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PDF file - 48K, Demethylation of a DNA region in NKp46 in control and HNSCC patient bloods assessed by qMSP

PDF file - 48K, Results of the NKp46 demethylation assay performed on HNSCC patient bloods stratified by tumor stage

PDF file - 60K, Bisulfite pyrosequencing of isolated human leukocytes

PDF file - 48K, Results of the NKp46 demethylation assay performed on HNSCC patient bloods stratified by tumor site

Purpose: 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.Experimental Design: 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.Results: 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 to 17.4). Cases did not show differences in NKp46 demethylation based on tumor site or stage.Conclusions: 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. Clin Cancer Res; 18(22); 6147–54. ©2012 AACR.

PDF file - 71K, Oligonucleotide sequences

PDF file - 46K, Comparison between NK cell level assessment by qMSP and fluorescence activated cell sorting (FACS)

Supplementary Tables 1-6 from Differentiation of Lung Adenocarcinoma, Pleural Mesothelioma, and Nonmalignant Pulmonary Tissues Using DNA Methylation Profiles

Supplementary Figure 2 from Epigenetic Profiles Distinguish Pleural Mesothelioma from Normal Pleura and Predict Lung Asbestos Burden and Clinical Outcome

Supplementary Table 1 from Epigenetic Profiles Distinguish Pleural Mesothelioma from Normal Pleura and Predict Lung Asbestos Burden and Clinical Outcome

Mechanisms of action of nonmutagenic carcinogens such as asbestos remain poorly characterized. As pleural mesothelioma is known to have limited numbers of genetic mutations, we aimed to characterize the relationships among gene-locus–specific methylation alterations, disease status, asbestos burden, and survival in this rapidly fatal asbestos-associated tumor. Methylation of 1505 CpG loci associated with 803 cancer-related genes were studied in 158 pleural mesotheliomas and 18 normal pleura. After false-discovery rate correction, 969 CpG loci were independently associated with disease status (Q < 0.05). Classifying samples based on CpG methylation profile with a mixture model approach, methylation classes discriminated tumor from normal pleura (permutation P < 0.0001). In a random forests classification, the overall misclassification error rate was 3.4%, with n = 1) of tumors misclassified as normal (P < 0.0001). Among tumors, methylation class membership was significantly associated with lung tissue asbestos body burden (P < 0.03), and significantly predicted survival (likelihood ratio P < 0.01). Consistent with prior work, asbestos burden was associated with an increased risk of death (hazard ratio, 1.4; 95% confidence interval, 1.1–1.8). Our results have shown that methylation profiles powerfully differentiate diseased pleura from nontumor pleura and that asbestos burden and methylation profiles are independent predictors of mesothelioma patient survival. We have added to the growing body of evidence that cellular epigenetic dysregulation is a critical mode of action for asbestos in the induction of pleural mesothelioma. Importantly, these findings hold great promise for using epigenetic profiling in the diagnosis and prognosis of human cancers. [Cancer Res 2009;69(1):227–34]

Pathologic differentiation of tissue of origin in tumors found in the lung can be challenging, with differentiation of mesothelioma and lung adenocarcinoma emblematic of this problem. Indeed, proper classification is essential for determination of treatment regimen for these diseases, making accurate and early diagnosis critical. Here, we investigate the potential of epigenetic profiles of lung adenocarcinoma, mesothelioma, and nonmalignant pulmonary tissues (n = 285) as differentiation markers in an analysis of DNA methylation at 1413 autosomal CpG loci associated with 773 cancer-related genes. Using an unsupervised recursively partitioned mixture modeling technique for all samples, the derived methylation profile classes were significantly associated with sample type (P < 0.0001). In a similar analysis restricted to tumors, methylation profile classes significantly predicted tumor type (P < 0.0001). Random forests classification of CpG methylation of tumors—which splits the data into training and test sets—accurately differentiated mesothelioma from lung adenocarcinoma over 99% of the time (P < 0.0001). In a locus-by-locus comparison of CpG methylation between tumor types, 1266 CpG loci had significantly different methylation between tumors following correction for multiple comparisons (Q < 0.05); 61% had higher methylation in adenocarcinoma. Using the CpG loci with significant differential methylation in a pathway analysis revealed significant enrichment of methylated gene-loci in Cell Cycle Regulation, DNA Damage Response, PTEN Signaling, and Apoptosis Signaling pathways in lung adenocarcinoma when compared with mesothelioma. Methylation profile–based differentiation of lung adenocarcinoma and mesothelioma is highly accurate, informs on the distinct etiologies of these diseases, and holds promise for clinical application. [Cancer Res 2009;69(15):6315–21]

