Abidemi Ajidahun

Age-Specific Seroprevalence of Merkel Cell Polyomavirus, BK Virus, and JC Virus

ABSTRACT We produced capsids of Merkel cell polyomavirus (MCPyV) in a baculovirus expression system and developed a virus-like particle (VLP) enzyme-linked immunosorbent assay (ELISA). To determine age-specific seroprevalence, serum samples were collected from 947 individuals attending hospital outpatient clinics and ranging in age from 1 to 93 years. To evaluate the association between exposure to MCPyV and Merkel cell cancer (MCC), plasma samples were obtained from 33 MCC patients and 37 controls. MCPyV seroprevalence was 45% in children under 10 years of age, increased to 60% in the next decade of life, and peaked at 81% among those 60 to 69 years of age. Levels of MCPyV capsid antibodies were positively correlated with age (P = 0.007). Virus specificity of MCPyV seroreactivity was supported by competitive inhibition of reactivity by MCPyV VLPs and not by BK polyomavirus (BKPyV) VLPs. MCPyV seroprevalence was greater among MCC patients (91%) than controls (68%; age-adjusted P value, 0.32); the mean level of MCPyV antibodies was also greater (P = 0.04). The age-specific seroprevalence of MCPyV shares with previously known polyomaviruses, BKPyV and JC polyomavirus (JCPyV), evidence of widespread exposure in human populations beginning early in life. MCPyV age-specific seroprevalence also has unique features. Seroprevalence among children is higher than that of JCPyV but lower than that of BKPyV. Among older adults, MCPyV seroprevalence remains high, while that of BKPyV declines and that of JCPyV continues to rise. In agreement with results from other studies, we found an association between MCPyV seropositivity and MCC, and higher levels of serum MCPyV capsid antibodies in MCC patients than in controls.

Race and prevalence of human papillomavirus infection among men residing in Brazil, Mexico and the United States.

Human papillomavirus (HPV) causes anal, penile and oropharyngeal cancers in men. Genital HPV prevalence in men appears to vary by world region with men residing in Asia having among the lowest prevalence. Unfortunately, there is little information on prevalence of HPV infection in men by race. The purpose of this study was to examine HPV prevalence by race across three countries. 3,909 men ages 18–70 years enrolled in an ongoing prospective cohort study of the natural history of HPV in men (The HIM Study) were included in the analysis. Participants completed risk factor questionnaires and samples were taken from the penile epithelium and scrotum for HPV detection. HPV testing of the combined DNA extract was conducted using PCR and genotyping. Asian/Pacific Islanders had the lowest HPV prevalence of 42.2% compared to Blacks (66.2%), and Whites (71.5%). The Asian/Pacific Islander race was strongly protective in univariate analysis (prevalence ratio (PR) = 0.59; 95% confidence interval (CI): 0.48–0.74) and multivariate analysis for any HPV infection (PR = 0.65; 95% CI: 0.52–0.8). Stratified analysis by lifetime number of female partners also showed strong inverse associations with the Asian/Pacific Islander race. We consistently observed the lowest prevalence of HPV infection among Asian/Pacific Islanders with moderate inverse associations even after various adjustments for potential confounding factors. Unmeasured behavioral factors, sexual mixing with low risk women, and/or race‐specific differences in the frequency of germline variations among immune regulating genes may underlie these associations. Further studies among Asian populations that incorporate measures of immuno‐genetics are needed to understand this phenomenon.

DNA Methylation Profiling across the Spectrum of HPV-Associated Anal Squamous Neoplasia

