Immune factors are thought to influence glioma risk and outcomes, but immune profiling studies to further our understanding of the immune response are limited by current immunodiagnostic methods. We developed a new assay to capture glioma immune biology based on quantitative methylation specific PCR (qMSP) of two T-cell genes (CD3Z: T-cells, and FOXP3: Tregs). Flow cytometry of T-cells correlated well with the CD3Z demethylation assay (r = 0.93; p < 2.2 × 10−16), demonstrating the validity of the assay. Furthermore, there was a high correlation between qMSP and immunohistochemistry (IHC) in quantifying tumor infiltrating T-cells (r = 0.85; p = 3.4 × 10−11). Applying our qMSP methods to archival whole blood from 65 glioblastoma multiforme (GBM) cases and 94 non-diseased controls, GBM cases had highly statistically significantly lower T-cells (p = 1.7 × 10−9) as well as Tregs (p = 5.2 × 10−11) and a modestly lower ratio of Tregs/T-cells (p = 0.024). Applying the methods to 120 excised glioma tumors, we observed that tumor infiltrating CD3+ T-cells were positively correlated with glioma tumor grade (p = 5.7 × 10−7), and that Tregs were enriched in tumors compared with peripheral blood indicating active chemoattraction of suppressive Tregs into the tumor compartment. Poorer patient survival was correlated with higher levels of tumor infiltrating T-cells (p = 0.01) and Tregs (p = 0.04). DNA methylation based immunodiagnostics represent a new generation of powerful laboratory tools offering many advantages over conventional methods that will facilitate large clinical epidemiologic studies and capitalize on stored archival blood and tissue banks.
DNA methylation; glioma; Tregs; T-cells; biomarkers
Natural killer (NK) cells are a key element of the innate immune system implicated in human cancer. To examine NK cell levels in archived bloods from a study of human head and neck squamous cell carcinoma (HNSCC), a new DNA-based quantification method was developed.
NK cell-specific DNA methylation was identified by analyzing DNA methylation and mRNA array data from purified blood leukocyte subtypes (NK, T, B, monocytes, granulocytes), and confirmed via pyrosequencing and quantitative methylation specific PCR (qMSP). NK cell levels in archived whole blood DNA from 122 HNSCC patients and 122 controls were assessed by qMSP.
Pyrosequencing and qMSP confirmed that a demethylated DNA region in NKp46 distinguishes NK cells from other leukocytes, and serves as a quantitative NK cell marker. Demethylation of NKp46 was significantly lower in HNSCC patient bloods compared with controls (p < 0.001). Individuals in the lowest NK tertile had over 5-fold risk of being a HNSCC case, controlling for age, gender, HPV16 status, cigarette smoking, alcohol consumption, and BMI (OR = 5.6, 95% CI: 2.0, 17.4). Cases did not show differences in NKp46 demethylation based on tumor site or stage.
The results of this study indicate a significant depression in NK cells in HNSCC patients that is unrelated to exposures associated with the disease. DNA methylation biomarkers of NK cells represent an alternative to conventional flow cytometry that can be applied in a wide variety of clinical and epidemiologic settings including archival blood specimens.
natural killer cells; NK cells; head and neck cancer; HNSCC; DNA methylation
Human papillomavirus (HPV) is an accepted cause of head and neck squamous cell carcinoma (HNSCC) and patients with HPV-associated HNSCC have a favorable prognosis. Currently there is no general guidance on the most appropriate biomarkers for clinical assessment of HPV in these malignancies. We compared PCR-based and serological HPV assays, as well as p16 immunohistochemistry, individually and in combination in a single population-based study to assess their associations with overall survival among HNSCC patients, and thus their potential value as biomarkers. HPV16 serology was determined for 488 patients, immunohistochemical detection of p16 expression in tumors was performed in a subset of 233 cases, and PCR-based methods to assess the presence of HPV16 DNA in a subset of 179 cases’ tumors. Considering each biomarker individually in the subset of patients studied for all endpoints, seropositivity for the E6 and E7 proteins was significantly associated with enhanced all-cause survival in oropharyngeal disease (HRE6/E7+ =0.1, 95%CI=0.02–0.3). Neither the presence of HPV16 DNA or p16 immunostaining was associated with significant enhanced overall survival in oropharyngeal disease ( HRDNA=0.9, 95% CI-0.3–2.9; HRp16=0.3, 95%CI=0.1–1.1). However, the combination of HPV positive DNA and E6 or E7 serology was associated with enhanced overall survival in oropharyngeal disease (HRDNA +/E6/E7+=0.1, 95%CI=0.02–1.0), while E6/E7 seronegative patients with evidence of HPV in tumor DNA did not show any evidence of favorable survival (HRDNA+/E6−/E7−=3.4, 95%CI = 0.6–18.1). Further, patients with p16 staining and E6 or E7 seropositivity had favorable survival from oropharyngeal disease (HRp16+/E6/E7+=0.1, 95%CI=0.02–0.4), while patients who were p16 positive and E6/E7 seronegative had significantly increased hazard of all causes of death (HRp16+/E6−/E7−=3.1, 95%CI=1.2–7.7). A stronger association of HPV presence with prognosis (assessed by all-cause survival) is observed when "HPV-associated" HNSCC is defined using tumor status (HPV DNA status or P16) and HPV E6/E7 serology in combination rather using tumor HPV status alone.
