Tumor detection by analysis of both symmetric- and hemi-methylation of plasma cell-free DNA.

Aberrant DNA methylation patterns have been used for cancer detection. However, DNA hemi-methylation, present at about 10% CpG dinucleotides, has been less well studied. Here we show that a majority of differentially hemi-methylated regions (DHMRs) in liver tumor DNA or plasma cells free (cf) DNA do not overlap with differentially methylated regions (DMRs) of the same samples, indicating that DHMRs could serve as independent biomarkers. Furthermore, we analyzed the cfDNA methylomes of 215 samples from individuals with liver or brain cancer and individuals without cancer (controls), and trained machine learning models using DMRs, DHMRs or both. The models incorporated with both DMRs and DHMRs show a superior performance compared to models trained with DMRs or DHMRs, with AUROC being 0.978, 0.990, and 0.983 in distinguishing control, liver and brain cancer, respectively, in a validation cohort. This study supports the potential of utilizing both DMRs and DHMRs for multi-cancer detection.

Genome-wide association study identifies high-impact susceptibility loci for HCC in North America.

BACKGROUND AND AIMS: Despite the substantial impact of environmental factors, individuals with a family history of liver cancer have an increased risk for HCC. However, genetic factors have not been studied systematically by genome-wide approaches in large numbers of individuals from European descent populations (EDP). APPROACH AND RESULTS: We conducted a 2-stage genome-wide association study (GWAS) on HCC not affected by HBV infections. A total of 1872 HCC cases and 2907 controls were included in the discovery stage, and 1200 HCC cases and 1832 controls in the validation. We analyzed the discovery and validation samples separately and then conducted a meta-analysis. All analyses were conducted in the presence and absence of HCV. The liability-scale heritability was 24.4% for overall HCC. Five regions with significant ORs (95% CI) were identified for nonviral HCC: 3p22.1, MOBP , rs9842969, (0.51, [0.40-0.65]); 5p15.33, TERT , rs2242652, (0.70, (0.62-0.79]); 19q13.11, TM6SF2 , rs58542926, (1.49, [1.29-1.72]); 19p13.11 MAU2 , rs58489806, (1.53, (1.33-1.75]); and 22q13.31, PNPLA3 , rs738409, (1.66, [1.51-1.83]). One region was identified for HCV-induced HCC: 6p21.31, human leukocyte antigen DQ beta 1, rs9275224, (0.79, [0.74-0.84]). A combination of homozygous variants of PNPLA3 and TERT showing a 6.5-fold higher risk for nonviral-related HCC compared to individuals lacking these genotypes. This observation suggests that gene-gene interactions may identify individuals at elevated risk for developing HCC. CONCLUSIONS: Our GWAS highlights novel genetic susceptibility of nonviral HCC among European descent populations from North America with substantial heritability. Selected genetic influences were observed for HCV-positive HCC. Our findings indicate the importance of genetic susceptibility to HCC development.

Blood DNA methylation and liver cancer in American Indians: evidence from the Strong Heart Study.

PURPOSE: Liver cancer incidence among American Indians/Alaska Natives has risen over the past 20 years. Peripheral blood DNA methylation may be associated with liver cancer and could be used as a biomarker for cancer risk. We evaluated the association of blood DNA methylation with risk of liver cancer. METHODS: We conducted a prospective cohort study in 2324 American Indians, between age 45 and 75 years, from Arizona, Oklahoma, North Dakota and South Dakota who participated in the Strong Heart Study between 1989 and 1991. Liver cancer deaths (n = 21) were ascertained using death certificates obtained through 2017. The mean follow-up duration (SD) for non-cases was 25.1 (5.6) years and for cases, 11.0 (8.8) years. DNA methylation was assessed from blood samples collected at baseline using MethylationEPIC BeadChip 850 K arrays. We used Cox regression models adjusted for age, sex, center, body mass index, low-density lipoprotein cholesterol, smoking, alcohol consumption, and immune cell proportions to examine the associations. RESULTS: We identified 9 CpG sites associated with liver cancer. cg16057201 annotated to MRFAP1) was hypermethylated among cases vs. non-cases (hazard ratio (HR) for one standard deviation increase in methylation was 1.25 (95% CI 1.14, 1.37). The other eight CpGs were hypomethylated and the corresponding HRs (95% CI) ranged from 0.58 (0.44, 0.75) for cg04967787 (annotated to PPRC1) to 0.77 (0.67, 0.88) for cg08550308. We also assessed 7 differentially methylated CpG sites associated with liver cancer in previous studies. The adjusted HR for cg15079934 (annotated to LPS1) was 1.93 (95% CI 1.10, 3.39). CONCLUSIONS: Blood DNA methylation may be associated with liver cancer mortality and may be altered during the development of liver cancer.

