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Aim: The aim of the study was to evaluate the use of global and gene-specific DNA methylation changes as potential biomarkers for gallbladder cancer (GBC) in a cohort from Chile., Material & Methods: DNA methylation was analyzed through an ELISA-based technique and quantitative methylation-specific PCR., Results: Global DNA Methylation Index (p = 0.02) and promoter methylation of SSBP2 (p = 0.01) and ESR1 (p = 0.05) were significantly different in GBC when compared with cholecystitis. Receiver curve operator analysis revealed promoter methylation of APC, CDKN2A, ESR1, PGP9.5 and SSBP2, together with the Global DNA Methylation Index, had 71% sensitivity, 95% specificity, a 0.97 area under the curve and a positive predictive value of 90%., Conclusion: Global and gene-specific DNA methylation may be useful biomarkers for GBC clinical assessment.

Methylation alterations of CpG islands, CpG island shores and first exons are key events in the formation and progression of human cancer, and an increasing number of differentially methylated regions and genes have been identified in breast cancer. Recent studies of the breast cancer methylome using deep sequencing and microarray platforms are providing a novel insight on the different roles aberrant methylation plays in molecular subtypes of breast cancer. Accumulating evidence from a subset of studies suggests that promoter methylation of tumor-suppressor genes associated with breast cancer can be quantified in circulating DNA. However, there is a paucity of studies that examine the combined presence of genetic and epigenetic alterations associated with breast cancer using blood-based assays. Dysregulation of DNA repair capacity (DRC) is a genetic risk factor for breast cancer that has been measured in lymphocytes. We isolated plasma DNA from 340 participants in a breast cancer case control project to study promoter methylation levels of five genes previously shown to be associated with breast cancer in frozen tissue and in cell line DNA: MAL, KIF1A, FKBP4, VGF and OGDHL. Methylation of at least one gene was found in 49% of the cases compared to 20% of the controls. Three of the four genes had receiver characteristic operator curve values of ≥ 0.50: MAL (0.64), KIF1A (0.51) and OGDHL (0.53). KIF1A promoter methylation was associated with breast cancer and inversely associated with DRC. This is the first evidence of a significant association between genetic and epigenetic alterations in breast cancer using blood-based tests. The potential diagnostic utility of these biomarkers and their relevance for breast cancer risk prediction should be examined in larger cohorts.

The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp™ Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after Nco I digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies.

Differentially methylated oral squamous cell carcinoma (OSCC) biomarkers, identified in vitro and validated in well-characterized surgical specimens, have shown poor clinical correlation in cohorts with different risk profiles. To overcome this lack of relevance, we used the HumanMethylation27 BeadChip, publicly available methylation and expression array data, and quantitative methylation specific PCR to uncover differential methylation in OSCC clinical samples with heterogeneous risk profiles. A two stage design consisting of discovery and prevalence screens was used to identify differential promoter methylation and deregulated pathways in patients diagnosed with OSCC and head and neck squamous cell carcinoma. Promoter methylation of KIF1A (κ = 0.64), HOXA9 (κ = 0.60), NID2 (κ = 0.60), and EDNRB (κ = 0.60) had a moderate to substantial agreement with clinical diagnosis in the discovery screen. HOXA9 had 68% sensitivity, 100% specificity, and a 0.81 Area Under the Curve (AUC). NID2 had 71% sensitivity, 100% specificity, and a 0.79 AUC. In the prevalence screen, HOXA9 (κ = 0.82) and NID2 (κ = 0.80) had an almost perfect agreement with histologic diagnosis. HOXA9 had 85% sensitivity, 97% specificity, and a 0.95 AUC. NID2 had 87% sensitivity, 95% specificity, and a 0.91 AUC. A HOXA9 and NID2 gene panel had 94% sensitivity, 97% specificity, and a 0.97 AUC. In saliva, from OSCC cases and controls, HOXA9 had 75% sensitivity, 53% specificity, and a 0.75 AUC. NID2 had 87% sensitivity, 21% specificity, and a 0.73 AUC. This phase I Biomarker Development Trial identified a panel of differentially methylated genes in normal and OSCC clinical samples from patients with heterogeneous risk profiles. This panel may be useful for early detection and cancer prevention studies.

Environmental exposures in-utero may alter the epigenome, thus impacting chromosomal stability and gene expression. We hypothesized that in utero exposures to maternal smoking and perfluoroalkyl compounds (PFCs) are associated with global DNA hypomethylation in umbilical cord serum. Our objective was to determine if global DNA methylation could be used as a biomarker of in utero exposures to maternal smoking and PFCs. Using an ELISA-based method, global DNA methylation was quantified in umbilical cord serum from 30 newborns with high (> 10 ng/ml, mean 123.8 ng/ml), low (range 1-10 ng/ml, mean 1.6 ng/ml) and very low (< 1 ng/ml, mean 0.06 ng/ml) cord serum cotinine levels. Y chromosome analysis was performed to rule out maternal DNA cross-contamination. Cord serum global DNA methylation showed an inverse dose response to serum cotinine levels (p< 0.001). Global DNA methylation levels in cord blood were the lowest among newborns with smoking mothers (mean=15.04%; 95% CI, 8.4, 21.7) when compared to babies of mothers who were second-hand smokers (21.1%; 95% CI, 16.6, 25.5) and non-smokers (mean=29.2%; 95% CI, 20.1, 38.1). Global DNA methylation was inversely correlated with serum PFOA (r= -0.72, p < 0.01) but not PFOS levels. Serum Y chromosome analyses did not detect maternal DNA cross-contamination. This study supports the use of global DNA methylation status as a biomarker of in utero exposure to cigarette smoke and PFCs.