human papillomavirus; head and neck cancer; p16 immunostaining
Global hypomethylation of repetitive DNA sequences is believed to occur early in tumorigenesis. There is a great interest in identifying factors that contribute to global DNA hypomethylation and associated cancer risk. We tested the hypothesis that plasma S-adenosylmethionine (SAM) level alone or in combination with genetic variation in DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was associated with global DNA methylation extent at long interspersed nucleotide element-1 (LINE-1) sequences.
Plasma SAM level and LINE-1 DNA methylation index were measured using stored blood samples collected from 440 healthy Singaporean Chinese adults during 1994-1999. Genetic polymorphisms of 13 loci in DNMT1, DNMT3A and DNMT3B were determined.
LINE-1 methylation index was significantly higher in men than in women (p = 0.001). LINE-1 methylation index was positively associated with plasma SAM levels (p ≤ 0.01), with a plateau at approximately 78% of LINE-1 methylation index (55 nmol/L plasma SAM) in men and 77% methylation index (50 nmol/L plasma SAM) in women. In men only, the T allele of DNMT1 rs21124724 was associated with a statistically significantly higher LINE-1 methylation index (ptrend = 0.001). The DNMT1 rs2114724 genotype modified the association between plasma SAM and LINE-1 methylation index at low levels of plasma SAM in men.
Circulating SAM level was associated with LINE-1 methylation status among healthy Chinese adults. The DNMT1 genetic polymorphism may exert a modifying effect on the association between SAM and LINE-1 methylation status in men, especially when plasma SAM level is low. Our findings support a link between plasma SAM and global DNA methylation status at LINE-1 sequences.
Blood leukocytes from patients with solid tumors exhibit complex and distinct cancer-associated patterns of DNA methylation. However, the biological mechanisms underlying these patterns remain poorly understood. Since epigenetic biomarkers offer significant clinical potential for cancer detection, we sought to address a mechanistic gap in recently published works, hypothesizing that blood-based epigenetic variation may be due to shifts in leukocyte populations.
We identified differentially methylated regions (DMRs) among leukocyte subtypes using epigenome-wide DNA methylation profiling of purified peripheral blood leukocyte subtypes from healthy donors. These leukocyte-tagging DMRs were then evaluated using epigenome-wide blood methylation data from three independent case-control studies of different cancers.
A substantial proportion of the top 50 leukocyte DMRs were significantly differentially methylated among head and neck squamous cell carcinoma (HNSCC) cases and ovarian cancer cases compared to cancer-free controls (48 and 47 out of 50, respectively). Methylation classes derived from leukocyte DMRs were significantly associated cancer case status (p < 0.001, p < 0.03, and p < 0.001) for all three cancer types: HNSCC, bladder cancer, and ovarian cancer, respectively and predicted cancer status with a high degree of accuracy (AUC = 0.82, 0.83, and 0.67).
These results suggest that shifts in leukocyte sub-populations may account for a considerable proportion of variability in peripheral-blood DNA methylation patterns of solid tumors.
This illustrates the potential utility of DNA methylation profiles for identifying shifts in leukocyte populations representative of disease, and that such profiles may represent powerful new diagnostic tools, applicable to a range of solid tumors.
DNA methylation; cancer; leukocytes; immune system; biomarkers
Individuals with allergies have a heightened Th2 (T helper 2) immunity which may provide advantages in controlling tumor growth. Inverse associations have been reported among individuals with allergies and risk of brain and pancreatic cancers.
We examined the relationship between allergies and risk of head and neck squamous cell carcinoma (HNSCC) in a population-based case-control study with 1014 cases and 1193 frequency-matched controls. Logistic regression models were used to estimate odds ratio (OR) and 95% confidence intervals (95% CI) controlling for age, sex, race, smoking history, alcohol consumption, and education. In addition, in a subset of the population, models were adjusted for HPV16 status.