Improved prediction of breast cancer risk based on phenotypic DNA damage repair capacity in peripheral blood B cells.

Background: Standard Breast Cancer (BC) risk prediction models based only on epidemiologic factors generally have quite poor performance, and there have been a number of risk scores proposed to improve them, such as AI-based mammographic information, polygenic risk scores and pathogenic variants. Even with these additions BC risk prediction performance is still at best moderate. In that decreased DNA repair capacity (DRC) is a major risk factor for development of cancer, we investigated the potential to improve BC risk prediction models by including a measured phenotypic DRC assay. Methods: Using blood samples from the Breast Cancer Family Registry we assessed the performance of phenotypic markers of DRC in 46 matched pairs of individuals, one from each pair with BC (with blood drawn before BC diagnosis) and the other from controls matched by age and time since blood draw. We assessed DRC in thawed cryopreserved peripheral blood mononuclear cells (PBMCs) by measuring γ-H2AX yields (a marker for DNA double-strand breaks) at multiple times from 1 to 20 hrs after a radiation challenge. The studies were performed using surface markers to discriminate between different PBMC subtypes. Results: The parameter Fres, the residual damage signal in PBMC B cells at 20 hrs post challenge, was the strongest predictor of breast cancer with an AUC (Area Under receiver-operator Curve) of 0.89 [95% Confidence Interval: 0.84-0.93] and a BC status prediction accuracy of 0.80. To illustrate the combined use of a phenotypic predictor with standard BC predictors, we combined Fres in B cells with age at blood draw, and found that the combination resulted in significantly greater BC predictive power (AUC of 0.97 [95% CI: 0.94-0.99]), an increase of 13 percentage points over age alone. Conclusions: If replicated in larger studies, these results suggest that inclusion of a fingerstick-based phenotypic DRC blood test has the potential to markedly improve BC risk prediction.

DNA repair phenotype and cancer risk: a systematic review and meta-analysis of 55 case-control studies.

DNA repair phenotype can be measured in blood and may be a potential biomarker of cancer risk. We conducted a systematic review and meta-analysis of epidemiological studies of DNA repair phenotype and cancer through March 2021. We used random-effects models to calculate pooled odds ratios (ORs) of cancer risk for those with the lowest DNA repair capacity compared with those with the highest capacity. We included 55 case-control studies that evaluated 12 different cancers using 10 different DNA repair assays. The pooled OR of cancer risk (all cancer types combined) was 2.92 (95% Confidence Interval (CI) 2.49, 3.43) for the lowest DNA repair. Lower DNA repair was associated with all studied cancer types, and pooled ORs (95% CI) ranged from 2.02 (1.43, 2.85) for skin cancer to 7.60 (3.26, 17.72) for liver cancer. All assays, except the homologous recombination repair assay, showed statistically significant associations with cancer. The effect size ranged from 1.90 (1.00, 3.60) for the etoposide-induced double-strand break assay to 5.06 (3.67, 6.99) for the γ-H2AX assay. The consistency and strength of the associations support the use of these phenotypic biomarkers; however large-scale prospective studies will be important for understanding their use related to age and screening initiation.

Impact of BRCA mutation on the survival and risk of contralateral breast cancer in Asian breast cancer patients.