Background Changes in host tumor genome DNA methylation patterns are among the molecular alterations associated with HPV-related carcinogenesis. However, there is little known about the epigenetic changes associated specifically with the development of anal squamous cell cancer (SCC). We sought to characterize broad methylation profiles across the spectrum of anal squamous neoplasia. Methodology/Principal Findings Twenty-nine formalin-fixed paraffin embedded samples from 24 patients were evaluated and included adjacent histologically normal anal mucosa (NM; n = 3), SCC-in situ (SCC-IS; n = 11) and invasive SCC (n = 15). Thirteen women and 11 men with a median age of 44 years (range 26–81) were included in the study. Using the SFP10 LiPA HPV-typing system, HPV was detected in at least one tissue from all patients with 93% (27/29) being positive for high-risk HPV types and 14 (93%) of 15 invasive SCC tissues testing positive for HPV 16. Bisulfite-modified DNA was interrogated for methylation at 1,505 CpG loci representing 807 genes using the Illumina GoldenGate Methylation Array. When comparing the progression from normal anal mucosa and SCC-IS to invasive SCC, 22 CpG loci representing 20 genes demonstrated significant differential methylation (p<0.01). The majority of differentially methylated gene targets occurred at or close to specific chromosomal locations such as previously described HPV methylation “hotspots” and viral integration sites. Conclusions We have identified a panel of differentially methlylated CpG loci across the spectrum of HPV-associated squamous neoplasia of the anus. To our knowledge, this is the first reported application of large-scale high throughput methylation analysis for the study of anal neoplasia. Our findings support further investigations into the role of host-genome methylation in HPV-associated anal carcinogenesis with implications towards enhanced diagnosis and screening strategies.

Expanding Epigenomics to Archived FFPE Tissues: An Evaluation of DNA Repair Methodologies

Background: Epigenome-wide association studies are emerging in the field of cancer epidemiology with the rapid development of large-scale methylation array platforms. Until recently, these methods were only valid for DNA from flash frozen (FF) tissues. Novel techniques for repairing DNA from formalin-fixed paraffin-embedded (FFPE) tissues have emerged; however, a direct comparison of FFPE DNA repair methods before analysis on genome-wide methylation array to matched FF tissues has not been conducted. Methods: We conducted a systematic performance comparison of two DNA repair methods (REPLI-g Ligase vs. Infinium HD Restore Kit) on FFPE-DNA compared with matched FF tissues on the Infinium 450K array. A threshold of discordant methylation between FF-FFPE pairs was set at Δβ > 0.3. The correlations of β-values from FF–FFPE pairs were compared across methods and experimental conditions. Results: The Illumina Restore kit outperformed the REPLI-g ligation method with respect to reproducibility of replicates (R2 > 0.970), highly correlated β-values between FF-FFPE (R2 > 0.888), and fewest discordant loci between FF-FFPE (≤0.61%). The performance of the Restore kit was validated in an independent set of 121 FFPE tissues. Conclusions: The Restore kit outperformed RELPI-g ligation in restoring FFPE-derived DNA before analysis on the Infinium 450K methylation array. Our findings provide critical guidance that may significantly enhance the breadth of diseases that can be studied by methylomic profiling. Impact: Epigenomic studies using FFPE tissues should now be considered among cancers that have not been fully characterized from an epigenomic standpoint. These findings promote novel epigenome-wide studies focused on cancer etiology, identification of novel biomarkers, and developing targeted therapies. See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.” Cancer Epidemiol Biomarkers Prev; 23(12); 2622–31. ©2014 AACR.

The interplay between histone deacetylases and c-Myc in the transcriptional suppression of HPP1 in colon cancer

HPP1 (hyperplastic polyposis protein 1), a tumor suppressor gene, is downregulated by promoter hypermethylation in a number of tumor types including colon cancer. c-Myc is also known to play a role in the suppression of HPP1 expression via binding to a promoter region cognate E-box site. The contribution of histone deacetylation as an additional epigenetic mechanism and its potential interplay with c-Myc in the transcriptional regulation of HPP1 are unknown. We have shown that the treatment of the HPP1-non-expressing colon cancer cell lines, HCT116 and DLD-1 with HDAC inhibitors results in re-expression of HPP1. RNAi-mediated knockdown of c-Myc as well as of HDAC2 and HDAC3 in HCT116 and of HDAC1 and HDAC3 in DLD-1 also resulted in significant re-expression of HPP1. Co-immunoprecipitation (IP), chromatin IP (ChIP), and sequential ChIP experiments demonstrated binding of c-Myc to the HPP1 promoter with recruitment of and direct interaction with HDAC3. In summary, we have demonstrated that c-Myc contributes to the epigenetic regulation of HPP1 via the dominant recruitment of HDAC3. Our findings may lead to a greater biologic understanding for the application of targeted use of HDAC inhibitors for anti-cancer therapy.