Individuals with allergies had a 19% lower risk of HNSCC (OR = 0.81, 95% CI = 0.67-0.98). Associations with allergies were stronger for laryngeal (OR = 0.66, 95% CI = 0.45-0.97) and oropharyngeal (OR =0.73, 95% CI=0.57-0.92) cancers, while no association was observed for oral cavity cancers (OR = 0.98, 95% CI = 0.76-1.26). History of asthma was not associated with overall HNSCC, but the association was statistically significant for oropharyngeal cancer (OR = 0.67, 95% CI = 0.44-0.99). HPV16 status did not confound or modify the associations with allergies.
Elevated Th2 immunity in individuals with history of allergies and asthma may reduce the risk of HNSCC. Additional research into related mechanisms may provide new insights into how to treat HNSCC.
These findings may provide new insight into biological pathways that could lead to a better understanding of the etiology of this disease.
allergies; atopy; head and neck cancer
Epigenetic alterations are a common event in lung cancer and their identification can serve to inform on the carcinogenic process and provide clinically relevant biomarkers. Using paired tumor and non-tumor lung tissues from 146 individuals from three independent populations we sought to identify common changes in DNA methylation associated with the development of non-small cell lung cancer. Pathologically normal lung tissue taken at the time of cancer resection was matched to tumorous lung tissue and together were probed for methylation using Illumina GoldenGate arrays in the discovery set (n = 47 pairs) followed by bisulfite pyrosequencing for validation sets (n = 99 pairs). For each matched pair the change in methylation at each CpG was calculated (the odds ratio), and these ratios were averaged across individuals and ranked by magnitude to identify the CpGs with the greatest change in methylation associated with tumor development. We identified the top gene-loci representing an increase in methylation (HOXA9, 10.3-fold and SOX1, 5.9-fold) and decrease in methylation (DDR1, 8.1-fold). In replication testing sets, methylation was higher in tumors for HOXA9 (p < 2.2 × 10−16) and SOX1 (p < 2.2 × 10−16) and lower for DDR1 (p < 2.2 × 10−16). The magnitude and strength of these changes were consistent across squamous cell and adenocarcinoma tumors. Our data indicate that the identified genes consistently have altered methylation in lung tumors. Our identified genes should be included in translational studies that aim to develop screening for early disease detection.
DNA Methylation; goldengate; lung cancer; molecular epidemiology; pyrosequencing
A genome-wide association study for upper aerodigestive tract cancers identified 19 candidate single-nucleotide polymorphisms (SNPs). We used these SNPs to investigate the potential gene-gene and gene-environment interactions in head and neck squamous cell carcinoma (HNSCC) risk.
The 19 variants were genotyped using Taqman (Applied Biosystems) assays among 575 cases and 676 controls in our population-based case-control study.
A restricted cubic spline model suggested both ADH1B and HEL308 modified the association between smoking pack-years and HNSCC. Classification and regression tree analysis demonstrated a higher order interaction between smoking status, ADH1B, FLJ13089 and FLJ35784 in HNSCC risk. Compared with ever smokers carrying ADH1B T/C+T/T genotypes, smokers carrying ADH1B C/C genotype and FLJ13089 A/G+A/A genotypes had a highest risk of HNSCC (OR=1.84).
Our results suggest that the risk associated with these variants may be specifically important amongst specific exposure groups.
post-genome wide association study; head and neck cancer; gene and environment interaction
Panels of prognostic biomarkers selected using candidate approaches often do not validate in independent populations, so additional strategies are needed to identify reliable classifiers. In this study, we used an array-based approach to measure DNA methylation and applied a novel method for grouping CpG dinucleotides according to well-characterized genomic sequence features. A hypermethylation profile among 13 CpG loci characterized by polycomb group target genes, mammalian interspersed repeats, and transcription factor binding sites (PcG/MIR/TFBS), was associated with reduced survival (hazard ratio: 3.98, p=0.001) in head and neck squamous cell carcinoma patients. This association was driven by CpGs associated with the TAP1 and ALDH3A1 genes, findings that were validated in an independent patient group (hazard ratio: 2.86, p=0.04). Together, the data not only elucidate new potential targets for therapeutic intervention in head and neck cancer, but also may aid in the identification of poor prognosis patients who may require more aggressive treatment regimens.