Defects in antigen presenting cell function have been implicated in glioma immunosuppression. We measured peripheral CCL22, a dendritic cell/macrophage derived T cell trafficking chemokine, in sera from 1,208 glioma cases and 976 controls to assess whether it might provide a biomarker of glioma risk, survival and immune dysfunction. Cluster models were used to examine the relationship between CCL22 and glioma risk. Patient survival was assessed using Cox regression models. We also examined the relationship between CCL22 levels and CD4 cell counts, as well as allergy history and IgE levels. CCL22 levels were significantly lower among glioma cases compared with controls (Mean ± SEM: 1.23 ± 0.03 ng/mL in cases vs. 1.60 ± 0.03 ng/mL in controls, p < 0.0001) and this difference remained significant even after controlling for other covariates in the cluster models (highest quartile versus lowest Odds Ratio = 0.21, p < 0.0001). CD4 cell counts were positively correlated with CCL22 in glioma cases (Spearman r(2) = 0.51, p < 0.01) and were significantly lower in cases compared with controls. Higher CCL22 levels were associated with longer survival in all cases combined and in GBM cases (hazard ratio(allcases) = 0.81; 95% CI: 0.72-0.91, p = 0.0003). CCL22 levels were not associated with IgE level or self-reported allergies. Circulating CCL22 levels are related to both glioma risk and survival duration independent of age, histology, grade and IDH mutation status. CCL22 should be considered a marker of immune status with potential prognostic value.

Glioma, the most common central nervous system cancer in adults, has poor prognosis. Here we identify a new SNP associated with glioma risk, rs1920116 (near TERC), that reached genome-wide significance (Pcombined= 8.3 × 10-9) in a meta-analysis of genome-wide association studies (GWAS) of high-grade glioma and replication data (1,644 cases and 7,736 controls). This region has previously been associated with mean leukocyte telomere length (LTL). We therefore examined the relationship between LTL and both this new risk locus and other previously established risk loci for glioma using data from a recent GWAS of LTL (n = 37,684 individuals). Alleles associated with glioma risk near TERC and TERT were strongly associated with longer LTL (P = 5.5 × 10-20and 4.4 × 10-19, respectively). In contrast, risk-associated alleles near RTEL1 were inconsistently associated with LTL, suggesting the presence of distinct causal alleles. No other risk loci for glioma were associated with LTL. The identification of risk alleles for glioma near TERC and TERT that also associate with telomere length implicates telomerase in gliomagenesis.© 2014 Nature America, Inc. All rights reserved.

