Median Methylation Research Articles

Genome-Wide Methylation Analysis of Patients with Diffuse Large B Cell Lymphoma Treated on the UK NCRI R-CHOP 14 Vs 21 Trial

Abstract Background: Epigenetic alterations are a hallmark of diffuse large B cell lymphoma (DLBCL). A high degree of methylation disruption and intra-tumor methylation heterogeneity have been linked to poor outcome in DLBCL. However, high-coverage methylation data within prospective trial cohorts are warranted to characterize clinically relevant methylation subgroups of DLBCL. Here, we investigate the methylome of DLBCL cases from a large multicentre Phase III trial and its interaction with clinical and molecular characteristics. Methods: DNA methylation was assessed with the Illumina Infinium 450k array in archived FFPE samples of 127 newly diagnosed DLBCL patients treated with R-CHOP chemotherapy on the UK NCRI R-CHOP14v21 trial. Status of BCL2-, BCL6-, and MYC-rearrangements and amplifications was available for 111 cases. Cell-of-origin classification was available for all cases, both according to the IHC Hans algorithm and the microarray-based classification tool using Illumina DASL technology (Barrans, Br J Haematol 2012). In addition, microarray data was used to extract mean expression values of the epigenetic modifiers EZH2, MLL2, CREBBP, EP300, DNMT3A, IDH1/2 and TET2 and patients in the lowest and highest expression quartiles were compared. Analyses were performed in R 3.2.3 using the RnBeads package. Differential methylation was analyzed on region level (tiling, genes, promoters, CpG islands). P values were adjusted for multiple testing using the false discovery rate method. Results: After normalization and quality control 425,623 probes were included in the analysis. Median methylation levels of probes showed a bimodal distribution (unmethylated/methylated) across samples, with CpG islands and shores being mainly unmethylated, and CpG shelves and open seas being predominantly methylated. No significant association of clinical prognostic factors (age, LDH level, extranodal involvement, stage, WHO performance status) and methylation could be observed. We did not find significant differential methylation between patients who relapsed after R-CHOP therapy and those who did not. However, more detailed outcome analyses using Cox regression models for progression- and overall survival on single-site level will be provided at the meeting. There was no evidence for a difference in methylation with regards to rearrangements or amplifications of MYC or BCL6, nor with presence of double-hit abnormalities. Interestingly, cases with BCL2 rearrangement showed differential methylation of 39 promoter regions compared to cases without this abnormality (all adj. P<0.05). A strong impact on methylation patterns was seen with regards to microarray-based cell-of-origin categories (but not according to the IHC classifier). GCB vs. ABC cases showed global hypermethylation, predominantly affecting CpG islands and shores. 388 promoter regions were differentially methylated between GCB and ABC subtypes, corresponding to 226 annotated genes or miRNAs, the majority (93%) being hypermethylated in GCB cases. Differentially methylated promoter regions in GCB lymphomas were enriched for signal transduction and cell-cell communication pathway genes (adj. P<0.05) and included EZH2 target genes such as CDKN1A and SOX9. mRNA expression levels of epigenetic modifiers was not associated with significant differences in methylation, apart from DNMT3A expression. Patients with high vs. low DNMT3Aexpression showed differential methylation of 3300 promoters, which were significantly enriched for KRAS-associated genes (P=3.68E-12), cancer pathway genes (e.g. Wnt-, FGF- and hedgehog signalling; P=1.45E-08), as well as cytokine signalling genes (P=1.96E-09). Data on recurrent somatic mutations of epigenetic modifiers and correlation of methylation and mRNA expression will be provided at the meeting. Conclusions: The cell-of-origin as well as expression levels of DNMT3A are main drivers of methylation variability in DLBCL, whereas other molecular features like MYC seem to have little impact on methylation patterns. This comprehensive dataset of genome-wide methylation profiles from a prospective trial cohort provides important information for identifying biologically and clinically relevant epigenetic subgroups of DLBCL. Disclosures Cunningham: Amgen: Research Funding; Astra-Zeneca: Research Funding; Bayer: Research Funding; Celgene: Research Funding; Merrimack: Research Funding; Medimmune: Research Funding.