Epigenetics; DNA Sequence; Head and Neck Carcinoma; Survival; Microarray
Reduced levels of global DNA methylation, assessed in peripheral blood, have been associated with bladder cancer risk in European and American populations. Similar data are lacking in Asian populations where genetic differences, lifestyle factors, and different environmental exposures may affect DNA methylation and its risk relationship with bladder cancer. The association between global DNA methylation measured at long interspersed nuclear element (LINE-1) repeat regions through bisulfite pyrosequencing in lymphocyte DNA and bladder cancer risk was examined in a case-control study of 510 bladder cancer patients and 528 healthy control subjects in Shanghai, China. In an initial analysis restricted to control subjects, LINE-1 methylation was elevated among men, those who frequently consumed cruciferous vegetables, and those with a null genotype for either glutathione S-transferase M1 (GSTM1) or GSTT1. In contrast, reduced LINE-1 methylation was found in current smokers with a high cytochrome P450 1A2 (CYP1A2) phenotype index. In a case-control analysis, there was no significant association of LINE-1 methylation with case status, although reduced LINE-1 methylation was associated with increased risk of bladder cancer among never smokers (P for trend = 0.03); analysis by tertile revealed odds ratios (ORs) of 1.91 (lowest tertile; 95% CI = 1.17–3.13) and 1.34 (middle tertile; 95% CI = 0.79–2.28) when compared to the highest tertile. This association was strongest among nonsmokers null for either the GSTM1 or GSTT1 genotype (P for trend = 0.006). Further research is needed to understand the relationships between methyl group availability and LINE-1 methylation in relation to bladder cancer risk.
Head and neck cancer accounts for an estimated 47,560 new cases and 11,480 deaths annually in the United States, the majority of which are squamous cell carcinomas (HNSCC). The overall 5 year survival is approximately 60% and declines with increasing stage at diagnosis, indicating a need for non-invasive tests that facilitate the detection of early disease. DNA methylation is a stable epigenetic modification that is amenable to measurement and readily available in peripheral blood. We used a semi-supervised recursively partitioned mixture model (SS-RPMM) approach to identify novel blood DNA methylation markers of HNSCC using genome-wide methylation array data for peripheral blood samples from 92 HNSCC cases and 92 cancer-free control subjects. To assess the performance of the resultant markers, we constructed receiver operating characteristic (RJC) curves and calculated the corresponding area under the curve (AUC). Cases and controls were best differentiated by a methylation profile of six CpG loci (associated with FGD4, SERPINF1, WDR39, IL27, HYAL2 and PLEKHA6), with an AUC of 0.73 (95% CI: 0.62–0.82). After adjustment for subject age, gender, smoking, alcohol consumption and HPV16 serostatus, the AUC increased to 0.85 (95% CI: 0.76–0.92). We have identified a novel blood-based methylation profile that is indicative of HNSCC with a high degree of accuracy. This profile demonstrates the potential of DNA methylation measured in blood for development of non-invasive applications for detection of head and neck cancer.
semi-supervised RPMM; HNSCC; epigenetics; biomarkers; Infinium; methylation array
Individuals diagnosed with non-melanoma skin cancer have a high risk of developing a second skin cancer diagnosis. We assessed whether a marker of immune function related to atopic allergy, IgE, was associated with diagnosis of subsequent squamous cell carcinoma (SCC) of the skin in patients with a previous skin cancer enrolled in a skin cancer prevention trial.
One hundred twelve cases with a repeat skin cancer diagnosis were compared to 227 controls, matched on age, sex, and study center. Total, respiratory, and food-specific IgE were measured in the baseline or year one (prior to diagnosis) sera samples for each subject.
IgE levels were higher in cases with a second SCC than controls (comparing the highest quartile to the lowest, ORtotal IgE=1.44; 95% CI:0.73–2.85; ORrespiratory IgE =2.43; 95% CI:1.16–5.06; ORfood IgE =2.53; 95%CI:1.19–5.35). The association between respiratory IgE and subsequent skin cancer was strongest among individuals with a tendency to sunburn (ORrespiratory IgE =3.82; 95%CI: 1.05–13.88) compared with those with a tendency to tan (ORrespiratory IgE = 0.95; 95%CI:0.20–4.76). Among 25 subjects with repeat IgE measurements taken over several years, IgE levels were remarkably stable (interclass coefficient = 0.90 for total IgE).
These results indicate that allergy or allergy-associated IgE may be indicative of an immune phenotype that enhances risk of SCC, possibly via immune-associate inflammatory mediators.
Our results indicate that controlling allergy and IgE levels may be a new avenue of skin cancer prevention in susceptible populations, and implicate immune mechanisms in skin carcinogenesis.
