Intratumoral presence of the genotoxic gut bacteria pks+ E. coli, Enterotoxigenic Bacteroides fragilis, and Fusobacterium nucleatum and their association with clinicopathological and molecular features of colorectal cancer.

BACKGROUND: This study aimed to investigate clinicopathological and molecular tumour features associated with intratumoral pks+ Escherichia coli (pks+E.coli+), pks+E.coli- (non-E.coli bacteria harbouring the pks island), Enterotoxigenic Bacteroides fragilis (ETBF) and Fusobacterium nucleatum (F. nucleatum). METHODS: We screened 1697 tumour-derived DNA samples from the Australasian Colorectal Cancer Family Registry, Melbourne Collaborative Cohort Study and the ANGELS study using targeted PCR. RESULTS: Pks+E.coli+ was associated with male sex (P < 0.01) and APC:c.835-8 A > G somatic mutation (P = 0.03). The association between pks+E.coli+ and APC:c.835-8 A > G was specific to early-onset CRCs (diagnosed<45years, P = 0.02). The APC:c.835-A > G was not associated with pks+E.coli- (P = 0.36). F. nucleatum was associated with DNA mismatch repair deficiency (MMRd), BRAF:c.1799T>A p.V600E mutation, CpG island methylator phenotype, proximal tumour location, and high levels of tumour infiltrating lymphocytes (Ps < 0.01). In the stratified analysis by MMRd subgroups, F. nucleatum was associated with Lynch syndrome, MLH1 methylated and double MMR somatic mutated MMRd subgroups (Ps < 0.01). CONCLUSION: Intratumoral pks+E.coli+ but not pks+E.coli- are associated with CRCs harbouring the APC:c.835-8 A > G somatic mutation, suggesting that this mutation is specifically related to DNA damage from colibactin-producing E.coli exposures. F. nucleatum was associated with both hereditary and sporadic MMRd subtypes, suggesting the MMRd tumour microenvironment is important for F. nucleatum colonisation irrespective of its cause.

Germline mutations in WNK2 could be associated with serrated polyposis syndrome.

BACKGROUND: Patients with serrated polyposis syndrome (SPS) have multiple and/or large serrated colonic polyps and higher risk for colorectal cancer. SPS inherited genetic basis is mostly unknown. We aimed to identify new germline predisposition factors for SPS by functionally evaluating a candidate gene and replicating it in additional SPS cohorts. METHODS: After a previous whole-exome sequencing in 39 SPS patients from 16 families (discovery cohort), we sequenced specific genes in an independent validation cohort of 211 unrelated SPS cases. Additional external replication was also available in 297 SPS cases. The WNK2 gene was disrupted in HT-29 cells by gene editing, and WNK2 variants were transfected using a lentiviral delivery system. Cells were analysed by immunoblots, real-time PCR and functional assays monitoring the mitogen-activated protein kinase (MAPK) pathway, cell cycle progression, survival and adhesion. RESULTS: We identified 2 rare germline variants in the WNK2 gene in the discovery cohort, 3 additional variants in the validation cohort and 10 other variants in the external cohorts. Variants c.2105C>T (p.Pro702Leu), c.4820C>T (p.Ala1607Val) and c.6157G>A (p.Val2053Ile) were functionally characterised, displaying higher levels of phospho-PAK1/2, phospho-ERK1/2, CCND1, clonogenic capacity and MMP2. CONCLUSION: After whole-exome sequencing in SPS cases with familial aggregation and replication of results in additional cohorts, we identified rare germline variants in the WNK2 gene. Functional studies suggested germline WNK2 variants affect protein function in the context of the MAPK pathway, a molecular hallmark in this disease.

Methylation scores for smoking, alcohol consumption and body mass index and risk of seven types of cancer.