Abstract P136: Dietary Salt and Non-Salt Components Have Substantial Effects on Genome-Wide DNA Methylation Patterns in Dahl SS Rats

Epigenetic modifications of the genome play a key role in the regulation of gene expression. It has been reported that epigenetic modifications of several genes are associated with hypertension. To investigate the potential role of genome-wide changes in DNA methylation in salt-induced hypertension, experiments were performed on inbred Dahl SS rats obtained from two colonies maintained at the Medical College of Wisconsin (i.e. MCWSS) and Charles River Laboratory (CRLSS). The colonies are genetically identical, but CRLSS rats were maintained on a whole grain diet containing 1% NaCl (CRLSS_LS) while MCWSS rats were fed casein-based AIN-76A chow containing 0.4% NaCl (MCWSS_LS) until both colonies were switched to an AIN-76A chow containing 4% NaCl for 14 days starting at 6 weeks of age (CRLSS_HS and MCWSS_HS, respectively). Mean arterial pressure and albumin excretion rate in MCWSS_HS rats were significantly greater (142±14 mmHg and 100±16 mg/day, n=6) than in CRLSS_HS rats (118±2 mmHg and 20±2 mg/day, n=7). Reduced representation bisulfite genome sequencing (RRBS) measured 5-Methylcytosine levels at single-base resolution in the renal outer medulla in the above groups, each with four biological replicates. For genomic regions located within CpG islands (CGI’s) and exhibiting differential methylation between LS and HS in each colony, HS diet increased median methylation levels several-fold in both MCWSS (7.45% vs. 0.35% for MCWSS_HS vs. MCWSS_LS, respectively, p = 2.84E-31) and CRLSS rats (7.62% vs. 1.21% for CRLSS_HS vs. CRLSS_LS, respectively, p = 1.65E-32). For genomic regions exhibiting differential methylation between MCWSS and CRLSS, MCWSS_HS rats (which exhibited higher blood pressure) had higher median methylation levels than CRLSS_HS rats (7.56% vs. 2.75%, p = 2.12E-9). We observed 156 hypermethylated and 241 hypomethylated regions within CGI’s of MCWSS_LS compared to CRLSS_LS. Examples of differentially methylated genes include the serine protease Prss2 , the transcription factor E2f1 , and the matrix protein Spock2 . These results suggest that sodium-dependent and independent dietary components could induce changes in DNA methylation that may predispose and participate in the development of hypertension and renal damage.

Within-pair differences of DNA methylation levels between monozygotic twins are different between male and female pairs.

BackgroundDNA methylation levels will be important for detection of epigenetic effects. However, there are few reports showing sex-related differences in the sensitivity to DNA methylation. To evaluate their sex-related individual differences in the sensitivity to methylation rigorously, we performed a systematic analysis of DNA methylation in monozygotic twins, an optimal model to evaluate them because the genetic backgrounds are the same.ResultsWe examined 30 male and 43 female older monozygotic twin pairs recruited from the registry established by the Center for Twin Research, Osaka University. Their methylation levels were determined using the Infinium HumanMethylation450 BeadChip Kit (Illumina), which interrogated 485577 highly informative CpG sites at the single-nucleotide resolution, and the median methylation level was calculated for each of the 25657 CpG islands. Within-pair differences of methylation levels (WPDMs) were greater in male pairs than female pairs for 86.0 % of autosomal CpG islands, but were higher in female pairs than male pairs for 76.7 % of X chromosomal CpG islands. Mean WPDMs of CpG islands in each autosomal chromosome were significantly higher in male pairs than in female whereas that in X chromosome was significantly higher in female pairs than in male. Multiple comparison indicated that WPDMs in three autosomal and two X-chromosomal CpG islands were significantly greater in male pairs, whereas those in 22 X-chromosomal CpG islands were significantly greater in female pairs.ConclusionSex-related differences were present in the WPDMs of CpG islands in individuals with the same genetic background. These differences may be associated with the sexual influences in susceptibility of some diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-016-0217-2) contains supplementary material, which is available to authorized users.

Global, cancer-specific microRNA cluster hypomethylation was functionally associated with the development of non-B non-C hepatocellular carcinoma.