PURPOSE: Breast cancer is increasing around the globe, including Asia. We aimed to examine the survival and risk of contralateral breast cancer (CBC) in Asian breast cancer patients with BRCA mutations. METHODS: A total of 128 breast cancer patients with germline BRCA mutations and 4,754 control breast cancer patients were enrolled. Data on clinical-pathologic characteristics, survival, and CBC were collected from the medical record. The rates of survival and CBC were estimated by Kaplan-Meier method. RESULTS: The mean age of onset in BRCA mutation carriers was significantly younger than control patients (BRCA vs. Non-BRCA: 43.9 vs. 53.2 years old). BRCA mutation carriers had a higher proportion of triple-negative breast cancer (TNBC) (52%) than control patients (12%, p < 0.001). The risk of CBC was significantly higher in BRCA mutation patients than in control cases (hazard ratio (HR) = 3.95, 95% CI 2.71-5.75); when stratified by genotype, the HRs (95%CI) were 4.84 (3.00-7.82) for BRCA1 and 3.13 (1.78-5.49) for BRCA2 carriers, respectively. Moreover, BRCA1 mutation patients with triple-negative breast cancer (TNBC) as their first breast cancer had the highest risk of CBC (HR = 5.55, 95% CI 3.29-9.34). However, we did not observe any differences in relapse-free survival and overall survival between mutation carriers and control patients. CONCLUSION: Our study suggest that BRCA patients had a significantly higher risk of developing CBC, particularly for BRCA1 mutation carriers with TNBC as the first breast cancer.

Improvement on recovery and reproducibility for quantifying urinary mono-hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs).

Efficient and reproducible measurements of multiple polycyclic aromatic hydrocarbon (PAH) metabolites in urinary samples are required to evaluate the complex health effects of PAH exposure. Here, we demonstrate a highly practical, automated off-line solid-phase extraction (SPE) of deconjugated hydroxylated PAHs followed by LC-MS/MS to simultaneously measure eight mono-hydroxylated PAH compounds: 1-hydroxynaphthalene, 2-hydroxynaphthalene, 2-hydroxyfluorene, 1-hydroxyphenanthrene, 2&3-hydroxyphenanthrene, 4-hydroxyphenanthrene and 1-hydroxypyrene. Initially, we observed low recovery rates (e.g., 16% for 1-hydroxypyrene) when using previously published methods. We optimized the procedure by choosing polymeric absorbent-based cartridges, automating the sample loading step by diluting samples with 15% methanol/sodium acetate, and most importantly, replacing acetonitrile with methanol as the eluting solvent. Optimized sample preparation has improved the recovery rates to more than 69% for analytes of interest. This improvement led to higher method sensitivity and detection frequency, especially for 1-hydroxypyrene, in all of 100 urine samples collected in the New York City site of the Legacy Girls Study. The limits of detection ranged from 7.6 pg/mL to 20.3 pg/mL using 1 mL of urine, compared to the 2 mL required in CDC, method 09-OD. The average coefficients of variance of quality control samples (n = 60) ranged between 7 and 21%; variance of repeated measurements (n = 45) was less than 10%. This efficient and reliable method for measuring PAH metabolites will greatly benefit epidemiology studies and biomonitoring programs.

Incidence of hepatocellular carcinoma in a community-based Taiwanese population without chronic HBV/HCV infection.

