseline and 16 months later. Median methylation levels were compared across CIN grades using the Mann-Whitney test and Cuzick test for trend. EPB41L3 methylation levels were dichotomized into 'high' and 'low' using the 66.7 percentile point of the distribution in the controls. Associations of EPB41L3 methylation with HIV-related factors were estimated by logistic regression. RESULTS: Among 94 WLHIV in Burkina Faso and 268 in South Africa, median methylation levels at baseline for EPB41L3 increased with increasing CIN grade in both countries (P-trend 5 years vs. ≤5 years; adjusted odds ratio (aOR) = 4.15, 95% CI 1.09-15.83, adjusted for age, CD4 count, high-risk HPV and CIN status], with low CD4 count in both countries (CD4 ≤200 vs. ≥350 cells/μl: aOR = 7.14, 95% CI 1.44-35.37 in Burkina Faso; aOR = 2.55, 95% CI 1.07-6.07 in South Africa), and with prolonged ART use in South Africa (ART >2 years vs. ART-naïve: aOR = 2.40, 95% CI: 1.23-4.69). CONCLUSION: Methylation of EPB41L3 DNA is elevated among WLHIV with CIN2+ and independently associated with lower CD4 count and ART use.

BACKGROUND Persistent infection %by with high risk human papillomavirus (hrHPV) types causes cervical cancer but most women who test positive are at very low risk of neoplasia. Strategies are needed which can retain high sensitivity of hrHPV testing but reduce the number of false-positives. We showed previously that a combination DNA methylation triage assay for HPV types 16, 18 and 31 and human gene EPB41L3 was useful to identify high grade cervical lesions. OBJECTIVE Assess whether measurement of DNA methylation in HPV type 33 can improve the previous classifier. METHODS A London colposcopy referral group of 1493 women of whom 556 (37%) had histologically-confirmed CIN (cervical intraepithelial neoplasia) 2 or 3 that included 114 HPV33 positive women with methylation measured for three L2 CpGs 5557, 5560 and 5566. Discrimination performance was assessed for the new classifier S5, built by adding HPV33 to the earlier classifier. RESULTS HPV33 methylation measurement improved prediction among HPV33 positive women. Receiver operating characteristic analyses showed an area under the curve (AUC) for HPV33 methylation of 0.68 (95% CI 0.57-0.78). The earlier risk score was significantly improved by HPV33 methytlation (AUC = 0.82 vs 0.80; P < 0.001). For 90% sensitivity the specificity for CIN2/3 was 49% (95% CI 46-52%). CONCLUSIONS Measurement of HPV33 DNA methylation contributes independent diagnostic information to EPB41L3 and HPV16, HPV18 and HPV31, and is superior to genotyping. Other HPV and human methylation target regions might be useful to further improve S5.

Aim: We investigated if methylation of candidate genes can be useful for predicting prostate cancer (PCa) specific death. Patients & methods: Methylation of PITX2, WNT5a, SPARC, EPB41L3 and TPM4 was investigated in a 1:2 case–control cohort comprising 45 men with cancer of Gleason score ≤7 who died (cases), and 90 men who were alive or died of other causes with survival time longer than the cases (controls). A univariate conditional logistic regression model was fitted by maximizing the likelihood of DNA methylation of each gene versus the primary end point. Results: A 10% increase in methylation of PITX2 was associated with PCa related death with OR 1.56 (95% CI: 1.17–2.08; p = 0.005). Conclusion: Our study strengthens prior findings that PITX2 methylation is useful as a biomarker of poor outcome of PCa and in addition we also suggest that it may be particularly useful in men with low Gleason score.

