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DNA methylation in peripheral blood is a potential biomarker of gastric cancer risk which could be used for early detection. We conducted a prospective case–control study nested within the Melbourne Collaborative Cohort Study. Genomic DNA was prepared from blood samples collected a median of 12 years before diagnosis for cases (N = 168). Controls (N = 163) were matched to cases on sex, year of birth, country of birth, and blood sample type using incidence density sampling. Genome-wide DNA methylation was measured using the Infinium HumanMethylation450K Beadchip. Global measures of DNA methylation were defined as the median methylation M value, calculated for each of 13 CpG subsets representing genomic function, mean methylation and location, and reliability of measurement. Conditional logistic regression was conducted to assess associations between these global measures of methylation and gastric cancer risk, adjusting for Helicobacter pylori and other potential confounders. We tested nonlinear associations using quintiles of the global measure distribution. A genome-wide association study of DNA methylation and gastric cancer risk was also conducted (N = 484,989 CpGs) using conditional logistic regression, adjusting for potential confounders. Differentially methylated regions (DMR) were investigated using the R package DMRcate. We found no evidence of associations with gastric cancer risk for individual CpGs or DMRs (P > 7.6 × 10−6). No evidence of association was observed with global measures of methylation (OR 1.07 per SD of overall median methylation; 95% confidence interval, 0.80–1.44; P = 0.65). We found no evidence that blood DNA methylation is prospectively associated with gastric cancer risk.Prevention Relevance: We studied DNA methylation in blood to try and predict who was at risk of gastric cancer before symptoms developed, by which stage survival is poor. We did not find any such markers, but the importance of early diagnosis in gastric cancer remains, and the search for markers continues.

Supplementary Data from Weight is More Informative than Body Mass Index for Predicting Postmenopausal Breast Cancer Risk: Prospective Family Study Cohort (ProF-SC)

Background: Self-reported information may not accurately capture smoking exposure. We aimed to evaluate whether smoking-associated DNA methylation markers improve urothelial cell carcinoma (UCC) risk prediction. Methods: Conditional logistic regression was used to assess associations between blood-based methylation and UCC risk using two matched case–control samples: 404 pairs from the Melbourne Collaborative Cohort Study (MCCS) and 440 pairs from the Women's Health Initiative (WHI) cohort. Results were pooled using fixed-effects meta-analysis. We developed methylation-based predictors of UCC and evaluated their prediction accuracy on two replication data sets using the area under the curve (AUC). Results: The meta-analysis identified associations (P < 4.7 × 10−5) for 29 of 1,061 smoking-associated methylation sites, but these were substantially attenuated after adjustment for self-reported smoking. Nominally significant associations (P < 0.05) were found for 387 (36%) and 86 (8%) of smoking-associated markers without/with adjustment for self-reported smoking, respectively, with same direction of association as with smoking for 387 (100%) and 79 (92%) markers. A Lasso-based predictor was associated with UCC risk in one replication data set in MCCS [N = 134; odds ratio per SD (OR) = 1.37; 95% CI, 1.00–1.90] after confounder adjustment; AUC = 0.66, compared with AUC = 0.64 without methylation information. Limited evidence of replication was found in the second testing data set in WHI (N = 440; OR = 1.09; 95% CI, 0.91–1.30). Conclusions: Combination of smoking-associated methylation marks may provide some improvement to UCC risk prediction. Our findings need further evaluation using larger data sets. Impact: DNA methylation may be associated with UCC risk beyond traditional smoking assessment and could contribute to some improvements in stratification of UCC risk in the general population.

While most epigenetic marks are reprogrammed during early embryogenesis, some studies have reported Mendelian-like inheritance of germline DNA methylation in particular in cancer susceptibility genes. For instance, individuals with MLH1 silenced throughout the soma fit the clinical criteria for hereditary nonpolyposis colorectal cancer that is indistinguishable from the syndrome resulting from germline mutations in MLH1. Research using multiple-case breast cancer families has shown that LINE-1 and Sat2 DNA methylation levels are lower in individuals with a strong family history. Family clustering of cancer could therefore be due to epigenetic as well as genetic and shared environmental factors. We have recently identified heritable methylation marks associated with breast and/or prostate cancer susceptibility by conducting a study involving 45 Australian multi-generational families with multiple cases of breast or prostate cancer who are not known to carry genetic mutations in cancer susceptibility genes. We developed and applied a new statistical method to identify heritable methylation marks based on complex segregation analysis and identified 24 and 41 methylation marks significantly associated with breast and prostate cancer risk respectively. Several marks across VTRNA2-1, a gene located in a differentially methylated region that is involved in imprinting and shows allele-specific methylation, were associated with heritable risk of both cancer types. A proportion of all identified marks were found to be associated with cancer risk in independent nested case-control studies (ie outside of the multiple-case family setting). We are expanding these successful studies to include additional families and to estimate HRs and age-specific cumulative risks of cancer associated with these marks to enable the incorporation of this information into clinical tools for risk prediction. Citation Format: Melissa C. Southey, Jihoon E. Joo, James G. Dowty, Roger L. Milne, EE Ming Wong, Pierre-Antoine Dugué, Dallas English, John L. Hopper, David E. Goldgar, Graham G. Giles. Heritable methylation marks associated with prostate and breast cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3314.