Epigenomic characterization of locally advanced anal cancer: A radiation therapy oncology group 98-11 specimen study

BACKGROUND: The Radiation Therapy Oncology Group 98-11 clinical trial demonstrated the superiority of standard 5-fluorouracil/mitomycin-C over 5-fluorouracil/cisplatin in combination with radiation in the treatment of anal squamous cell cancer. Tumor size (>5 cm) and lymph node metastases are associated with disease progression. There may be key molecular differences (eg, DNA methylation changes) in tumors at high risk for progression. OBJECTIVE: The objectives of this study were to determine whether there are differences in DNA methylation at individual CpG sites and within genes among locally advanced anal cancers, with large tumor size and/or nodal involvement, compared with those that are less advanced. DESIGN: This was a case-case study among 121 patients defined as high risk (tumor size >5 cm and/or nodal involvement; n = 59) or low risk (⩽5 cm, node negative; n = 62) within the mitomycin-C arm of the Radiation Therapy Oncology Group 98-11 trial. DNA methylation was measured using the Illumina HumanMethylation450 Array. SETTINGS: The study was conducted in a tertiary care cancer center in collaboration with a national clinical trials cooperative group. PATIENTS: The patients consisted of 74 women and 47 men with a median age of 54 years (range, 25-79 years). MAIN OUTCOME MEASURES: DNA methylation differences at individual CpG sites and within genes between low- and high-risk patients were compared using the Mann-Whitney test (p < 0.001). RESULTS: A total of 16 CpG loci were differentially methylated (14 increased and 2 decreased) in high- versus low-risk cases. Genes harboring differentially methylated CpG sites included known tumor suppressor genes and novel targets. LIMITATIONS: This study only included patients in the mitomycin-C arm with tumor tissue; however, this sample was representative of the trial. CONCLUSIONS: This is the first study to apply genome-wide methylation analysis to anal cancer. Biologically relevant differences in methylated targets were found to discriminate locally advanced from early anal cancer. Epigenetic events likely play a significant role in the progression of anal cancer and may serve as potential biomarkers.

Epigenomic characterization of locally advanced anal cancer

BACKGROUND: The Radiation Therapy Oncology Group 98-11 clinical trial demonstrated the superiority of standard 5-fluorouracil/mitomycin-C over 5-fluorouracil/cisplatin in combination with radiation in the treatment of anal squamous cell cancer. Tumor size (>5 cm) and lymph node metastases are associated with disease progression. There may be key molecular differences (eg, DNA methylation changes) in tumors at high risk for progression. OBJECTIVE: The objectives of this study were to determine whether there are differences in DNA methylation at individual CpG sites and within genes among locally advanced anal cancers, with large tumor size and/or nodal involvement, compared with those that are less advanced. DESIGN: This was a case-case study among 121 patients defined as high risk (tumor size >5 cm and/or nodal involvement; n = 59) or low risk (⩽5 cm, node negative; n = 62) within the mitomycin-C arm of the Radiation Therapy Oncology Group 98-11 trial. DNA methylation was measured using the Illumina HumanMethylation450 Array. SETTINGS: The study was conducted in a tertiary care cancer center in collaboration with a national clinical trials cooperative group. PATIENTS: The patients consisted of 74 women and 47 men with a median age of 54 years (range, 25-79 years). MAIN OUTCOME MEASURES: DNA methylation differences at individual CpG sites and within genes between low- and high-risk patients were compared using the Mann-Whitney test (p < 0.001). RESULTS: A total of 16 CpG loci were differentially methylated (14 increased and 2 decreased) in high- versus low-risk cases. Genes harboring differentially methylated CpG sites included known tumor suppressor genes and novel targets. LIMITATIONS: This study only included patients in the mitomycin-C arm with tumor tissue; however, this sample was representative of the trial. CONCLUSIONS: This is the first study to apply genome-wide methylation analysis to anal cancer. Biologically relevant differences in methylated targets were found to discriminate locally advanced from early anal cancer. Epigenetic events likely play a significant role in the progression of anal cancer and may serve as potential biomarkers.