Methylation of human papillomavirus (HPV) and host genes may predict cervical cancer risk. We examined the methylation status of selected sites in HPV16 and human genes in DNA extracted from exfoliated cervical cell samples of 244 women harboring HPV16-positive cancer or cervical intraepithelial neoplasia (CIN) or negative for intraepithelial lesions or malignancy (NILM). We quantified the methylation of CpG sites in the HPV16 L1 gene (CpG 6367 and 6389) and in the human genes EPB41L3 (CpG 438, 427, 425) and LMX1 (CpG 260, 262, 266, 274) following bisulfite treatment and pyrosequencing. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic utility of methylation level for the different sites and for a joint predictor score. Methylation in all sites significantly increased with lesion severity (p

Testing for high-risk (hr) types of human papillomavirus (HPV) is highly sensitive as a screening test of high-grade cervical intraepithelial neoplastic (CIN2/3) disease, the precursor of cervical cancer. However, it has a relatively low specificity. Our objective was to develop a prediction rule with a higher specificity, using combinations of human and HPV DNA methylation. Exfoliated cervical specimens from colposcopy-referral cohorts in London were analyzed for DNA methylation levels by pyrosequencing in the L1 and L2 regions of HPV16, HPV18, HPV31 and human genes EPB41L3, DPYS and MAL. Samples from 1,493 hrHPV-positive women were assessed and of these 556 were found to have CIN2/3 at biopsy; 556 tested positive for HPV16 (323 CIN2/3), 201 for HPV18 (73 CIN2/3) and 202 for HPV31 (98 CIN2/3). The prediction rule included EPB41L3 and HPV and had area under curve 0.80 (95% CI 0.78–0.82). For 90% sensitivity, specificity was 36% (33–40) and positive predictive value (PPV) was 46% (43–48). By HPV type, 90% sensitivity corresponded to the following specificities and PPV, respectively: HPV16, 38% (32–45) and 67% (63–71); HPV18, 53% (45–62) and 52% (45–59); HPV31, 39% (31–49) and 58% (51–65); HPV16, 18 or 31, 44% (40–49) and 62% (59–65) and other hrHPV 17% (14–21) and 21% (18–24). We conclude that a methylation assay in hrHPV-positive women might improve PPV with minimal sensitivity loss.

Background High risk human papillomavirus (HR-HPV) infection is common and only a small minority of infections become persistent and lead to cervical cancers. Women positive for HR-HPV usually require a second test to avoid unnecessary colposcopies and over treatment. Elevated DNA methylation of HR-HPV L1 and L2 genes in high grade disease has emerged as a promising molecular triage tool. Objectives Our aim was to accurately measure methylation levels at selected CpG positions in the HPV18, HPV31 and HPV33 genomes. We focused on the L2, L1, URR and E6 regions because these were previously shown to be interesting areas for study. Study design Pyrosequencing was used to measure methylation in 208 HPV18, 207 HPV31, and 126 HPV33 positive women selected from a London colposcopy referral population. Results After adjustment for multiple testing, at FDR 5%, elevated methylation was significantly associated with cervical intraepithelial neoplasia grades 2 or worse (CIN2+) in all investigated CpGs in HPV18 L2 and L1. Two of 6 L2 and 12 of 15 L1 sites in HPV31 and 6 of 8 L2 and 3 of 13 L1 sites in HPV33 showed significantly elevated methylation in CIN2+. Methylation of CpG sites in the URR and E6 region of the HPV types was low and most differences were not significant. Conclusion Elevated methylation of CpG sites in the L1 and L2 regions of HPV18, HPV31 and HPV33 is associated with CIN2+ and a panel test may be useful for triage of women with HR-HPV infections.

Clinically aggressive disease behavior is difficult to predict in men with low to intermediate clinical risk prostate cancer and methylation biomarkers may be a valuable adjunct for assessing the management of these patients. We set to evaluate the utility of DNA methylation to identify high risk disease in men currently considered as low or intermediate risk. DNA was extracted from formalin-fixed paraffin-embedded transurethral prostate resection tissues collected during the years 1990-96 in a watchful-waiting cohort of men in the UK. The primary end point was death of prostate cancer, assessed by reviewing cancer registry records from 2009. Methylation was quantified by pyrosequencing assays for six genes (HSPB1, CCND2, TIG1, DPYS, PITX2, and MAL) with established biomarker value in prostate cancer. A novel prognostic methylation score was developed by multivariate Cox modelling using the six methylation biomarkers in 385 men with low-and-intermediate clinical risk variables and its prognostic value compared to two previously defined clinically-derived risk scores. Methylation score was the most significant variable in univariate and bivariate analysis in men with low-to-intermediate CAPRA risk score. When combined with CAPRA score the hazard ratio was 2.02; 95% confidence interval, 1.40-2.92. For a methylation score sensitivity of 83% the specificity was 44%, while the maximum achieved sensitivity by CAPRA was 68% at a specificity of 44%. The derived methylation score is a strong predictor of aggressive prostate cancer that could have an important role in directing the management of patients with low-to-intermediate risk disease. The estimated areas under the curve (AUC) at 10 years of follow-up were 0.62 (95% CI: 0.51, 0.70) and 0.74 (95% CI: 0.65, 0.82) for CAPRA, and combined (CAPRA + methylation) risk score (CRS) respectively.