Methylation marks of exposure to health risk factors may be useful markers of cancer risk as they might better capture current and past exposures than questionnaires, and reflect different individual responses to exposure. We used data from seven case-control studies nested within the Melbourne Collaborative Cohort Study of blood DNA methylation and risk of colorectal, gastric, kidney, lung, prostate and urothelial cancer, and B-cell lymphoma (N cases = 3123). Methylation scores (MS) for smoking, body mass index (BMI), and alcohol consumption were calculated based on published data as weighted averages of methylation values. Rate ratios (RR) and 95% confidence intervals for association with cancer risk were estimated using conditional logistic regression and expressed per SD increase of the MS, with and without adjustment for health-related confounders. The contribution of MS to discriminate cases from controls was evaluated using the area under the curve (AUC). After confounder adjustment, we observed: large associations (RR = 1.5-1.7) with lung cancer risk for smoking MS; moderate associations (RR = 1.2-1.3) with urothelial cancer risk for smoking MS and with mature B-cell neoplasm risk for BMI and alcohol MS; moderate to small associations (RR = 1.1-1.2) for BMI and alcohol MS with several cancer types and cancer overall. Generally small AUC increases were observed after inclusion of several MS in the same model (colorectal, gastric, kidney, urothelial cancers: +3%; lung cancer: +7%; B-cell neoplasms: +8%). Methylation scores for smoking, BMI and alcohol consumption show independent associations with cancer risk, and may provide some improvements in risk prediction.

A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome.

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n = 135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n = 137; 80×CRCs, 33×ECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

Body Mass Index, sex, non-steroidal anti-inflammatory drug medications, smoking and alcohol are differentially associated with World Health Organisation criteria and colorectal cancer risk in people with Serrated Polyposis Syndrome: an Australian case-control study.

OBJECTIVE: The unknown aetiology of Serrated Polyposis Syndrome (SPS) impedes risk prediction and prevention. We investigated risk factors for SPS, overall and stratified by World Health Organization (WHO)2010 clinical criteria and by colorectal cancer (CRC). METHOD: A retrospective case-control study involving a cross-sectional analysis from 350 unrelated individuals with SPS from the Genetics of Colonic Polyposis Study and 714 controls from the Australasian Colorectal Cancer Family Registry. Univariate and multivariate logistic regression modelling was used to determine the association between risk factors and SPS and risk factors associated with CRC in SPS. RESULTS: Female biological sex (odds ratio (OR) = 4.54; 95%Confidence interval (CI) = 2.77-7.45), increasing body mass index (BMI) at age 20 years (OR = 1.09; 95%CI = 1.04-1.13), hormone replacement therapy (OR = 0.44; 95%CI = 0.20.98), and increasing weekly folate intake (OR = 0.82; 95%CI = 0.75-0.90) were associated with SPS by multivariate analysis. Increasing weekly calcium intake (OR = 0.79; 95%CI = 0.64-0.97) and smoking > 10 cigarettes daily (OR = 0.45; 95%CI = 0.23-0.86) were associated with WHO criterion I only. The consumption of 1-100 g of alcohol per week (OR = 0.39; 95%CI = 0.18-0.83) was associated with WHO criterion III only. Smoking 1-5 cigarettes daily (OR = 2.35; 95%CI = 1.09-5.05), weekly non-steroidal anti-inflammatory drug (NSAIDs) intake (OR = 0.88; 95%CI = 0.78-0.99), and increased height (OR = 1.09; 95% = 1.05-1.13), were associated with SPS fulfilling both WHO criteria I and III. Moreover, weekly NSAIDs intake (OR = 0.81; 95%CI = 0.67-0.98) was associated with a reduced likelihood of CRC in SPS. CONCLUSION: We identified novel risk and potential protective factors associated with SPS, some specific for certain WHO2010 criteria. Weekly use of NSAIDs may reduce the risk of CRC in people with SPS.

Evaluating the utility of tumour mutational signatures for identifying hereditary colorectal cancer and polyposis syndrome carriers.

OBJECTIVE: Germline pathogenic variants (PVs) in the DNA mismatch repair (MMR) genes and in the base excision repair gene MUTYH underlie hereditary colorectal cancer (CRC) and polyposis syndromes. We evaluated the robustness and discriminatory potential of tumour mutational signatures in CRCs for identifying germline PV carriers. DESIGN: Whole-exome sequencing of formalin-fixed paraffin-embedded (FFPE) CRC tissue was performed on 33 MMR germline PV carriers, 12 biallelic MUTYH germline PV carriers, 25 sporadic MLH1 methylated MMR-deficient CRCs (MMRd controls) and 160 sporadic MMR-proficient CRCs (MMRp controls) and included 498 TCGA CRC tumours. COSMIC V3 single base substitution (SBS) and indel (ID) mutational signatures were assessed for their ability to differentiate CRCs that developed in carriers from non-carriers. RESULTS: The combination of mutational signatures SBS18 and SBS36 contributing >30% of a CRC's signature profile was able to discriminate biallelic MUTYH carriers from all other non-carrier control CRCs with 100% accuracy (area under the curve (AUC) 1.0). SBS18 and SBS36 were associated with specific MUTYH variants p.Gly396Asp (p=0.025) and p.Tyr179Cys (p=5×10-5), respectively. The combination of ID2 and ID7 could discriminate the 33 MMR PV carrier CRCs from the MMRp control CRCs (AUC 0.99); however, SBS and ID signatures, alone or in combination, could not provide complete discrimination (AUC 0.79) between CRCs from MMR PV carriers and sporadic MMRd controls. CONCLUSION: Assessment of SBS and ID signatures can discriminate CRCs from biallelic MUTYH carriers and MMR PV carriers from non-carriers with high accuracy, demonstrating utility as a potential diagnostic and variant classification tool.