BackgroundWhile hepatitis B and C viral infection have been suppressed, non-B non-C hepatocellular carcinoma (NBNC-HCC) is considered to be rising in incidence terms in some developed countries where prevalence of those viral infections among HCC patients had been very high (such as Japan, Korea, and Italy). To elucidate critical molecular changes in NBNC-HCC, we integrated three large datasets relating to comprehensive array-based analysis of genome-wide DNA methylation (N = 43 pairs) and mRNA/miRNA expression (N = 15, and 24 pairs, respectively) via statistical modeling.ResultsHierarchical clustering of DNA methylation in miRNA coding regions clearly distinguished NBNC-HCC tissue samples from relevant background tissues, revealing a remarkable tumor-specific hypomethylation cluster. In addition, miRNA clusters were extremely hypomethylated in tumor samples (median methylation change for non-clustered miRNAs: -2.3%, clustered miRNAs: -24.6%). The proportion of CpGs hypomethylated in more than 90% of the samples was 55.9% of all CpGs within miRNA clusters, and the peak methylation level was drastically shifted from 84% to 39%. Following statistical adjustment, the difference in methylation levels within miRNA coding regions was positively associated with their expression change. Receiver operating characteristic (ROC) analysis revealed a great discriminatory ability in respect to cluster-miRNA methylation. Moreover, miRNA methylation change was negatively correlated with corresponding target gene expression amongst conserved and highly matched miRNA sites.ConclusionsWe observed a drastic negative shift of methylation levels in miRNA cluster regions. Changes in methylation status of miRNAs were more indicative of target gene expression and pathological diagnosis than respective miRNA expression changes, suggesting the importance of genome-wide miRNA methylation for tumor development. Our study dynamically summarized global miRNA hypomethylation and its genome-wide scale consequence in NBNC-HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0514-6) contains supplementary material, which is available to authorized users.

Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize.

An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types), z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control), normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25–30%) appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences.

Prediction efficiency of PITX2 DNA methylation for prostate cancer survival.

This study determined the level of PITX2 methylation in prostate cancer and benign tissues and its relationship with the postoperative survival rate. Forty-four patients with prostate cancer who underwent radical prostatectomy and 43 patients with benign prostatic hyperplasia were selected. DNA was extracted from the tissues and PITX2 methylation status was quantitatively analyzed by using the EpiTect MethyLight method. The median follow-up time of the patients was 63 months and was used to analyze the relationship between PITX2 methylation status with tumor stage and survival rates. Median PITX2 gene expression in benign tissues was 1.46, which was higher than that of tumor tissues with a median of 0.01 (P < 0.001). The median methylation in the controls was less than 0.001%, while the median methylation in the test group was 23.3% (P = 0.000). The number of patients with low methylation level in T2 stage was 15, which was more than that in T3 and T4 stages (8 patients); while the number of patients with high methylation levels in T2 stage was 6, which was less than that in T3 and T4 stages (15 patients) (P = 0.035). The PITX2 gene expression level in prostate cancer tissues was lower than that in benign tissues. A higher degree of PITX2 DNA methylation was associated with higher tumor stage and lower survival rates. PITX2 DNA methylation presents a good predictive value for prostate cancer survival.

Abstract 2953: Genomic DNA hypomethylation as an independent risk factor for renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is the most lethal genitourinary cancer. Cigarette smoking, hypertension and obesity are the major risk factors for RCC; however, much of the etiology of this disease remains to be elucidated. Genomic DNA hypomethylation is a hallmark of most cancer genomes, promoting genomic instability and cell transformation. In the present study, we sought to determine whether global DNA methylation in peripheral blood is associated with risk of RCC. A case control study consisting of 889 RCC cases and an equal number of age, gender, and ethnicity-matched controls was conducted. Genomic DNA was isolated from peripheral blood and global DNA methylation was measured as 5-mC% content using an antibody-based immune assay. Logistic regression was used to estimate odds ratio (OR) and 95% confidence interval (CI) for the association between DNA methylation level and the risk of RCC. The median age was 59.29±10.42 for cases and 59.39±10.30 for controls. Over two-thirds (67% in both cases and controls) were males, about half (50.2% in cases and 52.2% in controls) were never smokers, the vast majority (84.8% in both cases and controls) were Caucasian. BMI, physical activity, hypertension and family history of cancer were significantly different between cases and controls. RCC cases are more likely than controls to have high blood pressure level (p = 5.81E-15), higher BMI (p = 0.011), low physical activity (p = 1.48E-20), and a family history of cancer (other than kidney cancer) (p = 5.42E-07). The median methylation level in controls was 3.97, significantly higher than the median level of 3.64 in cases (p&amp;lt;0.001 Wilcoxon rank-sum test). In multivariate logistic regression analysis adjusted by age, gender, ethnicity, smoking status, and hypertension, individuals in the lowest tertile (T1) and middle tertile (T2) of 5-mC% had higher risks of RCC with ORs of 1.42 (95%CI 1.12-1.81) and 1.14 (95% CI 0.89-1.45), respectively, compared to individuals in the highest tertile (T3) of methylation (p for trend 0.004). When stratified by RCC risk factors, associations between hypomethylation and increased RCC risks were stronger among males (OR 1.64, 95%CI 1.21-2.21), younger individuals (OR 1.54, 95%CI 1.10-2.15), never-smokers (OR 1.61, 95%CI 1.14-2.28) and African-Americans (OR 2.75, 95%CI 0.75-10.08). These findings suggest for the first time that global DNA hypomethylation in peripheral blood is independently associated with RCC risk. The results may provide better understanding of the etiology of RCC tumorigenesis and in combination with modifiable risk factors and other potential biomarkers, may help identify high risk individuals for screening and early detection of RCC. This work was supported in part by the National Institutes of Health (grant R01 CA170298) and the Center for Translational and Public Health Genomics, Duncan Family Institute for Cancer Prevention, The University of Texas M D Anderson Cancer Center. Citation Format: Julia Mendoza Perez, Jian Gu, Nizar M. Tannir, Surena Matin, Jose A. Karam, David W. Chang, Luis A. Herrera, Christopher G. Wood, Xifeng Wu. Genomic DNA hypomethylation as an independent risk factor for renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2953. doi:10.1158/1538-7445.AM2015-2953