BACKGROUND & AIMS: In addition to HBV/HCV causing hepatocellular carcinoma (HCC), other risk factors including obesity and alcohol drinking also increase risk. We describe the cumulative risk of HCC and mortality from liver-related disease by selected modifiable risk factors among a non-hepatitis virus-infected population. METHODS: For a community-based cohort, residents aged 30-65 years living in 7 townships in Taiwan were recruited, and have been followed up since 1991. A total of 18,541 individuals were seronegative for markers of chronic infection of HBV/HCV and with no history of HCC at baseline. New non-HBV/HCV HCC cases and liver-related deaths were ascertained through data linkage to the National Cancer Registry and Death Certification System from 1 January 1991 through 31 December 2017. RESULTS: There were 207 HCC cases and 215 liver-related deaths identified. The incidence rate of non-HBV/HCV HCC was 47.2 per 100,000 person-years. The mortality rate of liver-related death was 49.0 per 100,000 person-years. Baseline information on alcohol consumption, heart disease, diabetes, elevated aspartate aminotransferase, and alanine aminotransferase predicted higher risks of HCC, with hazard ratios (HRs) (95% CIs) of 1.7 (1.1-2.5), 2.2 (1.1-4.1), 1.9 (1.0-3.5), 1.7 (1.1-2.4), and 1.6 (1.0-2.4), respectively. The HRs (95% CIs) of liver-related death were 2.3 (1.6-3.2) for alcohol consumption, 1.4 (1.1-1.9) for BMI ≥25 kg/m2, 2.2 (1.4-3.3) for elevated aspartate aminotransferase, and 1.5 (1.0-2.4) for elevated alanine aminotransferase. The HR (95% CI) was 8.1 (3.6-18.5) for those with diabetes and elevated aspartate aminotransferase. CONCLUSIONS: Individuals with elevated liver enzymes are at high risk of liver disease. Prevention and treatment of diabetes and heart disease are critical for non-hepatitis B, non-hepatitis C (NonB/C)-HCC. LAY SUMMARY: We followed up individuals with no chronic HBV or HCV infection and described the risk of hepatocellular carcinoma (HCC, the most common form of primary liver cancer) and mortality from liver-related disease by modifiable risk factors. This study estimated the incidence rate of HCC by selected lifestyle risk factors and chronic diseases conditions. Alcohol consumption, heart disease, diabetes, and abnormal blood liver function tests showed a strong association with HCC risk and mortality.

Environmental exposure and clinical correlates of hepatocellular carcinoma in New York City: a case only study.

In the U.S., Hepatocellular carcinoma (HCC) incidence rates have increased. We aimed to determine whether environmental exposure plays a role in the high incidence of HCC observed in New York City. We conducted a hospital-based case only study to examine the prevalence of aflatoxin B1 (AFB1)- and polycyclic aromatic hydrocarbon (PAH)-albumin adducts and the distribution of adducts by different characteristics of HCC patients. Blood samples were collected from 155 HCC patients for biomarker analyses. We observed that about 46% and 49% of cases had detectable AFB1- and PAH-albumin adducts, respectively. There were significant differences between AFB1-albumin adducts and selected factors such as HCV infection status (p = 0.04), diabetes (p = 0.03) and Barcelona Clinic Liver Cancer stage (p = 0.02). Cases with detectable PAH-albumin adducts had a smoking history compared with those with nondetectable levels (p = 0.04). The level of AFB1-albumin adducts was positively correlated with plasma bilirubin (rs = 0.32, p < 0.0001) and adiponectin concentrations (rs = 0.28, p = 0.0005). The level of aflatoxin B1-albumin adducts was negatively associated with blood albumin concentration (rs = - 0.28, p = 0.0009) and plasma DNA LINE-1 methylation (rs = - 0.16, p = 0.04). Our study provides additional evidence that environmental exposures including to aflatoxin might drive the high incidence of HCC observed in the New York City.

Reproductive and environmental exposures and the breast cancer risk in Taiwanese women.

Breast cancer (BC) incidence is increasing around the globe, including in Taiwan, though the cause of the increasing incidence is less clear. We followed up 11,296 Taiwanese females who did not have BC at baseline, and ascertained new invasive BC (N = 351) through data linkage to the National Cancer Registry from 1991 to 2018 to examine whether reproductive, lifestyle and environmental risk factors including polycyclic aromatic hydrocarbons (PAH) were associated with BC risk. We conducted a nested case-control study using baseline blood available from a total of 305 women with BC and 598 women without BC matched on time in cohort. We examined the association of PAH-albumin adducts and BC risk using conditional logistic regression models. Age at menarche (HR 0.6 (95% CI 0.5-0.9) for ≥ 15 vs. < 13 years) and multiparity were associated with BC risk (HR 2.0 (95% CI 1.4-2.8), 2.8 (1.9-4.2), and 2.4 (1.0-5.0) for 3-4, 1-2 and 0 live birth, compared with women ≥ 5 births). PAH-albumin adducts were not associated with BC risk. Given the increasing BC incidence in Taiwan, there is a need to identify environmental factors that are important to this population.