Alcohol consumption is associated with widespread changes in blood DNA methylation: Analysis of cross-sectional and longitudinal data.

DNA methylation may be one of the mechanisms by which alcohol consumption is associated with the risk of disease. We conducted a large-scale, cross-sectional, genome-wide DNA methylation association study of alcohol consumption and a longitudinal analysis of repeated measurements taken several years apart. Using the Illumina HumanMethylation450 BeadChip, DNA methylation was measured in blood samples from 5606 Melbourne Collaborative Cohort Study (MCCS) participants. For 1088 of them, these measures were repeated using blood samples collected a median of 11 years later. Associations between alcohol intake and blood DNA methylation were assessed using linear mixed-effects regression models. Independent data from the London Life Sciences Prospective Population (LOLIPOP) (N = 4042) and Cooperative Health Research in the Augsburg Region (KORA) (N = 1662) cohorts were used to replicate associations discovered in the MCCS. Cross-sectional analyses identified 1414 CpGs associated with alcohol intake at P < 10-7 , 1243 of which had not been reported previously. Of these novel associations, 1078 were replicated (P < .05) using LOLIPOP and KORA data. Using the MCCS data, we also replicated 403 of 518 previously reported associations. Interaction analyses suggested that associations were stronger for women, non-smokers, and participants genetically predisposed to consume less alcohol. Of the 1414 CpGs, 530 were differentially methylated (P < .05) in former compared with current drinkers. Longitudinal associations between the change in alcohol intake and the change in methylation were observed for 513 of the 1414 cross-sectional associations. Our study indicates that alcohol intake is associated with widespread changes in DNA methylation across the genome. Longitudinal analyses showed that the methylation status of alcohol-associated CpGs may change with alcohol consumption changes in adulthood.

VTRNA2-1: Genetic Variation, Heritable Methylation and Disease Association.

VTRNA2-1 is a metastable epiallele with accumulating evidence that methylation at this region is heritable, modifiable and associated with disease including risk and progression of cancer. This study investigated the influence of genetic variation and other factors such as age and adult lifestyle on blood DNA methylation in this region. We first sequenced the VTRNA2-1 gene region in multiple-case breast cancer families in which VTRNA2-1 methylation was identified as heritable and associated with breast cancer risk. Methylation quantitative trait loci (mQTL) were investigated using a prospective cohort study (4500 participants with genotyping and methylation data). The cis-mQTL analysis (334 variants ± 50 kb of the most heritable CpG site) identified 43 variants associated with VTRNA2-1 methylation (p < 1.5 × 10-4); however, these explained little of the methylation variation (R2 < 0.5% for each of these variants). No genetic variants elsewhere in the genome were found to strongly influence VTRNA2-1 methylation. SNP-based heritability estimates were consistent with the mQTL findings (h2 = 0, 95%CI: -0.14 to 0.14). We found no evidence that age, sex, country of birth, smoking, body mass index, alcohol consumption or diet influenced blood DNA methylation at VTRNA2-1. Genetic factors and adult lifestyle play a minimal role in explaining methylation variability at the heritable VTRNA2-1 cluster.

Inference about causation between body mass index and DNA methylation in blood from a twin family study.