Overlap between differentially methylated DNA regions in blood B lymphocytes and genetic at-risk loci in primary Sjögren's syndrome

BackgroundBeyond genetics, epigenetics alterations and especially those related to DNA methylation, play key roles in the pathogenesis of autoimmune diseases such as primary Sjögren's syndrome (pSS) and systemic lupus erythematosus....

The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells

Germ cells are vital for transmitting genetic information from one generation to the next and for maintaining the continuation of species. Here, we analyze the transcriptome of human primordial germ cells (PGCs) from the migrating stage to the gonadal stage at single-cell and single-base resolutions. Human PGCs show unique transcription patterns involving the simultaneous expression of both pluripotency genes and germline-specific genes, with a subset of them displaying developmental-stage-specific features. Furthermore, we analyze the DNA methylome of human PGCs and find global demethylation of their genomes. Approximately 10 to 11 weeks after gestation, the PGCs are nearly devoid of any DNA methylation, with only 7.8% and 6.0% of the median methylation levels in male and female PGCs, respectively. Our work paves the way toward deciphering the complex epigenetic reprogramming of the germline with the aim of restoring totipotency in fertilized oocytes.

Comparison of Genetic and Epigenetic Alterations of Primary Tumors and Matched Plasma Samples in Patients with Colorectal Cancer

BackgroundAlthough recent advances in circulating DNA analysis allow the prediction of tumor genomes by noninvasive means, some challenges remain, which limit the widespread introduction of cfDNA in cancer diagnostics. We analyzed the status of the two best characterized colorectal cancer (CRC) genetic and epigenetic alterations in a cohort of CRC patients, and then compared the degree to which the two patterns move from tissue to plasma in order to improve our understanding of biology modulating the concordance between tissues and plasma methylation and mutation profiles.MethodsPlasma and tumor tissues were collected from 85 patients (69±14 years, 56 males). KRAS and SEPT9 status was assessed by allele refractory mutation system quantitative PCR and quantitative methylation-specific PCR, respectively. Six of the most common point mutations at codon 12 and 13 were investigated for KRAS analysis.Results KRAS mutations and SEPT9 promoter methylation were present in 34% (29/85) and in 82% (70/85) of primary tumor tissue samples. Both genetic and epigenetic analyses of cfDNA revealed a high overall concordance and specificity compared with tumor-tissue analyses. Patients presenting with both genetic and epigenetic alterations in tissue specimens (31.8%, 27/85) were considered for further analyses. The median methylation rates in tumour tissues and plasma samples were 64.5% (12.2–99.8%) and 14.5% (0–45.5%), respectively. The median KRAS mutation load (for matched mutations) was 33.6% (1.8–86.3%) in tissues and 2.9% (0–17.3) in plasma samples. The plasma/tissue (p/t) ratio of SEPT9 methylation rate was significantly higher than the p/t ratio of KRAS mutation load, especially in early stage cancers (p=0.0108).ConclusionThe results of this study show a discrepant rate of epigenetic vs. genetic alterations moving from tissue to plasma. Many factors could affect mutation cfDNA analysis, including both presence of tumor clonal heterogeneity and strict compartmentalization of KRAS mutation profile. The present study highlights the importance of considering the nature of the alteration when analyzing tumor-derived cfDNA.