Circulating growth factor concentrations and breast cancer risk: a nested case-control study of IGF-1, IGFBP-3, and breast cancer in a family-based cohort.

BACKGROUND: Insulin-like growth factor 1 (IGF-1) and binding protein 3 (IGFBP-3) are associated with breast cancer in women at average risk of cancer. Less is known whether these biomarkers also predict risk in women with breast cancer family history. METHODS: We conducted a nested case-control study within the New York site of the Breast Cancer Family Registry (BCFR, n = 80 cases, 156 controls), a cohort enriched for breast cancer family history. Using conditional logistic regression, we estimated the association between IGF-1 and IGFBP-3 levels and breast cancer risk and examined whether this risk differed by predicted absolute breast cancer risk based on pedigree models. RESULTS: The overall association between IGF-1 or IGFBP-3 elevation (≥ median in controls) and breast cancer risk was elevated, but not statistically significant (IGF-1 OR = 1.37, 95% CI = 0.66-2.85; IGFBP-3 OR = 1.62, 95% CI = 0.81-3.24). Women with elevated predicted absolute 10-year risk ≥ 3.4% and elevated IGFBP-3 (≥ median) had more than a 3-fold increased risk compared to women with lower predicted absolute 10-year risk (< 3.4%) and low IGFBP-3 (OR = 3.47 95% CI = 1.04-11.6). CONCLUSIONS: These data offer some support that the overall magnitude of the associations between IGF-1 and IGFBP3 seen in average risk cohorts may be similar in women enriched with a strong breast cancer family history.

Influence of pubertal development on urinary oxidative stress biomarkers in adolescent girls in the New York LEGACY cohort.

Puberty is a time of intense growth and differentiation of breast tissue and a window of susceptibility (WOS) for breast cancer. Although oxidative stress markers have been associated with breast cancer risk, it is unclear whether oxidative stress levels are different during the pubertal WOS, and if so, whether these differences are related to breast cancer susceptibility. We measured urinary biomarkers of whole body oxidative stress (urinary F2-Isoprostanes and 8-oxodeoxyguanosine (8-oxodG)) in 158 girls (ages 6-13 years), 71 with and 87 without a breast cancer family history (BCFH) from a cohort of adolescent girls from the New York site of the LEGACY cohort (Lessons in Epidemiology and Genetics in Adults Cancer from Youth). We compared levels of urinary oxidative stress biomarkers (F2-Isoprostanes and 8-oxodG) across the pubertal window, defined by Tanner Stage (TS) of breast development, both cross-sectionally and longitudinally within girls over an 18-month follow up period. Urinary oxidative stress biomarkers were unrelated to pubertal stages in cross-sectional analyses after considering adjustments for body mass index (BMI) and BCFH. In our longitudinal analysis, we found that urinary 8-oxodG levels, but not F2-Isoprostane levels, increased with age in BCFH + girls (ß = 6.12, 95% CI = 0.08-12.16) compared to BCFH-girls. Higher BMI was associated with higher level of F2-Isoprostane in both cross-sectional (ß = 0.02, 95% CI = 0.0004-0.05) and longitudinal analysis (ß = 0.02, 95% CI = 0.0002-0.05). These findings support that higher BMI increases oxidative stress biomarkers over the pubertal window and that there are changes in 8-oxodG oxidative stress biomarkers in girls with a BCFH compared to girls without a BCFH.

High levels of global genome methylation in patients with retinoblastoma.