BACKGROUND: Several studies have reported DNA methylation in blood to be associated with body mass index (BMI), but few have investigated causal aspects of the association. We used a twin family design to assess this association at two life points and applied a novel analytical approach to appraise the evidence for causality. METHODS: The methylation profile of DNA from peripheral blood was measured for 479 Australian women from 130 twin families. Linear regression was used to estimate the associations of DNA methylation at ~410,000 cytosine-guanine dinucleotides (CpGs), and of the average DNA methylation at ~20,000 genes, with current BMI, BMI at age 18-21 years, and the change between the two (BMI change). A novel regression-based methodology for twins, Inference about Causation through Examination of Familial Confounding (ICE FALCON), was used to assess causation. RESULTS: At a 5% false discovery rate, nine, six and 12 CpGs at 24 loci were associated with current BMI, BMI at age 18-21 years and BMI change, respectively. The average DNA methylation of the BHLHE40 and SOCS3 loci was associated with current BMI, and of the PHGDH locus with BMI change. From the ICE FALCON analyses with BMI as the predictor and DNA methylation as the outcome, a woman's DNA methylation level was associated with her co-twin's BMI, and the association disappeared after conditioning on her own BMI, consistent with BMI causing DNA methylation. To the contrary, using DNA methylation as the predictor and BMI as the outcome, a woman's BMI was not associated with her co-twin's DNA methylation level, consistent with DNA methylation not causing BMI. CONCLUSION: For middle-aged women, peripheral blood DNA methylation at several genomic locations is associated with current BMI, BMI at age 18-21 years and BMI change. Our study suggests that BMI has a causal effect on peripheral blood DNA methylation.

Dietary Intake of Nutrients Involved in One-Carbon Metabolism and Risk of Gastric Cancer: A Prospective Study.

Dietary intakes of B vitamins and other components involved in one-carbon metabolism, which is necessary for DNA replication, DNA repair, and regulation of gene expression, may be associated with carcinogenesis. We investigated associations between intakes of 11 nutrients (thiamine, riboflavin, niacin, pantothenic acid, vitamin B6, biotin, folate, vitamin B12, methionine, choline, and betaine) and gastric cancer risk. A total of 159 incident gastric cancer cases were identified from the Melbourne Collaborative Cohort Study (N = 41,513) and matched with 159 controls on year of birth, sex, and country of birth using incidence density sampling. Dietary intakes of nutrients were estimated at baseline (1990-1994) using a 121-item food frequency questionnaire. Odds ratios (ORs) and 95% confidence intervals were estimated using conditional logistic regression models adjusting for Helicobacter pylori infection, and other potential confounders. We observed a positive association between intake of niacin and overall gastric cancer risk (OR = 1.33, 95%CI: 1.01-1.75 per SD increment). For thiamine, heterogeneity by subtype (cardia and non-cardia) was found (Phet = 0.05), with weak evidence of an inverse association with cardia cancer risk. Our results do not support increasing intakes of B vitamins or other nutrients involved in one-carbon metabolism to reduce gastric cancer risk in a well-nourished population.

Human fetoplacental arterial and venous endothelial cells are differentially programmed by gestational diabetes mellitus, resulting in cell-specific barrier function changes.

AIMS/HYPOTHESIS: An adverse intrauterine environment can result in permanent changes in the physiology of the offspring and predispose to diseases in adulthood. One such exposure, gestational diabetes mellitus (GDM), has been linked to development of metabolic disorders and cardiovascular disease in offspring. Epigenetic variation, including DNA methylation, is recognised as a leading mechanism underpinning fetal programming and we hypothesised that this plays a key role in fetoplacental endothelial dysfunction following exposure to GDM. Thus, we conducted a pilot epigenetic study to analyse concordant DNA methylation and gene expression changes in GDM-exposed fetoplacental endothelial cells. METHODS: Genome-wide methylation analysis of primary fetoplacental arterial endothelial cells (AEC) and venous endothelial cells (VEC) from healthy pregnancies and GDM-complicated pregnancies in parallel with transcriptome analysis identified methylation and expression changes. Most-affected pathways and functions were identified by Ingenuity Pathway Analysis and validated using functional assays. RESULTS: Transcriptome and methylation analyses identified variation in gene expression linked to GDM-associated DNA methylation in 408 genes in AEC and 159 genes in VEC, implying a direct functional link. Pathway analysis found that genes altered by exposure to GDM clustered to functions associated with 'cell morphology' and 'cellular movement' in healthy AEC and VEC. Further functional analysis demonstrated that GDM-exposed cells had altered actin organisation and barrier function. CONCLUSIONS/INTERPRETATION: Our data indicate that exposure to GDM programs atypical morphology and barrier function in fetoplacental endothelial cells by DNA methylation and gene expression change. The effects differ between AEC and VEC, indicating a stringent cell-specific sensitivity to adverse exposures associated with developmental programming in utero. DATA AVAILABILITY: DNA methylation and gene expression datasets generated and analysed during the current study are available at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database ( http://www.ncbi.nlm.nih.gov/geo ) under accession numbers GSE106099 and GSE103552, respectively.