Quantitative DNA methylation analysis of candidate genes in cervical cancer.

Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

DNA methylation level of OPCML and SFRP1: a potential diagnostic biomarker of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium which is caused by liver fluke infection. The clinical symptoms of CCA were revealed as the disease progresses to advanced stage. Thus, specific diagnostic biomarkers are important for this fatal disease. We applied methylation-sensitive high-resolution melting (MS-HRM) to quantify DNA methylation levels of opioid binding protein/cell adhesion molecule-like gene (OPCML) and Secreted frizzled-related protein 1 (SFRP1) in 73 primary CCA and 10 adjacent normal tissues and evaluated the sensitivity, specificity, and accuracy of the assay. The median methylation level of OPCML in CCA was 38.7 % (ranged from 0 to 82.2 %) and of SFRP1 was 31.5 % (ranged from 0 to 86.2 %). Methylation cutoff values of OPCML and SFRP1 derived from adjacent normal tissue were 6.90 and 10.44 %, respectively. With these cutoff values, the area under curve (AUC) of OPCML was 0.932 (95 % CI 0.878-0.986) and of SFRP1 was 0.951 (95 % CI 0.905-0.996). The sensitivity, specificity, and accuracy of OPCML were 89.04, 100, and 90.36 %, respectively, and of SFRP1 were 83.56, 100, and 85.54 %, respectively. In conclusion, the DNA methylation levels of OPCML and SFRP1 could be potential biomarkers for diagnosis of CCA with high specificity, sensitivity, and accuracy, in particular for biopsy specimens. Further validation in noninvasive samples such as serum or plasma is warranted for clinical applicability, especially as early diagnostic biomarkers.

LINE1 methylation levels in pre-diagnostic leukocyte DNA and future renal cell carcinoma risk

Higher levels of LINE1 methylation in blood DNA have been associated with increased kidney cancer risk using post-diagnostically collected samples; however, this association has never been examined using pre-diagnostic samples. We examined the association between LINE1 %5mC and renal cell carcinoma (RCC) risk using pre-diagnostic blood DNA from the United States-based, Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) (215 cases/436 controls), and the Alpha-tocopherol, Beta-carotene Cancer Prevention Study (ATBC) of Finnish male smokers (191 cases/575 controls). Logistic regression adjusted for age at blood draw, study center, pack-years of smoking, body mass index, hypertension, dietary alcohol intake, family history of cancer, and sex was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) using cohort and sex-specific methylation categories. In PLCO, higher, although non-significant, RCC risk was observed for participants at or above median methylation level (M2) compared to those below the median (M1) (OR: 1.37, 95% CI: 0.96–1.95). The association was stronger in males (M2 vs. M1, OR: 1.54, 95% CI: 1.00–2.39) and statistically significant among male smokers (M2 vs. M1, OR: 2.60, 95% CI: 1.46–4.63). A significant interaction for smoking was also detected (P-interaction: 0.01). No association was found among females or female smokers. Findings for male smokers were replicated in ATBC (M2 vs. M1, OR: 1.31, 95% CI: 1.07–1.60). In a pooled analysis of PLCO and ATBC male smokers (281cases/755controls), the OR among subjects at or above median methylation level (M2) compared to those below the median (M1) was 1.89 (95% CI: 1.34–2.67, P-value: 3 x 10–4); a trend was also observed by methylation quartile (P-trend: 0.002). These findings suggest that higher LINE1 methylation levels measured prior to cancer diagnosis may be a biomarker of future RCC risk among male smokers.

Methylation status of blood leukocyte DNA and risk of gastric cancer in a high-risk Chinese population.

