Bridget Riggs

Quantitative DNA methylation analysis of candidate genes in cervical cancer.

Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

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.

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.

A positive association between umbilical cord RBC folate and fetal TL at birth supports a potential for fetal reprogramming

Telomere length (TL) has been studied extensively in adults; however, limited information exists regarding maternal influences on TL in utero. The objective of this study was to investigate the relationship between fetal red blood cell (RBC) folate levels, a surrogate measure for maternal folate levels, and TL. We hypothesized that umbilical cord RBC folate concentrations would positively correlate with fetal TL. Data for this analysis were collected as part of a prospective cohort study that recruited pregnant women upon admission into labor and delivery. Cord blood was collected for 96 maternal-fetal dyads, and DNA analysis was performed using quantitative polymerase chain reaction. The telomere to single copy gene ratio method was used to determine TL, and RBC folate levels were measured. Statistical analysis was conducted by incorporating a bootstrapping approach into generalized linear modeling-based analyses. Consistent significant positive correlations were observed between RBC folate and TL (telomere to single copy gene ratio) with 9880 of the 10000 (98.8%) iterations performed having a P value less than .05. Our study shows a positive association between umbilical cord RBC folate and fetal TL at birth. These findings may provide a pathway of understanding and preventing adult-onset disease and mortality through intrauterine reprogramming.

Homocysteine Levels Are not Related to Telomere Length in Cord Blood Leukocytes of Newborns.

Objective Elevated homocysteine (HC) levels and/or shortened telomere length (TL) are associated with adverse medical conditions. Our objective is to investigate the relationship between HC and TL in cord blood leukocytes of newborns. Study Design This is a nested study from a prospective cohort from 2011 to 2012 in pregnant women admitted for delivery at a university-affiliated hospital. Cord blood was collected at delivery and genomic DNA was analyzed using quantitative PCR. The telomere-to-single copy gene ratio method was employed to quantify TL. Newborn HC levels were measured. generalized linear regression modeling (GLM) and bootstrap statistical analyses were performed. Results Seventy-seven maternal-fetal dyads with a mean gestational age of 39 weeks were included. The distribution of the coefficient of homocysteine showed most values greater than zero demonstrating that homocysteine had a positive relationship with TL. In 915 of 10,000 (9.15%) iterations, the p-value was < 0.05 demonstrating a positive effect. Conclusion Increasing newborn concentrations of HC are not associated with decreasing TL. Larger, prospective studies are needed to confirm these findings and long-term implications.

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.

Evidence of altered brain regulatory gene expression in tobacco-exposed fetuses

Abstract Aim: We sought to determine the association between prenatal smoking status and expression of fetal brain regulatory genes. Methods: At delivery, we collected information from parturient women on prenatal smoking habits and analyzed salivary cotinine levels. We obtained neonatal umbilical cord blood and extracted total RNA. We then employed the quantitative polymerase chain reaction (QPCR) analyses and the comparative CT method to calculate the relative gene expression of selected fetal brain regulatory genes responsible for (1) brain growth (brain-derived neutrotrophic factor, BDNF), (2) myelination (proteolipidic protein 1, PLP1 and myelin basic protein, MBP), and (3) neuronal migration and cell-cell interactions during fetal brain development or RLN. The χ2-test, analysis of variance (ANOVA), and the Grubb test were used to evaluate the relationship between prenatal smoking status and relative gene expression levels. Further analysis using bootstrapping was performed to assess the precision of our estimates. Results: Of the 39 maternal-infant dyads included in this study, 25.6% were non-smokers, 43.6% were passive smokers and 30.8% were active smokers. The results showed down-regulation of the selected fetal brain regulatory genes among active smokers. Conclusions: These findings represent preliminary evidence in humans that intrauterine tobacco exposure impacts fetal brain programming. Future studies are warranted to examine whether our findings represent potential mechanisms through which adverse childhood/adult-onset cognitive and behavioral outcomes that have been previously linked to intrauterine exposure occur.

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.

Abstract 4930: Comparing the methylation status in cervical squamous intraepithelial lesions using quantitative real-time methylation specific PCR (QMSP) and pyrosequencing

Background: Tumor suppressor genes, adenomatous polyposis coli (APC) and retinoic acid receptor b-2 (RARB) have been shown to be methylated early in cervical carcinogenesis; however results vary across studies. There are several approaches that can be taken to assess cervical methylation status, each of which may influence the results. Quantitative real-time methylation specific PCR (QMSP) amplifies methylated DNA and quantifies target methylation level relative to house-keeping genes (e.g. b-actin). Pyrosequencing, on the other hand, amplifies bisulfite converted genomic DNA using primers independent of methylation status and quantifies the percentage of methylated CpG dinucleotides with single base resolution within a targeted loci. Objectives: The purpose of this study was to determine the prevalence of methylation in two tumor suppressor genes, APC and RARB in liquid-based cytology specimens diagnosed as low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL) using QMSP and pyrosequencing. Methods: We examined 63 LSIL and 32 HSIL residual liquid-based cytology specimens. Methylation of APC and RARB was measured using QMSP and pyrosequencing in the same genomic region. HPV genotyping was conducted using the Roche Linear Array HPV Genotyping Test which enables PCR-based identification of 37 high- and low-risk HPV genotypes. Results: QMSP detected APC methylation more often than pyrosequencing (36% vs. 6%), with QMSP identifying all pyrosequencing positive samples. RARB was rarely methylated in SILs in both assays (QMSP vs. Pyrosequencing: 4% vs. 3%) and there was no overlap in methylation between the methods. Overall, there were no differences in methylation for APC between HPV positive and HPV negative samples and LSIL and HSIL. RARB was significantly more likely to be methylated in HSIL using QMSP but not when measured by pyrosequencing, however there was no difference among HPV positive and HPV negative specimens. Conclusions: The use of different methylation detection methods on exfoliated cervical specimens provided different methylation prevalence estimates. Exfoliated cervical specimens (e.g. Pap Smears) contain a high proportion of normal cervical cells and relatively few abnormal cells. This heterogeneity may lead to misclassification of lesion methylation status when using methylation independent assays. Methylation biomarker detection in exfoliated cells collected for clinical use is critical for the translation to clinical testing; however, detection methods need to be sensitive enough to identify biomarkers in a high background of normal cells. While QMSP does not provide methylation frequency at individual CpG sites, this study suggests QMSP may be well suited for the assessment of methylation within heterogeneous samples. Larger studies are needed to confirm these results. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4930.