BACKGROUND: Heat shock protein 27 (Hsp-27) encoded by gene HSPB1 is a critical regulator of the behavioral phenotype of human prostate cancer (PCa) cells, enhanced expression being associated with highly aggressive disease and poor clinical outcome. In contrast, the protein is not expressed in PCas of low malignant potential. To gain insight into the mechanism regulating its expression, we tested the hypothesis that differential methylation of CpG islands within HSPB1 controls transcription and subsequent translation of the gene. METHODS: We studied prostate epithelial cell lines and tissue biopsies, including 59 BPH and 415 PCas, of which 367 were a cohort of men with up to 20 years of follow-up. Methylation across the gene (DNA methylation (DNAme)) was assayed by pyrosequencing. Hsp-27 expression was assessed by western blot and immunohistochemistry. RESULTS: In cancer tissues, methylation increased in a 3' direction (P < 0.0001) whereas in benign hyperplasia methylation was constantly below 5%, a cutoff giving a specificity of 100% and sensitivity of 50%. Although methylation of the promoter region was significantly discriminating between benign and malignant prostatic epithelia, it compared poorly with methylation of the first intron. The prognostic value of HSPB1 DNAme was confirmed by both univariate (hazard ratio 1.77 per 50% increment, P = 0.02) and multivariate models. Interaction between HSPB1 methylation and Gleason score revealed high DNAme to be a reliable prognostic marker of poor outcome in men with low Gleason score (P = 0.014). CONCLUSIONS: Our data indicate CpG methylation of the first HSPB1 intron to be an important biomarker that identifies aggressive PCas otherwise regarded as low risk by current clinical criteria but that, biologically, require immediate active management.

Background: The need for new prognostic factors in breast cancer is ever increasing as breast cancer management evolves. Aberrant DNA methylation plays a pivotal role in cancer development and progression; DNA methylation-based biomarkers may provide independent prognostic information. We used pyrosequencing to investigate the prognostic potential of quantitative DNA methylation of a large set of candidate genes in a Korean single-institution series of operable breast cancer. Methods: Absolute DNA methylation in 20 candidate genes from an initial set of 30 genes was measured by pyrosequencing of bisulfite converted DNA in 121 fresh frozen breast cancer cases. Survival analyses used continuous and categorized (quintile- based) gene methylation data with time to recurrence (TTR) as an endpoint. Prognostic abilities of gene-only and risk-score models were explored. Results: Median follow-up was 5.1 years; 25 recurrences (21%) were observed. Nodal status, methylation of TWIST1, SLIT2 (both as continuous and categorized variables) and APC, HLA-A, NKX2-5, SERPINB5, SFN (as categorized variables) were significantly prognostic; grade showed a prognostic trend. A multivariate model containing nodal status, grade and TWIST1 was ab estfi t( p< 0.001) in stepwise regression; risk-score based on this model separated patients into 3 distinct risk-groups ( p< 0.001). A gene-only model based on TWIST1 and SFN also classified patients into distinct risk-groups (p = 0.009). Conclusions: This study shows that accurate quantitative measurement of DNA methylation by pyrosequencing identifies a small set of genes with independent prognostic potential in breast cancer. These genes complement the current clinico-pathological prognostic factors and appear to be potential biomarkers that warrant further validation.