To evaluate the relationship between methylation status of blood leukocyte DNA and risk of gastric cancer, a population-based study was conducted in Linqu County. Methylation levels of IGFII and N33 were determined by quantitative methylation-specific PCR. The temporal trend of methylation levels during gastric cancer development was investigated in 133 gastric cancer cases from two cohorts with pre- and/or post-gastric cancer samples. As the references of pre-GCs, 204 intestinal metaplasia (IM) or dysplasia (DYS) subjects who did not progress to gastric cancer during the follow-up period were selected. Meanwhile, 285 subjects with superficial gastritis/chronic atrophic gastritis (SG/CAG) were also selected as controls. IGFII median methylation level was significantly higher in gastric cancer cases than those with SG/CAG (61.47% vs. 49.73%; P < 0.001). IGFII and N33 methylation levels were elevated at least 5 years ahead of clinical gastric cancer diagnosis comparing with SG/CAG (63.38% vs. 49.73% for IGFII, 9.12% vs. 5.70% for N33; all P < 0.001). Furthermore, the frequency of hypermethylated IGFII was markedly increased in IM or DYS subjects who progressed to gastric cancer in contrast to those who remained with IM and DYS, and adjusted ORs were 12.52 [95% confidence interval (CI), 3.81-41.15] for IM and 10.12 (95% CI, 2.68-38.22) for DYS. Similar results were also found for N33 in subjects with IM (OR, 3.77; 95% CI, 1.20-11.86). Our findings suggested that hypermethylated IGFII and N33 in blood leukocyte DNA were associated with risk of gastric cancer in a Chinese population. IGFII and N33 methylation status may be related to gastric carcinogenesis.

Highlights of This Issue

To gauge the effectiveness of human papillomavirus (HPV) vaccines, Joura and colleagues estimated the prevalence and incidence of 14 HPV types in clinical swabs and then used predefined algorithms to attribute HPV types to different cervical lesions. Among 10,656 women enrolled in the placebo arms of phase 3 clinical trials of the 9-valent vaccine, seven high-risk HPV types that are targeted by the 9-valent investigational HPV vaccine were attributed to 43–55% of CIN1, 70–78% of CIN2, 85–91% of CIN3, and 95–100% of adenocarcinoma in situ (AIS) lesions. If the 9-valent HPV vaccination programs are effectively implemented, the majority of CIN2 and CIN3 lesions worldwide could be prevented.HIV-infected (HIV+) men face cancer treatment disparities which impact outcomes. Murphy and colleagues used electronic chart reviews to conduct a retrospective cohort study of HIV+ cases with prostate cancer. Most HIV+ patients with prostate cancer (95.3%) received antiretroviral therapy and 87.1% were virally suppressed. The authors report that prostate cancer in HIV+ men in the US is appropriately treated but more research is needed on prostate cancer survival indices and etiologies in the HIV/AIDS population.Zhang and colleagues evaluated the relationship between methylation status of blood leukocyte DNA and risk of gastric cancer in a population-based study. Methylation levels of IGFII and N33 were determined by quantitative methylation-specific PCR. IGFII median methylation level was significantly higher in gastric cancer cases. Both IGFII and N33 methylation levels were elevated at least 5 years ahead of clinical gastric cancer diagnosis. These findings suggest that hypermethylated IGFII and N33 in blood leukocyte DNA are associated with risk of gastric cancer in a Chinese population.Despite substantial evidence on the inverse association between circulating 25-hydroxyvitamin D [25(OH)D] levels and colorectal cancer, it is unclear whether this relationship is due to confounding by inflammation. Song and colleagues reevaluated the association between plasma 25(OH)D and colorectal cancer risk and accounted for inflammatory markers in a prospective case–control study nested within the Nurses' Health Study and Health Professionals Follow-up Study. Plasma 25(OH)D was associated with reduced risk of colorectal cancer but additional adjustment for inflammatory markers (C-reactive protein, interleukin-6, and soluble tumor necrosis factor receptor 2) did not change the results. These findings suggest that confounding by inflammation does not account for the inverse association between plasma 25(OH)D and colorectal cancer.