Retinoblastoma is a tumor of the embryonic neural retina in young children. The DNA methyltransferase 1 (DNMT1) gene has been demonstrated to be transcriptionally activated in cells lacking retinoblastoma 1 (RB1). Thus, there is a direct interaction between DNMT1 and RB1 in vivo. The present study hypothesized that uncontrolled DNMT1, DNMT2 and DNMT3 expression may lead to a high level of global genome methylation causing a second hit or where both alleles are altered, in RB1 and/or inactivation of other genes in retinal cells. To test this, the global genome methylation levels were analyzed in 69 patients with retinoblastoma, as well as 26 healthy siblings and 18 healthy unrelated children as the control groups. Peripheral blood and tumor tissue samples were obtained from 32 patients. The expression levels of DNMT genes were also determined in cell lines. Based on the median levels of global genome methylation in patients, higher genome-wide methylation levels in peripheral blood were associated with a 3.33-fold increased risk for retinoblastoma in patients compared with all healthy controls (95% confidence interval, 0.98-11.35; P<0.0001). The level of global genome methylation and the expression of DNMT genes were increased in the WERI-RB-1 cell line, which has a mutated RB1 gene, compared with a wild-type RB1-expressing cell line. These results supported the hypothesis that epigenetic alterations, as well as mutations in RB1, may be associated with the oncogenesis and inheritance of retinoblastoma. The repression of genes that interact with RB1, such as the DNMT gene family, may be important in patients with retinoblastoma with alterations in RB1, and may serve a role in the treatment and regression of retinoblastoma.

DDT exposure during pregnancy and DNA methylation alterations in female offspring in the Child Health and Development Study.

Studies measuring dichlorodiphenyltrichloroethane (DDT) exposure during key windows of susceptibility including the intrauterine period suggest that DDT exposure is associated with breast cancer risk. We hypothesized that prenatal DDT exposure is associated with DNA methylation. Using prospective data from 316 daughters in the Child Health and Development Study, we examined the association between prenatal exposure to DDTs and DNA methylation in blood collected in midlife (mean age: 49 years). To identify differentially methylated regions (DMRs) associated with markers of DDTs (p,p'-DDT and the primary metabolite of p,p'-DDT, p,p'-DDE, and o,p'-DDT, the primary constituents of technical DDT), we measured methylation in 30 genes important to breast cancer. We observed DDT DMRs in three genes, CCDC85A, CYP1A1 and ZFPM2, each of which has been previously implicated in pubertal development and breast cancer susceptibility. These findings suggest prenatal DDT exposure may have life-long consequence through alteration in genes relevant to breast cancer.

Iron-sulfur protein NFU2 is required for branched-chain amino acid synthesis in Arabidopsis roots.

Numerous proteins require a metallic co-factor for their function. In plastids, the maturation of iron-sulfur (Fe-S) proteins necessitates a complex assembly machinery. In this study, we focused on Arabidopsis thaliana NFU1, NFU2, and NFU3, which participate in the final steps of the maturation process. According to the strong photosynthetic defects observed in high chlorophyll fluorescence 101 (hcf101), nfu2, and nfu3 plants, we determined that NFU2 and NFU3, but not NFU1, act immediately upstream of HCF101 for the maturation of [Fe4S4]-containing photosystem I subunits. An additional function of NFU2 in the maturation of the [Fe2S2] cluster of a dihydroxyacid dehydratase was obvious from the accumulation of precursors of the branched-chain amino acid synthesis pathway in roots of nfu2 plants and from the rescue of the primary root growth defect by supplying branched-chain amino acids. The absence of NFU3 in roots precluded any compensation. Overall, unlike their eukaryotic and prokaryotic counterparts, which are specific to [Fe4S4] proteins, NFU2 and NFU3 contribute to the maturation of both [Fe2S2] and [Fe4S4] proteins, either as a relay in conjunction with other proteins such as HCF101 or by directly delivering Fe-S clusters to client proteins. Considering the low number of Fe-S cluster transfer proteins relative to final acceptors, additional targets probably await identification.

Aflatoxin B1 exposure increases the risk of hepatocellular carcinoma associated with hepatitis C virus infection or alcohol consumption.