We explored the association of human papillomavirus type 16 (HPV16) DNA methylation with age, viral load, viral persistence and risk of incident and prevalent high grade CIN (CIN2+) in serially collected specimens from the Guanacaste, Costa Rica cohort. 273 exfoliated cervical cell specimens (diagnostic and pre-diagnostic) were selected: (1) 92 with HPV16 DNA clearance (controls), (2) 72 with HPV16 DNA persistence (without CIN2+) and (3) 109 with CIN2+. DNA was extracted, bisulfite converted and methylation was quantified using pyrosequencing assays at 66 CpGs across the HPV genome. The Kruskal-Wallis test was used to determine significant differences among groups, and receiver operating characteristic curve analyses were used to evaluate how well methylation identified women with CIN2+. In diagnostic specimens, 88% of CpG sites had significantly higher methylation levels in CIN2+ after correction for multiple tests compared with controls. The highest area under the ROC curve (AUC) was 0.82 for CpG site 6457 in L1, and a diagnostic sensitivity of 91% corresponded to a specificity of 60% for CIN2+. Prospectively, 17% of CpG sites had significantly higher methylation in pre-diagnostic CIN2+ specimens (median time of 3 years before diagnosis) versus controls. The strongest pre-diagnostic CpG site was 6367 in L1 with an AUC of 0.76. Age-stratified analyses suggested that women older than the median age of 28 years have an increased risk of precancer associated with high methylation. Higher methylation in CIN2+ cases was not explained by higher viral load. We conclude that elevated levels of HPV16 DNA methylation may be useful to predict concurrently diagnosed as well as future CIN2+.

Two novel human papillomaviruses (HPVs), HPV93 and HPV96, with genomes of 7450 and 7438 bp, respectively, are described. The L1 open reading frame of HPV93 showed highest identity to HPV24 (79 %) and that of HPV96 had highest identity to HPV92 (71 %). Real-time PCR for HPV92, 93 and 96 on stripped biopsies from tumours and healthy skin from 269 immunocompetent patients found HPV DNA in 2.6 % of tumours and in 0.4 % of healthy skin samples. Double infections were observed in two tumours. HPV92 was detected in four, HPV93 in two and HPV96 in three tumours. The range of viral loads spanned from one copy per 45 cells to one copy per 10 000 cells. The E7 proteins of HPV92, 93 and 96 were found to bind the retinoblastoma protein (pRb). These results suggest a possible role for these HPV types in skin carcinogenesis that deserves further study.

Background Prostate cancer has a variable clinical behaviour with frequently unpredictable outcome. DNA methylation plays an important role in determining the biology of cancer but prognostic information is scanty. We assessed the potential of gene-specific DNA methylation changes to predict death from prostate cancer in a cohort of untreated men in the UK. Methods This was a population-based study in which cases were identified from six cancer registries in Great Britain. DNA was extracted from formalin-fixed paraffin wax-embedded transurethral prostate resection tissues collected during 1990-96 from men with clinically-localised cancer who chose not to be treated for at least 6 months following diagnosis. The primary end point was death from prostate cancer. Outcomes were determined through medical records and cancer registry records. Pyrosequencing was used to quantify methylation in 13 candidate genes with established or suggested roles in cancer. Univariate and multivariate Cox models were used to identify possible predictors for prostate cancer-related death. Results Of 367 men, 99 died from prostate cancer during a median of 9.5 years follow-up (max = 20). Univariately, 12 genes were significantly associated with prostate cancer mortality, hazard ratios ranged between 1.09 and 1.28 per decile increase in methylation. Stepwise Cox regression modelling suggested that the methylation of genes HSPB1, CCND2 and DPYS contributed objective prognostic information to Gleason score and PSA with respect to cancer-related death during follow-up (p = 0.006). Conclusion Methylation of 13 genes was analysed in 367 men with localised prostate cancer who were conservatively treated and stratified with respect to death from prostate cancer and those who survived or died of other causes. Of the 13 genes analysed, differential methylation of HSPB1, CCND2 and DPYS provided independent prognostic information. Assessment of gene-methylation may provide independent objective information that can be used to segregate prostate cancers at diagnosis into predicted behavioural groups. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-655) contains supplementary material, which is available to authorized users.

Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase-dependent apoptotic pathway and heterozygous knockout of ZDHHC14 increased [CORRECTED] cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down-regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer.