Obtaining high quality transcriptome data from formalin-fixed, paraffin-embedded diagnostic prostate tumor specimens.

Prognostic genomic biomarkers that can be measured at diagnosis to aid choice of treatment options are unavailable for most common cancers. This is due in part to the poor quality and quantity of available diagnostic specimens for discovery research and to limitations in genomic technologies. Recent technical advances now enable high-density molecular analyses using suboptimal biological specimens. Here we describe the optimization of a transcriptome-specific protocol for use with formalin-fixed, paraffin-embedded (FFPE) diagnostic prostate cancer (PrCa) specimens. We applied the Ion AmpliSeq Transcriptome Human Gene Expression Kit (AmpliSeq Kit) to RNA samples extracted from 36 tumor-enriched and 16 adjacent normal tissues (ADJNT) from 37 FFPE PrCa specimens over a series of eight pilot studies, incorporating protocol modifications from Pilots 2 to 5. Data quality were measured by (1) the total number of mapped reads; (2) the percentage of reads that mapped to AmpliSeq target regions (OnTarget%); (3) the percentage of genes on the AmpliSeq panel with a read count ≥10 (TargetsDetected%); and (4) comparing the gene read-count distribution of the prostate tissue samples with the median gene read-count distribution of cell line-derived RNA samples. Modifications incorporated into Pilot study 5 provided gene expression data equivalent to cell line-derived RNA samples. These modifications included the use of freshly cut slides for macrodissection; increased tissue section thickness (8 µm); RNA extraction using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (ThermoFisher); 18 target amplification cycles; and processing six samples per Ion PI chip. This protocol will facilitate the discovery of prognostic biomarkers for cancer by allowing researchers to exploit previously underutilized diagnostic FFPE specimens.

DNA methylation-based biological aging and cancer risk and survival: Pooled analysis of seven prospective studies.

The association between aging and cancer is complex. Recent studies have developed measures of biological aging based on DNA methylation and called them "age acceleration." We aimed to assess the associations of age acceleration with risk of and survival from seven common cancers. Seven case-control studies of DNA methylation and colorectal, gastric, kidney, lung, prostate and urothelial cancer and B-cell lymphoma nested in the Melbourne Collaborative Cohort Study were conducted. Cancer cases, vital status and cause of death were ascertained through linkage with cancer and death registries. Conditional logistic regression and Cox models were used to estimate odds ratios (OR) and hazard ratios (HR) and 95% confidence intervals (CI) for associations of five age acceleration measures derived from the Human Methylation 450 K Beadchip assay with cancer risk (N = 3,216 cases) and survival (N = 1,726 deaths), respectively. Epigenetic aging was associated with increased cancer risk, ranging from 4% to 9% per five-year age acceleration for the 5 measures considered. Heterogeneity by study was observed, with stronger associations for risk of kidney cancer and B-cell lymphoma. An associated increased risk of death following cancer diagnosis ranged from 2% to 6% per five-year age acceleration, with no evidence of heterogeneity by cancer site. Cancer risk and mortality were increased by 15-30% for the fourth versus first quartile of age acceleration. DNA methylation-based measures of biological aging are associated with increased cancer risk and shorter cancer survival, independently of major health risk factors.

Association of DNA Methylation-Based Biological Age With Health Risk Factors and Overall and Cause-Specific Mortality.

Measures of biological age based on blood DNA methylation, referred to as age acceleration (AA), have been developed. We examined whether AA was associated with health risk factors and overall and cause-specific mortality. At baseline (1990-1994), blood samples were drawn from 2,818 participants in the Melbourne Collaborative Cohort Study (Melbourne, Victoria, Australia). DNA methylation was determined using the Infinium HumanMethylation450 BeadChip array (Illumina Inc., San Diego, California). Mixed-effects models were used to examine the association of AA with health risk factors. Cox models were used to assess the association of AA with mortality. A total of 831 deaths were observed during a median 10.7 years of follow-up. Associations of AA were observed with male sex, Greek nationality (country of birth), smoking, obesity, diabetes, lower education, and meat intake. AA measures were associated with increased mortality, and this was only partly accounted for by known determinants of health (hazard ratios were attenuated by 20%-40%). Weak evidence of heterogeneity in the association was observed by sex (P = 0.06) and cause of death (P = 0.07) but not by other factors. DNA-methylation-based AA measures are associated with several major health risk factors, but these do not fully explain the association between AA and mortality. Future research should investigate what genetic and environmental factors determine AA.