Abstract 579: HPP1's tumor suppressive effects are mediated by ectodomain shedding but not by juxtacrine signaling

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: The tumor suppressor gene; HPP1 is downregulated in over 80% of colorectal cancers (CRC) and has potential as a serum and stool-based biomarker. HPP1 is a secreted transmembrane protein that contains an Epidermal Growth Factor (EGF) - like domain, two follistatin modules, and a cytosolic tail with a potential G-protein activating motif. The soluble ectodomain of HPP1 gene may undergo cleavage and functions as an EGF-like ligand which may bind to erbB family receptors. We sought to investigate the importance of cleavage and the possiblity of a juxtacrine role of the flanking EGF-like domain in the context of HPP1s tumor suppressive function.. Methods: An HPP1 double deletion construct (DD) lacking the juxtamembrane stalk cleavage site and an additional 4 amino acid residues (299-320) that constitutes the critical portion of the EGF-like domain was developed by PCR-based overlap extension mutagenesis. HPP1 non-expressing HCT116 cell lines were transfected with either wild-type HPP1 (WT), empty vector (EV) or double deletion construct (DD). Effects on proliferation (MTT assay) and anchorage-indpendent growth (soft agar assay) were evaluated. Results: We have previously shown that overexpression of HPP1 results in substantial reduction in proliferation, growth in soft agar, and tumorigenicity. We have also identified that transfection of HPP1 with a deleted stalk sequence cleavage site (amino acid residues 303-320) results in a near-complete attenuation of its tumor suppressive effects. In order to determine whether there is a contributory juxtacrine effect of non-cleaved HPP1, we transfected the double deletion construct with a non-functioning EGF-like domain. Successful transfections of EV, WT, and DD were confirmed by RT-PCR. DD cells had similar proliferative potential and growth in soft agar when compared to EV cells (p = 0.609 and p= 0.263, respectively). When compared to WT, DD cells demonstrated a loss of HPP1s growth suppressive impact on cellular proliferation (optical density; 0.16± 0.03 vs. 0.31±0.04, p= 0.0001) and colony formation in soft agar (14.7 ± 5.5 vs. 278 ± 95.0, p= 0.009). Conclusion: HPP1s tumor suppressive effects in colorectal cancer require cleavage and ectodomain shedding. However, it is unlikely that HPP1s biologic functions are mediated via an additional juxtacrine mechanism in this setting. Citation Format: Abul Elahi, Abidemi Ajidahun, Whalen Clark, Jonathan Hernandez, Leigh Ann Humphries, David Shibata. HPP1s tumor suppressive effects are mediated by ectodomain shedding but not by juxtacrine signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 579. doi:10.1158/1538-7445.AM2014-579

Abstract 3996: ErbB4 is critical for tumor suppressive HPP1/STAT signaling in colon cancer.