Background. Environmental and genetic factors play an important role in the etiology of breast cancer; however the potential epigenetic component to the risk remains largely unexplored. DNA methylation in blood cells could serve as a biomarker for exposure and breast cancer risk. Several small blood-based DNA methylation studies have reported risk associations with individual CpGs and average methylation levels; however, these findings require validation in larger prospective cohort studies. To investigate the role of blood DNA methylation on breast cancer risk, we conducted a meta-analysis of four prospective cohort studies, including a total of 1,941 cases and 1,952 controls, the largest study of blood DNA methylation and breast cancer risk to date. Methods. We assessed associations with methylation at 365,589 CpGs present in the HumanMethylation450 Beadchip, after excluding CpGs that did not pass quality controls in all studies. Each of the four cohorts (the Melbourne Collaborative Cohort Study, the Italian and IARC cohorts of the European Prospective Investigation into Cancer and Nutrition and the Prostate, Lung, Colorectal and Ovarian screening trial) estimated odds ratios (ORs) and 95% confidence intervals (CI) for the association between each individual CpG and breast cancer risk. In addition, each study assessed the association between average methylation measures and breast cancer risk, adjusted and unadjusted for cell-type composition. Study-specific ORs were combined using fixed-effect meta-analysis with inverse variance weights. The false discovery rate (q-value) was used to account for multiple testing. Results. Methylation measured at individual CpGs in blood DNA was not associated with breast cancer risk (q-value>0.6). In addition, higher average methylation level was not associated with risk of breast cancer (OR=0.94, 95% CI=0.85, 1.04; P=0.25; P for study heterogeneity=0.83). We found no evidence of modification of this association by age at diagnosis, time since blood collection or CpG location (P-heterogeneity>0.05). Conclusions. Overall, our data indicates that DNA methylation measured in blood prior to breast cancer diagnosis is unlikely to be associated with substantial breast cancer risk. Larger studies are needed to determine if modest to weak associations exist between blood DNA methylation and breast cancer risk. Citation Format: Clara Bodelon, Srikant Ambatipudi, Pierre-Antoine Dugué, Annelie Johansson, Joshua N. Sampson, Melissa C. Southey, Graham G. Giles, Silvia Polidoro, Zdenko Herceg, James M. Flanagan, Roger L. Milne, Montserrat Garcia-Closas. Genome-wide blood DNA methylation and breast cancer risk: A meta-analysis of four prospective studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3230.

While most epigenetic marks are reprogrammed during early embryogenesis, some studies have reported Mendelian-like inheritance of germline DNA methylation in particular cancer susceptibility genes. We aimed to identify heritable methylation marks associated with breast cancer susceptibility by studying 25 Australian multi-generational families with multiple cases of breast cancer who were not known to carry mutations in breast cancer susceptibility genes. Peripheral blood DNA methylation was measured at approximately 480,000 genetic loci (methylation sites) for 210 members of these families using the Infinium HumanMethylation450 BeadArray. We developed and applied a new statistical method to identify heritable methylation marks based on complex segregation analysis. Carrier probabilities were estimated for the 1000 most heritable methylation marks, based on family structure but not disease status, and Cox proportional hazards survival analysis was used to test for associations between these carrier probabilities and breast cancer. After correcting for multiple testing, we identified 11 methylation marks whose corresponding carriers probabilities were significantly associated with breast cancer. Three marks clustered within 200 base pairs of the noncoding RNA miR886. We found evidence for these marks to be associated with aggressive (but not non-aggressive) prostate cancer risk in a population-based prostate cancer study. The association with breast cancer risk was replicated for three of the eleven marks in an independent population-based breast cancer study. Our work demonstrates the efficacy of our novel approach for identifying heritable methylation marks associated with breast cancer. This methodology may provide insights into other disease settings where known lifestyle exposures and genetic risk variants do not fully explain the familial relative risk. Citation Format: Melissa C. Southey, Jihoon E. Joo, James G. Dowty, Roger L. Milne, EE Ming Wong, Pierre-Antoine Dugué, Dallas English, John L. Hopper, Daivd E. Goldgar, Graham G. Giles. Heritable methylation marks associated with breast cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3357. doi:10.1158/1538-7445.AM2017-3357