Cigarette smoking is a risk factor for colorectal cancer. Putative colorectal procarcinogens in tobacco smoke include polycyclic aromatic hydrocarbons and heterocyclic aromatic amines that are known substrates of glutathione S-transferases (GSTs). This study examined the influence of functional GST gene polymorphisms on the smoking–colorectal cancer association in a population known to be minimally exposed to dietary sources of these procarcinogens. Incident cases of colorectal cancer (n = 480) and matched controls (n = 1167) were selected from the Singapore Chinese Health Study, a population-based prospective cohort of 63 257 men and women who have been followed since 1993. We determined the deletion polymorphisms of GSTM1 and GSTT1 and the functional polymorphism at codon 105 of GSTP1 for each subject. A three level composite GST index was used to examine if GST profile affected a smoker’s risk of developing colorectal cancer. While there was no statistically significant association between cigarette smoking and colorectal cancer risk among subjects absent of any at-risk GST genotypes, smokers possessing two to three at-risk GST genotypes exhibited a statistically significant increased risk of colorectal cancer compared with non-smokers (P = 0.0002). In this latter stratum, heavy smokers exhibited a >5-fold increased risk relative to never-smokers (odds ratio, 5.43; 95% confidence interval, 2.22–13.23). Subjects with one at-risk GST genotype displayed a statistically significant but weaker association with smoking. These findings suggest that GST gene polymorphisms influence interindividual susceptibility to smoking-associated colorectal cancer. Our data indicate an important role for GST enzymes in the detoxification of colorectal carcinogens in tobacco smoke.
Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) are potentially chemopreventive.
We examined the relation between NSAID use and non-melanoma skin cancer in a population-based case-control study.
NSAID and analgesic use was analyzed in 1484 subjects: 535 squamous cell carcinoma (SCC), 487 basal cell carcinoma (BCC), and 462 controls.
Use of NSAIDs, particularly aspirin, related to reduced odds ratios (OR) of SCC especially tumors positive for p53 (OR = 0.29; 95% CI= 0.11-0.79) or with PTCH loss of heterozygosity (OR = 0.35; 95% CI = 0.13 – 0.96). Although not considered an NSAID, decreased odds ratios of both BCC and SCC were observed in relation to use of paracetamol. Risk of BCC was unrelated to NSAID use.
Self reported drug use.
This study supports the hypothesis that NSAIDs, aspirin in particular, may reduce risk of SCC and additionally may affect specific molecular subtypes of SCC.
NSAIDS; non-melanoma skin cancer; basal cell carcinoma; squamous cell carcinoma; p53; PTCH; case-control study
Malignant pleural mesothelioma (MPM) remains a cancer of poor prognosis. It is hoped that implementation of effective screening biomarkers will lead to earlier diagnoses and improved outcomes. Serum-measured soluble mesothelin-related peptide (SMRP) has been demonstrated to have excellent specificity for MPM, but poor sensitivity precludes its use as a screening biomarker. Using a case series of MPM patients from the International Mesothelioma Program at the Brigham and Women's hospital, we sought to determine whether epigenetic change at the MSLN gene in patient tumors is responsible for the poor sensitivity of SMRP. We identified three potential target regions for CpG methylation silencing in the MSLN promoter, one of which was amenable to bisulfite pyrosequencing and located 214 bp upstream of the transcription start site. MSLN promoter methylation was significantly higher in normal pleura than tumor tissue (p < 6.0 × 10−9). Next, we compared cases according to serum SMRP status and observed that MSLN methylation was significantly higher among tumors from patients testing negative for SMRP (<1.5 nM) versus those that were SMRP positive (p < 0.03). These results demonstrate that MSLN is normally methylated in the pleura, and that methylation is lost in most tumors. However, in a subset of tumors methylation is retained, and this mechanism explains the poor sensitivity of the SMRP assay. These results may lead to additional biomarker targets that will resolve the poor sensitivity of the SMRP assay and allow implementation of screening among exposed populations.