Methylation of human papillomavirus (HPV) and host genes may predict cervical cancer risk. We examined the methylation status of selected sites in HPV16 and human genes in DNA extracted from exfoliated cervical cell samples of 244 women harboring HPV16-positive cancer or cervical intraepithelial neoplasia (CIN) or negative for intraepithelial lesions or malignancy (NILM). We quantified the methylation of CpG sites in the HPV16 L1 gene (CpG 6367 and 6389) and in the human genes EPB41L3 (CpG 438, 427, 425) and LMX1 (CpG 260, 262, 266, 274) following bisulfite treatment and pyrosequencing. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic utility of methylation level for the different sites and for a joint predictor score. Methylation in all sites significantly increased with lesion severity (p 0.0001). Area under the curve (AUC) was highest among the CIN2/3 vs. cancer ranging from 0.786 to 0.853 among the different sites. Site-specific methylation levels strongly discriminated CIN2/3 from NILM/CIN1 and cancer from CIN2/3 (range of odds ratios [OR]: 3.69-12.76, range of lower 95% confidence bounds: 1.03-4.01). When methylation levels were mutually adjusted for each other EPB41L3 was the only independent predictor of CIN2/3 vs. NILM/CIN1 contrasts (OR = 9.94, 95%CI: 2.46-40.27). High methylation levels of viral and host genes are common among precancerous and cancer lesions and can serve as independent risk biomarkers. Methylation of host genes LMX1 and EPB41L3 and of the viral HPV16 L1 sites has the potential to distinguish among precancerous lesions and to distinguish the latter from invasive disease.

DNA methylation changes in human papillomavirus type 16 (HPV16) DNA are common and might be important for identifying women at increased risk of cervical cancer. Using recently published data from Costa Rica we developed a classification score to differentiate women with cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) from those with no evident high-grade lesions. Here, we aim to investigate the performance of the score using data from the UK. Exfoliated cervical cells at baseline and 6-months follow-up were analyzed in 84 women selected from a randomized clinical trial of women undergoing surveillance for low-grade cytology. Selection of women for the methylation study was based on detectable HPV16 in the baseline sample. Purified DNA was bisulfite converted, amplified and pyrosequenced at selected CpG sites in the viral genome (URR, E6, L1 and L2), with blinding of laboratory personnel to the clinical data. The primary measure was a predefined score combining the mean methylation in L1 and any methylation in L2. At the second follow-up visit, 73/84 (87%) women were HPV16 positive and of these 25 had a histopathological diagnosis of CIN2/3. The score was significantly associated with CIN2/3 (area under curve = 0.74, p = 0.002). For a cutoff with 92% sensitivity, colposcopy could have been avoided in 40% (95% CI 27–54%) of HPV16 positive women without CIN2/3; positive predictive value was 44% (32–58%) and negative predictive value was 90% (71–97%). We conclude that quantitative DNA methylation assays could help to improve triage among HPV16 positive women.

Aberrant DNA methylation plays a pivotal role in carcinogenesis and its mapping is likely to provide biomarkers for improved diagnostic and risk assessment in prostate cancer (PCa). We quantified and compared absolute methylation levels among 28 candidate genes in 48 PCa and 29 benign prostate hyperplasia (BPH) samples using the pyrosequencing (PSQ) method to identify genes with diagnostic and prognostic potential. RARB, HIN1, BCL2, GSTP1, CCND2, EGFR5, APC, RASSF1A, MDR1, NKX2-5, CDH13, DPYS, PTGS2, EDNRB, MAL, PDLIM4, HLAa, ESR1 and TIG1 were highly methylated in PCa compared to BPH (p < 0.001), while SERPINB5, CDH1, TWIST1, DAPK1, THRB, MCAM, SLIT2, CDKN2a and SFN were not. RARB methylation above 21% completely distinguished PCa from BPH. Separation based on methylation level of SFN, SLIT2 and SERPINB5 distinguished low and high Gleason score cancers, e.g. SFN and SERPINB5 together correctly classified 81% and 77% of high and low Gleason score cancers respectively. Several genes including CDH1 previously reported as methylation markers in PCa were not confirmed in our study. Increasing age was positively associated with gene methylation (p < 0.0001). Accurate quantitative measurement of gene methylation in PCa appears promising and further validation of genes like RARB, HIN1, BCL2, APC and GSTP1 is warranted for diagnostic potential and SFN, SLIT2 and SERPINB5 for prognostic potential.