Glioma, the most common central nervous system cancer in adults, generally has poor prognosis. Glioblastoma, the most common and most aggressive form of glioma, has a median patient survival time of just 15 months from diagnosis under current standard of care.1 While prognosis is better for low-grade astrocytic tumors and tumors with an oligodendroglial component, over time these tumors all progress to high-grade glioma.2 Gliomagenesis is a complex and multifaceted process influenced by both inherited and acquired genetic variation. Glioma risk loci in 7 genes have been confirmed in genome-wide association studies.3–6 Several of these risk loci are found in genes previously implicated in gliomagenesis due to their mutation in glioma-associated hereditary cancer syndromes (TP53, CDKN2B/ANRIL) or their alteration in glioma tumors (TP53, CDKN2B/ANRIL, EGFR).7–9 Risk loci in 2 genes involved in telomerase structure and function (TERT [telomerase reverse transcriptase] and RTEL1 [regulator of telomere elongation helicase 1]) had not been implicated in gliomagenesis prior to genome-wide association studies, but telomerase activation has been observed in ∼90% of all human cancers.10 Telomeres act as a protective cap at the end of chromosomes but are progressively shortened during mitotic divisions.11 Telomere depletion ultimately leads to replicative senescence, limiting the proliferative capacity of cells. With activation of telomerase, an enzyme that adds DNA sequence repeats to telomeres, dividing cells can replace lost telomeric DNA and continue proliferating.10 TERT is a key component of human telomerase, and RTEL1 is needed to allow telomerase-dependent telomere extension to proceed effectively.12,13 Of the tumors that do not maintain telomere length through activation of telomerase, a significant subset activates a secondary pathway: alternative lengthening of telomeres (ALT).14 Inheriting an increased number of glioma risk alleles might be expected to decrease age at diagnosis among glioma patients by reducing the number of somatic mutations an individual must acquire to initiate tumor formation or by facilitating the accumulation of such mutations. To investigate this, we examined the associations of known glioma risk loci with age at diagnosis in glioma patients from the University of California, San Francisco (UCSF) and the Mayo Clinic. Caucasian glioma patients were genotyped at single nucleotide polymorphisms (SNPs) in 7 genes associated with glioma risk in previous genome-wide association studies, including variants in TERT, EGFR, CCDC26, CDKN2B/ANRIL, PHLDB1, TP53, and RTEL1. Associations were also calculated within histologic subgroups, stratified by tumor IDH­-mutation status, and pooled across study sites. Interactions between age and risk SNPs were also examined in case-control comparisons.

Geomorphological classification of the Fujian province was done based on remote-sensing imaging, digital elevation maps, and slope gradient data acquired by ArcGIS 9.2. The engineering geological units in the Fujian province were divided into five types by the geomorphologic shape and genesis. The relationship among geomorphological type, engineering geological unit, and site category was determined using engineering geological data and site category data. Then, after making a preliminary site classification adjusted by drill hole data, the site was classified into four types: I0, I1, II, and III according to site classification standards of equivalent shear-wave velocity and overburden thickness. The results showed that the site categories in the Fujian province mainly consist of type II, which accounts for 85.26 % of the land area. The percentage of I0 was the smallest, which accounts for only 2.44 % of the total area.

We and others first reported several inherited variants that increase glioma risk in 2009.1,2 Additional confirmation and new risk loci have been identified since then.3–8 We9 and Simon et al.10 showed that the glioma risk loci in 8q24 and 11q23 first identified by Shete et al.1 were associated with risk of lower grade infiltrating gliomas but not with glioblastomas. It is now well established that mutation in the isocitrate dehydrogenase (IDH) 1 and 2 genes leads to genome-wide histone and DNA methylation changes that result in abnormal gene expression and gliomagenesis, defining a distinct subclass of gliomas.11,12 IDH mutations occur in ∼50%–80% of grade II-III gliomas and secondary glioblastomas but in fewer than 10% of primary glioblastomas.13–17 Tumor IDH mutations are associated with younger age of onset and better overall survival among patients with gliomas of all grades and histologies18,19 and are also associated with other somatic, genetic, and epigenetic alterations.17,20 Given these observations, we hypothesized that the 8q24 and 11q23 glioma risk loci might be specific to IDH-mutated, but not to IDH-wild-type gliomas. In a separate recent publication,21 we showed that the 8q24 locus is specifically associated with risk for oligodendroglial tumors and IDH-mutated astrocytomas of all grades. Here, we report that the 11q23 glioma risk variant rs498872 T allele is associated with risk of IDH-mutated gliomas but not with risk of IDH-wild-type gliomas, regardless of grade or histology.