SMRP; MSLN; mesothelioma; methylation; screening
Polycyclic aromatic hydrocarbons (PAH) are believed to be among the principal causative agents for lung cancer in smokers, but no epidemiologic studies have evaluated the relationship of PAH uptake and metabolism to lung cancer. In this study, we quantified prediagnostic urinary levels of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydro-phenanthrene (PheT), a validated biomarker of PAH uptake and metabolism, as well as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), and cotinine and its glucuronides (total cotinine), validated biomarkers of uptake of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and nicotine, respectively, in relation to lung cancer risk among current smokers in a nested case–control study within a cohort of 18,244 Chinese men in Shanghai, China. Urinary levels of PheT, total NNAL, and total cotinine were significantly higher in cases than controls (N = 476 matched pairs). ORs (95% confidence intervals) for lung cancer in the second, third, fourth, and fifth quintiles of PheT were 1.70 (1.00–2.88), 1.07 (0.62–1.84), 1.48 (0.86–2.53), and 2.34 (1.33–4.11), respectively, relative to the lowest quartile (Ptrend = 0.023) after adjustment for self-reported smoking intensity and duration and urinary total NNAL and total cotinine. This study also confirmed that urinary total NNAL and total cotinine are independently related to lung cancer risk.
In order to properly comprehend the epigenetic dysregulation that occurs during the course of disease, there is a need to characterize the epigenetic variability in healthy individuals that arises in response to aging and exposures, and to understand such variation within the biological context of the DNA sequence. We analyzed the methylation of 26,486 autosomal CpG loci in blood from 205 healthy subjects, using three complementary approaches to assess the association between methylation, age or exposures and local sequence features, such as CpG island status, repeat sequences, location within a polycomb target gene or proximity to a transcription factor binding site. We clustered CpGs (1) using unsupervised recursively partitioned mixture modeling (RPMM) and (2) bioinformatically-informed methods and (3) also employed a marginal model-based (non-clustering) approach. We observed associations between age and methylation and hair dye use and methylation, where the direction and magnitude was contingent on the local sequence features of the CpGs. Our results demonstrate that CpGs are differentially methylated dependent upon the genomic features of the sequence in which they are embedded, and that CpG methylation is associated with age and hair dye use in a CpG context-dependent manner in healthy individuals.
methylation array; epigenetics; exposures; aging; Infinium; hair dye
Authors Scott M. Langevin and E. Andres Houseman contributed equally and should be listed as joint first authors in Epigenetics Volume 6, Issue 7 in the manuscript: Langevin SM, Houseman EA, Christensen BC, Wiencke JK, Nelson HH, Karagas MR, et al. The influence of aging, environmental exposures and local sequence features on the variation of DNA methylation in blood. Epigenetics 2011; 6:908-19.
S-adenosylmethionine (SAM) is a primary methyl donor for the methylation of many molecules including DNA. DNA methylation is believed to play an important role in functions of cells and genes. Dietary, genetic and metabolic factors that influence systematic SAM levels are not fully understood. We conducted cross-sectional analysis to evaluate associations between plasma concentrations of one-carbon metabolism nutrients and metabolites and plasma SAM concentrations using healthy individuals within the Singapore Chinese Health Study. Plasma SAM, betaine, choline, folate, total homocysteine (Hcy), methionine, S-adenosylhomocysteine (SAH), vitamin B6 and vitamin B12 concentrations were quantified. Genotypes of methionine adenosyltransferases (MAT1A, MAT2A and MAT2B) were also determined. Linear regression and path analysis were performed to depict the directed dependencies in one-carbon metabolism. Age and body mass index were positively associated while cigarette smoking were inversely associated with plasma SAM concentrations. Plasma choline, methionine and SAH were positively and strongly associated with plasma SAM after adjustment for confounders. Plasma betaine and folate were positively associated with plasma SAM only in men. Men carrying the variant MAT1A genotypes had lower plasma SAM concentrations than men carrying the wild type genotype (p for gene x gender interaction = 0.02). This effect modification by gender was restricted to individuals with low plasma methionine. In conclusion, plasma choline, methionine and SAH were strongly associated with plasma SAM concentrations. The MAT1A genetic polymorphism may impact plasma SAM concentrations in men with low plasma methionine concentrations.
One-carbon metabolism; plasma SAM; MAT genetic polymorphism; path analysis
There has been a long-standing need in biomedical research for a method that quantifies the normally mixed composition of leukocytes beyond what is possible by simple histological or flow cytometric assessments. The latter is restricted by the labile nature of protein epitopes, requirements for cell processing, and timely cell analysis. In a diverse array of diseases and following numerous immune-toxic exposures, leukocyte composition will critically inform the underlying immuno-biology to most chronic medical conditions. Emerging research demonstrates that DNA methylation is responsible for cellular differentiation, and when measured in whole peripheral blood, serves to distinguish cancer cases from controls.