BACKGROUND: Hepatocarcinogenicity of aflatoxin B1 (AFB1) has rarely been studied in populations with hepatitis C virus (HCV) infection and those without hepatitis B virus (HBV) and HCV infection (non-B-non-C). This case-control study nested in a community-based cohort aimed to investigate the HCC risk associated with AFB1 in HCV-infected and non-B-non-C participants. METHODS: Baseline serum AFB1-albumin adduct levels were measured in 100 HCC cases and 1767 controls seronegative for anti-HCV and HBsAg (non-B-non-C), and another 103 HCC cases and 176 controls who were anti-HCV-seropositive and HBsAg-seronegative. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated using logistic regression. RESULTS: In 20 years of follow-up, the follow-up time to newly developed HCC was significantly shorter in participants with higher serum AFB1-albumin adduct levels in non-B-non-C (p = 0.0162) and HCV-infected participants (p < 0.0001). Within 8 years of follow-up, HCV infection and AFB1 exposure were independent risk factors for HCC. Elevated serum AFB1-albumin adduct levels were significantly associated with an increased risk of HCC newly developed within 8 years of follow-up in non-B-non-C participants with habitual alcohol consumption [crude OR (95% CI) for high vs. low/undetectable levels, 4.22 (1.16-15.37)] and HCV-infected participants [3.39 (1.31-8.77)], but not in non-B-non-C participants without alcohol drinking habit. AFB1 exposure remained an independent risk predictor for HCV-related HCC after adjustment for other HCC predictors (multivariate-adjusted OR [95% CI], 3.65 [1.32-10.10]). CONCLUSIONS: AFB1 exposure contributes to the development of HCC in participants with significant risk factors for cirrhosis including alcohol and HCV infection.

Breast cancer family history and allele-specific DNA methylation in the legacy girls study.

Family history, a well-established risk factor for breast cancer, can have both genetic and environmental contributions. Shared environment in families as well as epigenetic changes that also may be influenced by shared genetics and environment may also explain familial clustering of cancers. Epigenetic regulation, such as DNA methylation, can change the activity of a DNA segment without a change in the sequence; environmental exposures experienced across the life course can induce such changes. However, genetic-epigenetic interactions, detected as methylation quantitative trait loci (mQTLs; a.k.a. meQTLs) and haplotype-dependent allele-specific methylation (hap-ASM), can also contribute to inter-individual differences in DNA methylation patterns. To identify differentially methylated regions (DMRs) associated with breast cancer susceptibility, we examined differences in white blood cell DNA methylation in 29 candidate genes in 426 girls (ages 6-13 years) from the LEGACY Girls Study, 239 with and 187 without a breast cancer family history (BCFH). We measured methylation by targeted massively parallel bisulfite sequencing (bis-seq) and observed BCFH DMRs in two genes: ESR1 (Δ4.9%, P = 0.003) and SEC16B (Δ3.6%, P = 0.026), each of which has been previously implicated in breast cancer susceptibility and pubertal development. These DMRs showed high inter-individual variability in methylation, suggesting the presence of mQTLs/hap-ASM. Using single nucleotide polymorphisms data in the bis-seq amplicon, we found strong hap-ASM in SEC16B (with allele specific-differences ranging from 42% to 74%). These findings suggest that differential methylation in genes relevant to breast cancer susceptibility may be present early in life, and that inherited genetic factors underlie some of these epigenetic differences.

Maternal cigarette smoking during pregnancy and offspring DNA methylation in midlife.

Maternal smoking in pregnancy (MSP) has been associated with DNA methylation in specific CpG sites (CpGs) in infants and children. We investigated whether MSP, independent of own personal active smoking, was associated with midlife DNA methylation in CpGs that were previously identified in studies of MSP-DNA methylation in children. We used data on MSP collected from pregnant mothers of 89 adult women born in 1959-1964 and measured DNA methylation in blood (granulocytes) collected in 2001-2007 (mean age: 43 years). Seventeen CpGs were differentially methylated by MSP, with multiple CpGs mapping to CYP1A1, MYO1G, AHRR, and GFI1. These associations were consistent in direction with prior studies (e.g., MSP associated with more and less methylation in AHRR and CYP1A1, respectively) and, with the exception of AHRR CpGs, were not substantially altered by adjustment for active smoking. These preliminary results confirm prior prospective reports that MSP influences the offspring DNA methylation, and extends the timeframe to midlife, and suggest that these effects may persist into adulthood, independently of active smoking.