Molecular Profiling of Small Intestinal Neuroendocrine Tumours

ABSTRACT Aim: Aberrant DNA methylation is known to play an important role in the pathogenesis of many human cancers, however little is known about its role in small intestinal neuroendocrine tumour (SI NET) development. We report the first unbiased genome wide DNA methylation analysis of a large cohort of SI NET, aiming to identify key epigenetic changes specific to SI NET which may contribute to tumorigenesis. Methods: Illumina Infinium HumanMethylation450 Array (interrogating 99% of RefSeq genes) analysis was performed on DNA extracted from macrodissected SI NET primary tumours (n = 49) and normal small intestine (SI)(n = 21). Publicly available methylation data on >600 samples from The Cancer Genome Atlas was also assessed for comparison (colorectal, pancreatic and gastric adenocarcinoma and healthy tissue). Gene expression was determined using the Illumina DASL array on RNA extracted from SI NET primary tumours (n = 32) and normal SI (n = 6). Analysis was performed using ChAMP and limma R packages. A Bonferroni adjusted significance threshold value of p Results: Comparison of SI NET with normal SI identified a total of 130,083 significant methylation variable positions, including 1841 sites hypermethylated by >30% in tumour compared to normal tissue. 626 genes were found to have significant >3fold differential expression between SI NET and normal SI. Integrated analysis identified a group of 11 candidate genes where altered methylation and expression was significant and concordant (downregulated: CDX1, FBP1, C20orf54, GATA5; upregulated: PTPRN, PCSK1, PRLHR, CELSR3, GIPR, LMX1B, SCGN). Hypermethylation of GIPR (gastic inhibitory polypeptide receptor) was most significant (SI NET median methylation 0.67 vs normal SI 0.29 p Conclusions: This study is the first comprehensive analysis of the epigenetic profile of SI NET and identifies hypermethylation of GIPR as a potential novel biomarker. Novel radioligands targeting GIPR have been developed for use as imaging tools and it is a promising target for novel therapeutic agents. Disclosure: All authors have declared no conflicts of interest.

Epigenetic alterations in metastatic cutaneous carcinoma

Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the 2 most common cutaneous carcinomas. Molecular profiles predicting metastasis of these cancers have not been identified. Epigenetic profiles of 37 primary cases of cutaneous SCC and BCC were quantified via the Illumina Goldengate Cancer Panel. Differential protein expression by metastatic potential was analyzed in 110 total cases by immunohistochemical (IHC) staining. Unsupervised hierarchical clustering analysis revealed that metastatic BCCs had a methylation profile resembling cutaneous SCCs. Metastatic cutaneous SCCs were found to be hypermethylated at FRZB (median methylation: 46.7% vs 4.7%; p = 4 × 10(-5) ). Metastatic BCCs were found to be hypomethylated at MYCL2 (median methylation: 3.8% vs 83.4%; p = 1.9 × 10(-6) ). Immunohistochemical staining revealed few differences between metastatic and nonmetastatic cancers. Metastatic primary BCCs and cutaneous SCCs had distinct epigenetic profiles when compared to their nonmetastatic counterparts. Epigenetic profiling may prove useful in future diagnosis and prevention of advanced nonmelanoma skin cancers.

Abstract P261: Methylation Status of VKORC1 and Warfarin Dose

Introduction: It is well known that genetic variants, especially in the VKORC1 and CYP2C9 genes, can be associated with warfarin maintenance dose. However, currently known genetic variants fail to explain a large portion of the observed variability in warfarin dose, especially in African American patients. Methylation is a heritable epigenetic modification in which conversion of cytosine residues within “CpG islands” to 5-methylcytosine is associated with reduced gene expression. To our knowledge, the effect of methylation status on warfarin dose has never been studied. Hypothesis: We hypothesized that higher rates of methylation near the start region of VKORC1 in whole blood would be associated with lower required maintenance dose in warfarin patients. Methods: We conducted a prospective cohort study of patients initiating warfarin at three anticoagulation clinics. All individuals with available methylation data who reached maintenance dose were included in the analysis (N = 237). Methylation status was assessed in the region from 1500bp upstream to 500bp downstream of the VKORC1 start site using the Sequenom EpiTYPER assay on DNA extracted from whole blood. Linear regression was used to assess the association between methylation levels at individual CpG sites (66 total) and required warfarin maintenance dose. Visual inspection of univariable results was used to identify potentially important CpG clusters, which were subsequently examined using univariable and multivariable linear regression, with the latter adjusting for the effects of race, presence of VKORC1 variant, and other well-known predictors of warfarin dose. Results: Two of 66 CpG sites were nominally associated with lower maintenance dose requirement on univariable analysis. These CpG sites were identified as part of a CpG cluster located approximately 330 to 226bp upstream of the VKORC1 start site. Median methylation levels in this cluster was 16% (IQR 15%, 17%). A 1% increase in methylation in this cluster was associated with a 2.8 mg/wk reduction in weekly warfarin maintenance dose (P = 0.02). No significant interaction between methylation status and African American race was observed (P = 0.77). This association was substantially attenuated with the inclusion of VKORC1 variant status in the model (P = 0.33). Conclusions: Preliminary evidence suggests that methylation levels in a CpG cluster in VKORC1 may be moderately associated with warfarin maintenance dose, although this association may not be independent of VKORC1 variant status. However, these results require confirmation from additional epidemiological studies and possibly functional assays in liver tissue.