Heritable methylation marks associated with breast and prostate cancer risk.

BACKGROUND: DNA methylation can mimic the effects of germline mutations in cancer predisposition genes. Recently, we identified twenty-four heritable methylation marks associated with breast cancer risk. As breast and prostate cancer share genetic risk factors, including rare, high-risk mutations (eg, in BRCA2), we hypothesized that some of these heritable methylation marks might also be associated with the risk of prostate cancer. METHODS: We studied 869 incident prostate cancers (430 aggressive and 439 non-aggressive) and 869 matched controls nested within a prospective cohort study. DNA methylation was measured in pre-diagnostic blood samples using the Illumina Infinium HM450K BeadChip. Conditional logistic regression models, adjusted for prostate cancer risk factors and blood cell composition, were used to estimate odds ratios and 95% confidence intervals for the association between the 24 methylation marks and the risk of prostate cancer. RESULTS: Five methylation marks within the VTRNA2-1 promoter region (cg06536614, cg00124993, cg26328633, cg25340688, and cg26896946), and one in the body of CLGN (cg22901919) were associated with the risk of prostate cancer. In stratified analyses, the five VTRNA2-1 marks were associated with the risk of aggressive prostate cancer. CONCLUSIONS: This work highlights a potentially important new area of investigation for prostate cancer susceptibility and adds to our knowledge about shared risk factors for breast and prostate cancer.

Genome-wide DNA methylation assessment of 'BRCA1-like' early-onset breast cancer: Data from the Australian Breast Cancer Family Registry.

Breast cancers arising in women carrying a germline mutation in BRCA1 are typically high-grade, early-onset and have distinct morphological features (BRCA1-like). However, the majority of early-onset breast cancers of this morphological type are not associated with germline BRCA1 mutations or constitutional BRCA1 promoter methylation. We aimed to assess DNA methylation across the genome for associations with the "BRCA1-like" morphology. Genome-wide methylation in blood-derived DNA was measured using the Infinium HumanMethylation450K BeadChip assay for women under the age of 40 years participating in the Australian Breast Cancer Family Study (ABCFS) diagnosed with: i) BRCA1-like breast cancer (n = 30); and ii) breast cancer without BRCA1-like morphological features (non BRCA1-like; n = 30), and age-matched unaffected women (controls; n = 30). Corresponding tumour-derived DNA from 43 of the affected women was also assessed. Methylation of blood-derived DNA was found to be elevated across 17 consecutive marks in the BRCA1 promoter region and decreased at several other genomic regions (including TWIST2 and CTBP1) for 7 women (23%) diagnosed with BRCA1-like breast cancer compared with women in the other groups. Corresponding tumour-derived DNA available from 5 of these 7 women had elevated methylation within the BRCA1 and SPHK2 promoter region and decreased methylation within the ADAP1, IGF2BP3 and SPATA13 promoter region when compared with the other breast tumours. These methylation marks could be biomarkers of risk for BRCA1-like breast cancer, and could be responsible in part for their distinctive morphological features and biology. As such, they may assist with prevention and targeted therapies for this cancer subtype.

DNA methylation changes measured in pre-diagnostic peripheral blood samples are associated with smoking and lung cancer risk.