Plasma DNA methylation marker and hepatocellular carcinoma risk prediction model for the general population.

Metastases in the later stages of hepatocellular carcinoma (HCC) cause the majority of deaths associated with the disease, making early detection crucial to patient survival. Risk models assessing HCC risk in the general population can be used for risk stratification for further HCC surveillance, however, none have been validated externally. Methylation of circulating DNA shows potential for non-invasive diagnosis of HCC. We conducted a prospective case-control study nested within a community-based cohort. We measured methylation levels in six genes (CDKN2A, RASSF1A, STEAP4, TBX2, VIM and ZNF154) which were identified in our previous work, using pre-diagnostic plasma DNA from 237 HCC cases and 257 matched controls. We found TBX2 hypermethylation was associated with increased HCC risk, with ORs (95% CI) of 3.2 (1.8-6.0). The associations were mainly among high-risk subjects; among subjects infected with HBV/HCV, the OR (95% CI) of TBX2 methylation was 5.3 (2.2-12.7). Among subjects with high risk scores, the ORs (95% CIs) were 7.8 (1.5-38.6) for Wen-HCC model ≥16, 5.8 (2.2-15.5) for Hung-HCC ≥15 and 7.5 (2.2-26.0) for Michikawa-HCC ≥8. Adding TBX2 methylation improved the accuracy of risk models for a high-risk population, with the area under the curve (AUC) of 76% for Wen-HCC score with TBX2 methylation compared with 69% with Wen-HCC alone. The AUCs were 63% for Hung-HCC score plus TBX2 methylation, and 53% for Hung-HCC alone, 65% for Michikawa-HCC score plus TBX2 methylation and 58% for Michikawa-HCC alone. Our findings suggest the potential increase in risk assessment discrimination and accuracy from incorporation of DNA methylation.

Global Level of Plasma DNA Methylation is Associated with Overall Survival in Patients with Hepatocellular Carcinoma.

BACKGROUND: The impact of folate deficiency on global DNA methylation is uncertain. It also is unclear whether global DNA methylation is associated with outcome in HCC. LINE-1 methylation levels, as a surrogate marker of global methylation, may be influenced by folate deficiency. However, the interaction between LINE-1 methylation and folate level on overall survival (OS) in hepatocellular carcinoma (HCC) patients is unknown. We evaluated whether LINE-1 hypomethylation and folate deficiency are associated with HCC prognosis. METHODS: We prospectively recruited 172 HCC patients between 2008 and 2012. LINE-1 methylation levels in plasma and white blood cells (WBC) were measured by pyrosequencing, and plasma folate levels by a radioprotein-binding assay. RESULTS: Patients with plasma LINE-1 methylation <70.0% (hypomethylation) had significantly worse OS compared with those with ≥70.0% methylation (hypermethylation) [hazard ratio (HR) = 1.77; 95% confidence interval (CI) 1.12-2.79; P = 0.015]. HCC patients with lower plasma folate levels also had worse survival (<27.7 vs. ≥27.7 nmol/L; HR = 1.96; 95% CI, 1.24-3.09; P = 0.004). Furthermore, survival was poor in patients in whom both plasma LINE-1 methylation and folate levels were low compared with those patients in whom both levels were high (HR = 3.36; 95%CI, 1.77-6.40; P < 0.001). This interaction neared statistical significance (P = 0.057). No significant association was found between WBC LINE-1 methylation levels and survival. CONCLUSIONS: These findings suggest that both lower plasma levels of LINE-1 methylation and folate are associated with worse survival in HCC patients.