The relationship between FHIT gene promoter methylation and lung cancer risk: a meta-analysis

背景与目的抑癌基因启动子序列DNA甲基化与该基因在肿瘤细胞中表达下调有关。FHIT（fragile histindine triad）基因被认为是重要的肿瘤抑制基因，文献报道其启动子序列DNA在肿瘤组织中常发生甲基化改变。本研究采用meta分析的方法探讨FHIT基因启动子序列DNA甲基化与肺癌的相关性。方法检索Pubmed、CNKI及万方数据库，收集公开发表的关于FHIT基因启动子序列DNA甲基化与肺癌相关性的论文。对比分析肺癌组织与其配对的癌旁/正常肺组织、支气管盥洗液或血浆中FHIT基因启动子序列DNA甲基化发生率有无差别。以优势比（odd ratio, OR）为效应量，采用STATA 11.0统计软件进行合并分析。结果最终纳入符合要求的文献11篇，肺癌组织、癌旁/正常肺组织、支气管盥洗液和血浆中FHIT基因启动子序列甲基化率分别为Pmedian=40.0%（0-68.3%）、Pmedian=8.7%（0-35.0%）、Pmedian=33.3%（17.1%-38.3%）和Pmedian=35.9%（31.1%-50.8%）。肺癌中FHIT基因启动子序列甲基化率明显高于正常肺组织（OR=5.82, 95%CI: 3.74-9.06, P < 0.05），而与支气管盥洗液和血浆比较差别无统计学意义，（OR=1.55, 95%CI: 0.89-2.70, P > 0.05; OR=1.41, 95%CI: 0.90-2.20, P > 0.05）。结论与癌旁/正常肺组织相比患者肺癌组织中FHIT基因启动子序列DNA甲基化率明显增高，该基因的启动子甲基化与肺癌的发生可能存在相关性。

Epigenetic Influences on Associations between Air Pollutants and Lung Function in Elderly Men: The Normative Aging Study

Background: Few studies have been performed on pulmonary effects of air pollution in the elderly—a vulnerable population with low reserve capacity—and mechanisms and susceptibility factors for potential effects are unclear.Objectives: We evaluated the lag structure of air pollutant associations with lung function and potential effect modification by DNA methylation (< or ≥ median) at 26 individual CpG sites in nine candidate genes in a well-characterized cohort of elderly men.Methods: We measured forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV1), and blood DNA methylation one to four times between 1999 and 2009 in 776 men from the Normative Aging Study. Air pollution was measured at fixed monitors 4 hr to 28 days before lung function tests. We used linear mixed-effects models to estimate the main effects of air pollutants and effect modification by DNA methylation.Results: An interquartile range (IQR) increase in subchronic exposure (3 to 28 days cumulated), but not in acute exposure (during the previous 4 hr, or the current or previous day), to black carbon, total and nontraffic particles with aerodynamic diameter ≤ 2.5 μm (PM2.5), carbon monoxide, and nitrogen dioxide was associated with a 1–5% decrease in FVC and FEV1 (p < 0.05). Slope estimates were greater for FVC than FEV1, and increased with cumulative exposure. The estimates slopes for air pollutants (28 days cumulated) were higher in participants with low (< median) methylation in TLR2 at position 2 and position 5 and high (≥ median) methylation in GCR.Conclusions: Subchronic exposure to traffic-related pollutants was associated with significantly reduced lung function in the elderly; nontraffic pollutants (particles, ozone) had weaker associations. Epigenetic mechanisms related to inflammation and immunity may influence these associations.Citation: Lepeule J, Bind MAC, Baccarelli AA, Koutrakis P, Tarantini L, Litonjua A, Sparrow D, Vokonas P, Schwartz JD. 2014. Epigenetic influences on associations between air pollutants and lung function in elderly men: the Normative Aging Study. Environ Health Perspect 122:566–572; http://dx.doi.org/10.1289/ehp.1206458