DNA methylation changes are associated with cigarette smoking. We used the Illumina Infinium HumanMethylation450 array to determine whether methylation in DNA from pre-diagnostic, peripheral blood samples is associated with lung cancer risk. We used a case-control study nested within the EPIC-Italy cohort and a study within the MCCS cohort as discovery sets (a total of 552 case-control pairs). We validated the top signals in 429 case-control pairs from another 3 studies. We identified six CpGs for which hypomethylation was associated with lung cancer risk: cg05575921 in the AHRR gene (p-valuepooled = 4 × 10-17 ), cg03636183 in the F2RL3 gene (p-valuepooled = 2 × 10 - 13 ), cg21566642 and cg05951221 in 2q37.1 (p-valuepooled = 7 × 10-16 and 1 × 10-11 respectively), cg06126421 in 6p21.33 (p-valuepooled = 2 × 10-15 ) and cg23387569 in 12q14.1 (p-valuepooled = 5 × 10-7 ). For cg05951221 and cg23387569 the strength of association was virtually identical in never and current smokers. For all these CpGs except for cg23387569, the methylation levels were different across smoking categories in controls (p-valuesheterogeneity ≤ 1.8 x10 - 7 ), were lowest for current smokers and increased with time since quitting for former smokers. We observed a gain in discrimination between cases and controls measured by the area under the ROC curve of at least 8% (p-values ≥ 0.003) in former smokers by adding methylation at the 6 CpGs into risk prediction models including smoking status and number of pack-years. Our findings provide convincing evidence that smoking and possibly other factors lead to DNA methylation changes measurable in peripheral blood that may improve prediction of lung cancer risk.

Genome-Wide Measures of Peripheral Blood Dna Methylation and Prostate Cancer Risk in a Prospective Nested Case-Control Study.

BACKGROUND: Global measures of peripheral blood DNA methylation have been associated with risk of some malignancies, including breast, bladder, and gastric cancer. Here, we examined genome-wide measures of peripheral blood DNA methylation in prostate cancer and its non-aggressive and aggressive disease forms. METHODS: We used a matched, case-control study of 687 incident prostate cancer samples, nested within a larger prospective cohort study. DNA methylation was measured in pre-diagnostic, peripheral blood samples using the Illumina Infinium HM450K BeadChip. Genome-wide measures of DNA methylation were computed as the median M-value of all CpG sites and according to CpG site location and regulatory function. We used conditional logistic regression to test for associations between genome-wide measures of DNA methylation and risk of prostate cancer and its subtypes, and by time between blood draw and diagnosis. RESULTS: We observed no associations between the genome-wide measure of DNA methylation based on all CpG sites and risk of prostate cancer or aggressive disease. Risk of non-aggressive disease was associated with higher methylation of CpG islands (OR = 0.80; 95%CI = 0.68-0.94), promoter regions (OR = 0.79; 95%CI = 0.66-0.93), and high density CpG regions (OR = 0.80; 95%CI = 0.68-0.94). Additionally, higher methylation of all CpGs (OR = 0.66; 95%CI = 0.48-0.89), CpG shores (OR = 0.62; 95%CI = 0.45-0.84), and regulatory regions (OR = 0.68; 95% CI = 0.51-0.91) was associated with a reduced risk of overall prostate cancer within 5 years of blood draw but not thereafter. CONCLUSIONS: A reduced risk of overall prostate cancer within 5 years of blood draw and non-aggressive prostate cancer was associated with higher genome-wide methylation of peripheral blood DNA. While these data have no immediate clinical utility, with further work they may provide insight into the early events of prostate carcinogenesis. Prostate 77:471-478, 2017. © 2017 Wiley Periodicals, Inc.

Genome-wide measures of DNA methylation in peripheral blood and the risk of urothelial cell carcinoma: a prospective nested case-control study.

BACKGROUND: Global DNA methylation has been reported to be associated with urothelial cell carcinoma (UCC) by studies using blood samples collected at diagnosis. Using the Illumina HumanMethylation450 assay, we derived genome-wide measures of blood DNA methylation and assessed them for their prospective association with UCC risk. METHODS: We used 439 case-control pairs from the Melbourne Collaborative Cohort Study matched on age, sex, country of birth, DNA sample type, and collection period. Conditional logistic regression was used to compute odds ratios (OR) of UCC risk per s.d. of each genome-wide measure of DNA methylation and 95% confidence intervals (CIs), adjusted for potential confounders. We also investigated associations by disease subtype, sex, smoking, and time since blood collection. RESULTS: The risk of superficial UCC was decreased for individuals with higher levels of our genome-wide DNA methylation measure (OR=0.71, 95% CI: 0.54-0.94; P=0.02). This association was particularly strong for current smokers at sample collection (OR=0.47, 95% CI: 0.27-0.83). Intermediate levels of our genome-wide measure were associated with decreased risk of invasive UCC. Some variation was observed between UCC subtypes and the location and regulatory function of the CpGs included in the genome-wide measures of methylation. CONCLUSIONS: Higher levels of our genome-wide DNA methylation measure were associated with decreased risk of superficial UCC and intermediate levels were associated with reduced risk of invasive disease. These findings require replication by other prospective studies.