Here we present a method, similar to regression calibration, for inferring changes in the distribution of white blood cells between different subpopulations (e.g. cases and controls) using DNA methylation signatures, in combination with a previously obtained external validation set consisting of signatures from purified leukocyte samples. We validate the fundamental idea in a cell mixture reconstruction experiment, then demonstrate our method on DNA methylation data sets from several studies, including data from a Head and Neck Squamous Cell Carcinoma (HNSCC) study and an ovarian cancer study. Our method produces results consistent with prior biological findings, thereby validating the approach.
Our method, in combination with an appropriate external validation set, promises new opportunities for large-scale immunological studies of both disease states and noxious exposures.
While a possible link between assisted reproductive technology (ART) and rare imprinting disorders has been found, it is not clear if this is indicative of subtler disruptions of epigenetic mechanisms. Results from previous studies have been mixed, but some methylation differences have been observed.
Children conceived through ART and children conceived spontaneously were recruited for this cross-sectional study. Information about reproductive history, demographic factors, birth characteristics, and infertility treatment was obtained from maternal interview and medical records. Peripheral blood lymphocytes and buccal cell samples were collected from participating children. Methylation analysis was performed on five loci using pyrosequencing. Statistical analysis of methylation differences was performed using linear regression with generalized estimating equations. Results are reported as differences with 95% confidence intervals (CI).
A total of 67 ART children and 31 spontaneously conceived (SC) children participated. No significant difference in methylation in lymphocyte samples was observed between groups for any loci. Possible differences were found in buccal cell samples for IGF2 DMR0 (Difference: 2.07; 95% confidence interval (CI): -0.28, 4.42; p = 0.08) and IGF2R (Difference: -2.79; 95% CI: -5.74, 0.16; p = 0.06). Subgroup analysis indicated potential lower methylation in those whose parents used ART for unexplained infertility.
Observed differences in methylation between the ART and SC groups were small for all loci in the two sample types examined and no statistical differences were observed. It is still unclear whether or not small differences observed in several studies represent a real difference between groups and if this difference is biologically meaningful. Larger studies with long term follow-up are needed to fully answer these questions.
Assisted reproductive technology; Epigenetics; Imprinting
We explored the associations of aberrant DNA methylation patterns in 12 candidate genes with adult glioma subtype, patient survival, and gene expression of enhancer of zeste human homolog 2 (EZH2) and insulin-like growth factor-binding protein 2 (IGFBP2). We analyzed 154 primary glioma tumors (37 astrocytoma II and III, 52 primary glioblastoma multiforme (GBM), 11 secondary GBM, 54 oligodendroglioma/oligoastrocytoma II and III) and 13 nonmalignant brain tissues for aberrant methylation with quantitative methylation-specific PCR (qMS-PCR) and for EZH2 and IGFBP2 expression with quantitative reverse transcription PCR (qRT-PCR). Global methylation was assessed by measuring long interspersed nuclear element-1 (LINE1) methylation. Unsupervised clustering analyses yielded 3 methylation patterns (classes). Class 1 (MGMT, PTEN, RASSF1A, TMS1, ZNF342, EMP3, SOCS1, RFX1) was highly methylated in 82% (75/91) of lower-grade astrocytic and oligodendroglial tumors, 73% (8/11) of secondary GBMs, and 12% (6/52) of primary GBMs. The primary GBMs in this class were early onset (median age 37 years). Class 2 (HOXA9 and SLIT2) was highly methylated in 37% (19/52) of primary GBMs. None of the 10 genes for class 3 that were differentially methylated in classes 1 and 2 were hypermethylated in 92% (12/13) of nonmalignant brain tissues and 52% (27/52) of primary GBMs. Class 1 tumors had elevated EZH2 expression but not elevated IGFBP2; class 2 tumors had both high IGFBP2 and high EZH2 expressions. The gene-specific hypermethylation class correlated with higher levels of global LINE1 methylation and longer patient survival times. These findings indicate a generalized hypermethylation phenotype in glioma linked to improved survival and low IGFBP2. DNA methylation markers are useful in characterizing distinct glioma subtypes and may hold promise for clinical applications.