Keratoacanthoma (KA) is difficult to histologically distinguish from squamous cell carcinoma (SCC). Therefore, although KA is a benign self-resolving skin lesion, KA is commonly treated as SCC. Biomarkers to distinguish KA and SCC would thus be desirable. In search for specific markers, paraffin-embedded tissue samples from 25 SCC and 64 KA were arranged in a tissue microarray (TMA) and stained for immunologic cell-markers CD3, CD20 and CD68 as well as for proteins considered of relevance in tumorgenesis, namely NF kappaB/p65, I kappaB-alpha, STAT3, p53, TRAP-1, pRB, phosphorylated pRb, Cyld, p21, p16(INK4), Survivin, Bcl-xL, Caspase 3, Bak, FLK-1/VEGF-r2 and Ki-67. In addition, the tumors were tested for presence of human papillomavirus by PCR. We detected that the two lesions differed significantly in expression of Bcl-xL which was present in 84% of the SCC compared with only 15% in the KA (p < 0.001). The lower expression of the antiapoptotic protein Bcl-xL in KA is consistent with a possible role of apoptosis in the regression of KA.

Recent studies have suggested an association between human papillomaviruses (HPVs), particularly species 2 members of the genusBetapapillomavirus, and squamous cell carcinoma (SCC) of the skin. As most of these viruses are uncharacterized, molecular characterization and epidemiology are needed to advance our understanding of their significance in carcinogenesis. This study determined the complete genomes of four betapapillomaviruses of species 2 from skin lesions designated HPV-107, -110 and -111 and FA75[KI88-03], an isolate of an unpublished HPV type, and analysed their prevalence and viral loads in biopsies from SCC, actinic keratosis (AK), basal cell carcinoma, seborrhoeic keratosis and the healthy skin of 263 immunocompetent patients by HPV type-specific real-time PCR assays. Seventeen patients (6.5 %) harboured at least one of the four HPV types in their lesion, whereas seven patients (2.7 %) harboured one or more of the HPV types in healthy skin. Overall, the four viruses were more common in AK than in healthy skin (odds ratio 5.0, 95 % confidence interval 1.4–17.5), but the prevalence and viral loads were low. This characterization of HPV-107, -110 and -111 and FA75[KI88-03] expands the heterogeneity of members of species 2 of the genusBetapapillomavirus. However, as these types were found in only a few samples and in low amounts, a possible role in carcinogenesis remains elusive.

The interaction between UV-B irradiation and cutaneous human papillomaviruses (HPV) has been suggested to be of relevance for the development of non-melanoma skin cancers. We investigated the activity within the upstream regulatory region (URR) of the HPV types 8, 38, 92, 93 and 96, as well as their responsiveness to UV-B irradiation and cellular differentiation. Promoter activities were higher in HaCaT than in SiHa cells, corresponding to the HPV tissue tropism. Transcriptional start sites were mapped at P(92) (HPV-38), P(45) (HPV-92), P(7439) (HPV-93) and P(256) (HPV-96). Transcription from HPV-8, 93 and 96 URR was up-regulated by cellular differentiation, linking the activity of these HPVs to the cellular state. UV-B irradiation activated HPV-8 but inhibited HPV-38 and HPV-93 whereas HPV-92 and 96 were not affected. As there are variable UV-B responses among the HPV types, further studies of interactions between UV-B and HPV need to consider the HPV type.