Background: HPP1 (also known as TMEFF2) encodes a cleavable transmembrane protein that is thought to be a tumor suppressive EGF-like ligand that activates STAT1 and 2-associated pathways. From phylogenetic analyses, it is postulated that HPP1 exerts its effects via erbB4. We sought to characterize the role of erbB4 in the context of the HPP1/STAT tumor suppressive signaling axis. Methods: Transient knockdown of erbB4 was achieved using siRNA in stable HPP1-overexpressing HCT116 transfectants. The impact of erbB4 manipulation on STAT signaling was assessed by expression and activation analyses using RT-PCR and Western blotting. Subsequent alterations in cell proliferation and anchorage-independent growth were evaluated by MTT and soft agar assays, respectively. Results: We have previously demonstrated that HPP1’s tumor suppressive effects are associated with activation of suppressive STAT1 and 2 and downregulation of oncogenic STAT3 and 5. Abrogation of erbB4 expression resulted in a reversal of this profile with a reduction in activated STAT1 and 2 and increased phosphorylation of STAT3 and 5. Moreover, inhibition of ErbB4 receptor in HPP1 over-expressing transfectants resulted in an attenuation of tumor suppressive behavior with increased cell proliferation (Optical Density: 0.12±0.11 vs. 0.35 ±0.040; p≤0.001) and colony formation in soft agar (385±25 vs. 66±30 colonies; p≤0.001) as compared to scrambled siRNA control transfectants. Conclusion: The EGF-like domain of HPP1 via erbB4 receptor signaling is essential for its tumor suppressive effects. Knockdown of erbB4 elicits a reversal of HPP1-mediated effects on cell behavior and STAT-family activation status. Therapeutic targeting of the erbB family of receptors is of great interest and our findings may lead to a greater understanding of the complex nature of their associated signaling pathways. Citation Format: Abul Elahi, Whalen Clark, Jonathan Hernandez, Leigh Ann Humphries, Jian Wang, Abidemi Ajidahun, David Shibata. ErbB4 is critical for tumor suppressive HPP1/STAT signaling in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3996. doi:10.1158/1538-7445.AM2013-3996

Abstract PR08: Identification of novel methylated genomic regions in invasive cervical cancer using a genome-wide approach

Introduction: In the US, an estimated 35,000 cancer cases per year are attributable to Human papillomavirus (HPV), including cancers of the cervix, anus and oropharynx. Despite the availability of an HPV-targeted vaccine, issues with low uptake and significant lag time from vaccination to cancer prevention will result in cervical cancer continuing to be a substantial burden to US and world-wide populations. It is becoming increasingly clear that epigenetic events play a critical role in HPV-associated carcinogenesis, including changes in host genome DNA methylation patterns. In the cervix, methylation patterns have been shown to be altered; however, there have been inconclusive findings as to the most informative methylation targets in cervical cancer. We hypothesize that there are novel epigenetic alterations in HPV-associated cervical cancer and these changes may be early detection biomarkers in pre-invasive cervical lesions. In this study, we sought to identify epigenetic differences in HPV-positive cervical cancers compared to HPV-positive normal tissue using genome-wide methylation arrays and to explore if these differences can be identified in cervical intraepithelial neoplasia (CIN) 3 lesions. Methods: DNA was extracted from 24 frozen cervical tissues (9 normal, 5 CIN 3 and 10 invasive cancers) following pathology review and macrodissection. Bisulfite-modified DNA was interrogated for methylation at over 450,000 CpG loci using the Illumina HumanMethylation450 BeadChip array. HPV genotyping was conducted using SFP10 LiPA HPV-typing system. Differential methylation between normal and tumors at individual CpG loci was determined by t-test with significance set by a false discovery rate (or q-value) and final sets were restricted to those with a mean difference in β-values≥0.3. Next, using a window-based approach, we examined clusters of CpG loci within gene coding regions or CpG islands. Finally, a binary classification strategy was used for each gene set of probes, where a probe was defined as methylated if the β-values were above the minimum point in the bi-modal histogram. Sensitivity and specificity for each gene at the set methylation cut-point was determined. Results: A total of 24 HPV positive tissues were examined in this study (9 normal, 5 CIN 3 and 10 invasive cancers). A total of 321 CpG loci displayed differential methylation with mean difference ≥0.5 in tumors compared to normal (FDR corrected q Conclusions: We have identified several novel methylated CpG loci and genomic regions in HPV-associated cervical cancer, some of which were also found to be altered in CIN 3 lesions. Validation of these novel CpG loci in a larger population of women with cervical dysplasia and cancer is warranted. A number of these novel methylated sites may emerge as promising biomarkers for the early detection of cervical cancer and possibly, for other HPV-associated malignancies. This abstract is also presented as Poster C24. Citation Format: Erin M. Siegel, Anders Berglund, Bridget Riggs, Steven Eschrich, Maddox Kristen, Abidemi Ajidahun, David Shibata, Kevin D. Brown. Identification of novel methylated genomic regions in invasive cervical cancer using a genome-wide approach. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr PR08.