glioma; DNA methylation; EZH2; Polycomb; PI3K/Akt
Arsenic is a carcinogen that contaminates drinking water worldwide. Accumulating evidence suggests that both exposure and genetic factors may influence susceptibility to arsenic-induced malignancies. We sought to identify novel susceptibility loci for arsenic-related bladder cancer in a US population with low to moderate drinking water levels of arsenic. We first screened a subset of bladder cancer cases using a panel of approximately 10,000 non-synonymous single nucleotide polymorphisms (SNPs). Top ranking hits on the SNP array then were considered for further analysis in our population-based case–control study (n = 832 cases and 1,191 controls). SNPs in the fibrous sheath interacting protein 1 (FSIP1) gene (rs10152640) and the solute carrier family 39, member 2 (SLC39A2) in the ZIP gene family of metal transporters (rs2234636) were detected as potential hits in the initial scan and validated in the full case–control study. The adjusted odds ratio (OR) for the FSIP1 polymorphism was 2.57 [95% confidence interval (CI) 1.13, 5.85] for heterozygote variants (AG) and 12.20 (95% CI 2.51, 59.30) for homozygote variants (GG) compared to homozygote wild types (AA) in the high arsenic group (greater than the 90th percentile), and unrelated in the low arsenic group (equal to or below the 90th percentile) (P for interaction = 0.002). For the SLC39A2 polymorphism, the adjusted ORs were 2.96 (95% CI 1.23, 7.15) and 2.91 (95% CI 1.00, 8.52) for heterozygote (TC) and homozygote (CC) variants compared to homozygote wild types (TT), respectively, and close to one in the low arsenic group (P for interaction = 0.03). Our findings suggest novel variants that may influence risk of arsenic-associated bladder cancer and those who may be at greatest risk from this widespread exposure.
P53 is a key regulatory molecule in the cellular response to ultraviolet radiation, and TP53 mutation is the most common alteration in non-melanoma skin cancer. The MDM2 oncogene negatively regulates p53 protein levels, and both genes have functional polymorphisms that may modify skin cancer risk. Furthermore, prior research suggests that TP53 mutations preferentially occur on the arginine allele to selectively inactivate the p63 pathway. We tested these hypotheses of susceptibility and preferential mutation in non-melanoma skin cancer. The TP53 Arg72Pro and MDM2 309 polymorphisms were genotyped in a population-based case–control study of non-melanoma skin cancer, and TP53 alteration (mutation and immunohistochemistry staining) was evaluated in case tumors. In 902 cases of basal cell carcinoma (BCC), 676 cases of squamous cell carcinoma (SCC) and 812 controls, no association was found between the TP53 polymorphism and risk of non-melanoma skin cancer [odds ratio (OR)BCC 0.98, 95% confidence interval (CI) 0.80–1.20; ORSCC 0.93, 95% CI 0.75–1.16]. However, carriers of the MDM2 SNP309 G allele did have an elevated risk of non-melanoma skin cancer (ORBCC 1.15, 95% CI 0.93–1.42; ORSCC 1.29, 95% CI 1.02–1.63). We observed an association between TP53 alterations in the tumors and constitutive TP53 genotype (P < 0.01), with alterations preferentially occurring on the proline allele. Collectively, these data highlight the significant effects of genotype on gene-specific mutation events in carcinogenesis.
Although much is known about molecular and chromosomal characteristics that distinguish glioma histological subtypes, DNA methylation patterns of gliomas and their association with other tumor features such as mutation of isocitrate dehydrogenase (IDH) genes have only recently begun to be investigated.
DNA methylation of glioblastomas, astrocytomas, oligodendrogliomas, oligoastrocytomas, ependymomas, and pilocytic astrocytomas (n = 131) from the Brain Tumor Research Center at the University of California San Francisco, as well as nontumor brain tissues (n = 7), was assessed with the Illumina GoldenGate methylation array. Methylation data were subjected to recursively partitioned mixture modeling (RPMM) to derive methylation classes. Differential DNA methylation between tumor and nontumor was also assessed. The association between methylation class and IDH mutation (IDH1 and IDH2) was tested using univariate and multivariable analysis for tumors (n = 95) with available substrate for sequencing. Survival of glioma patients carrying mutant IDH (n = 57) was compared with patients carrying wild-type IDH (n = 38) using a multivariable Cox proportional hazards model and Kaplan–Meier analysis. All statistical tests were two-sided.
We observed a statistically significant association between RPMM methylation class and glioma histological subtype (P < 2.2 × 10−16). Compared with nontumor brain tissues, across glioma tumor histological subtypes, the differential methylation ratios of CpG loci were statistically significantly different (permutation P < .0001). Methylation class was strongly associated with IDH mutation in gliomas (P = 3.0 × 10−16). Compared with glioma patients whose tumors harbored wild-type IDH, patients whose tumors harbored mutant IDH showed statistically significantly improved survival (hazard ratio of death = 0.27, 95% confidence interval = 0.10 to 0.72).
The homogeneity of methylation classes for gliomas with IDH mutation, despite their histological diversity, suggests that IDH mutation is associated with a distinct DNA methylation phenotype and an altered metabolic profile in glioma.