Objectifs Evaluer les niveaux de methylation de l’ADN de genes humain EPB41L3 parmi les femmes africaines seropositives pour le VIH-1 et co-infectees par des genotypes HPV a haut-risque (HR-HPV), avec ou sans lesions du col uterin ; et evaluer l’influence des facteurs lies au VIH sur la methylation du gene EPB41L3. Population d’etude et methodes La methylation du gene humain EPB41L3 a ete mesure par pyrosequencage parmi 132 echantillons cervicaux obtenus lors d’une etude de cohorte de femmes seropositives au VIH-1 âgees de 25–50 ans suivies au Burkina Faso et en Afrique du Sud (etude HARP). Cette sous-etude inclut 41 femmes du Burkina Faso et 91 femmes d’Afrique du Sud, dont 83 femmes avec pathologie cervicale (CIN2+ a l’histologie) et 49 femmes sans pathologie detectee. Resultats Le niveau (pourcentage) de methylation du gene EPB41L3 etait plus eleve parmi les cas de CIN2+ compares aux cas ≤ CIN 1 (mediane = 2,6 % (IQR 0,0 - 14,4) chez les CIN2+ compare a 0,0 % (IQR 0,0 - 2,2) chez les cas ≤ CIN1, Mann Whitney U p-test = 0,0003). La methylation du gene EPB41L3 permettait globalement une bonne separation des cas de CIN2+ de ceux avec ≤ CIN 1, avec une valeur AUC (Area Under the Curve) de 0,69 (IC95 % 0,59 - 0,78). Cette performance etait meilleure en ce qui concerne les echantillons du Burkina Faso (AUC = 0,79, IC95 % 0,65 - 0,94) compares a ceux d’Afrique du Sud (AUC = 0,63, IC95 % 0,51 - 0,76) (p = 0,099). La methylation etait aussi significativement plus elevee pour les cas de CIN2+ compares aux cas ≤ CIN1 parmi les femmes plus âgees (≥ 35 ans, p = 0,01), celles sous antiretroviraux depuis une plus courte periode (≤ 24 mois, p = 0,05) et celles ayant des taux de lymphocytes CD4+ ≤ 350 cellules/mm 3 (p = 0,005). Conclusion La methylation du gene humain EPB41L3 se revele etre un biomarqueur potentiel des lesions cervicales chez les femmes seropositives au VIH et co-infectees par les HR-HPV, bien que les differences specifiques liees aux populations indiquent que des facteurs ethniques ou demographiques voire virologiques lies au VIH doivent peut-etre etre pris en consideration.

Tumor suppressor genes (TSGs) play critical roles in preventing tumorigenesis and they are frequently inactivated in tumours. Recently developed high-density microarrays can detect subchromosomal deletions, recurrence of which usually indicates the location of TSGs within the deleted region. We analyzed testicular germ cell tumour (TGCT) clinical samples using SNP arrays and found a frequent small deletion on the region 6q25.3 containing only one known gene, ZDHHC14. While its cellular function is unknown, ZDHHC14 belongs to the recently discovered DHHC family, which are predicted to be involved in protein palmitoylation, a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Consistently, we found a dramatic under-expression of ZDHHC14 mRNA and protein in TGCTs, and this associated with chemoresistance. Oncomine database mining showed that ZDHHC14 is also under-expressed in lymphoma, liposarcoma, brain, kidney, lung and colorectal cancers. Thus, it appears that ZDHHC14 downregulation may be involved in other cancers. We studied ZDHHC14 expression in prostate cancer (PCa), detecting a decrease at mRNA and protein level. We also detected that ZDHHC14 mRNA was downregulated in a pilot study on breast cancer samples. As genomic loss of the ZDHHC14 region was only detected in a small number of PCa samples, we checked whether promoter hypermethylation was the cause for ZDHHC14 downregulation. However, no changes in methylation status were found. We then sequenced the whole genomic region surrounding ZDHHC14 by next generation sequencing in TGCTs and PCa and found several mutations in the promoter, the coding region, as well as in intronic regions. Finally, we tested the function of ZDHHC14 in cell-based studies. We generated a 293 T-REx tetracycline inducible ZDHHC14 overexpressing stable cell line, which showed that ZDHHC14 overexpression decreased cell viability. The induction of apoptosis by ZDHHC14 overexpression was detected both by FACS and caspase 7 and PARP cleavage analyses. This was confirmed by transient ZDHHC14 overexpression in the PCa cell line 22RV1. In vivo we xenografted mice using both tetracycline inducible ZDHHC14 overexpressing 293 T-REx cells and control cells transfected with the empty vector. ZDHHC14 expression was induced by tetracycline at the beginning of inoculation and we detected that ZDHHC14 overexpression blocked tumour initiation completely. In conclusion, these results implicate ZDHHC14 as a tumour suppressor gene commonly inactivated in human cancers, indicating that it might exert its tumor suppressor role through the induction of programmed cell death. This is the first study showing the involvement of ZDHHC14 in a specific pathway, the classic caspase-dependent apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4858. doi:1538-7445.AM2012-4858