Genomewide association studies (GWAS) and candidate-gene studies have implicated single-nucleotide polymorphisms (SNPs) in at least 45 different genes as putative glioma risk factors. Attempts to validate these associations have yielded variable results and few genetic risk factors have been consistently replicated. We conducted a case-control study of Caucasian glioma cases and controls from the University of California San Francisco (810 cases, 512 controls) and the Mayo Clinic (852 cases, 789 controls) in an attempt to replicate previously reported genetic risk factors for glioma. Sixty SNPs selected from the literature (eight from GWAS and 52 from candidate-gene studies) were successfully genotyped on an Illumina custom genotyping panel. Eight SNPs in/near seven different genes (TERT, EGFR, CCDC26, CDKN2A, PHLDB1, RTEL1, TP53) were significantly associated with glioma risk in the combined dataset (P < 0.05), with all associations in the same direction as in previous reports. Several SNP associations showed considerable differences across histologic subtype. All eight successfully replicated associations were first identified by GWAS, although none of the putative risk SNPs from candidate-gene studies was associated in the full case-control sample (all P values > 0.05). Although several confirmed associations are located near genes long known to be involved in gliomagenesis (e.g., EGFR, CDKN2A, TP53), these associations were first discovered by the GWAS approach and are in noncoding regions. These results highlight that the deficiencies of the candidate-gene approach lay in selecting both appropriate genes and relevant SNPs within these genes.

Purpose: 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. Experimental Design: 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. Results: 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 to 17.4). Cases did not show differences in NKp46 demethylation based on tumor site or stage. Conclusions: 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. Clin Cancer Res; 18(22); 6147–54. ©2012 AACR.

The epigenetic changes during B-cell development relevant to both normal function and hematologic malignancy are incompletely understood. We examined DNA methylation and RNA expression status during early B-cell development by sorting multiple replicates of four separate stages of pre-B cells derived from normal human fetal bone marrow and applied high-dimension DNA methylation scanning and expression arrays. Features of promoter and gene body DNA methylation were strongly correlated with RNA expression in multipotent progenitors (MPPs) both in a static state and throughout differentiation. As MPPs commit to pre-B cells, a predominantly demethylating phenotype ensues, with 79% of the 2966 differentially methylated regions observed involving demethylation. Demethylation events were more often gene body associated rather than promoter associated; predominantly located outside of CpG islands; and closely associated with EBF1, E2F, PAX5 and other functional transcription factor (TF) sites related to B-cell development. Such demethylation events were accompanied by TF occupancy. After commitment, DNA methylation changes appeared to play a smaller role in B-cell development. We identified a distinct development-dependent demethylation signature which has gene expression regulatory properties for pre-B cells, and provide a catalog reference for the epigenetic changes that occur in pre-B-cell leukemia and other B-cell-related diseases.

Variants at 8q24.21 have been shown to be associated with glioma development. By means of tag SNP genotyping and imputation, pooled next-generation sequencing using long-range PCR and subsequent validation SNP genotyping, we identified seven low-frequency SNPs at 8q24.21 that were strongly associated with glioma risk (P=1×10(-25) to 1×10(-14)). The most strongly associated SNP, rs55705857, remained highly significant after individual adjustment for the other top six SNPs and two previously published SNPs. After stratifying by histological and tumor genetic subtype, the most significant associations of rs55705857 were with oligodendroglial tumors and gliomas with mutant IDH1 or IDH2 (odds ratio (OR)=5.1, P=1.1×10(-31) and OR=4.8, P=6.6×10(-22), respectively). Strong associations were observed for astrocytomas with mutated IDH1 or IDH2 (grades 2-4) (OR=5.16-6.66, P=4.7×10(-12) to 2.2×10(-8)) but not for astrocytomas with wild-type IDH1 and IDH2 (smallest P=0.26). The conserved sequence block that includes rs55705857 is consistently modeled as a microRNA.

The signaling pathways that underlie the pathogenesis of pediatric gliomas are poorly understood. We characterized the PI3K/Akt/mTOR pathway in pediatric gliomas of all grades. Using immunohistochemistry, we assessed activation of the PI3K/Akt/mTOR pathway by evaluating the downstream signaling molecules phospho(p)-S6, phospho(p)-4BP1, and phospho(p)-PRAS40; PTEN; and PTEN promoter methylation, as well as the MIB labeling index. We correlated these findings with the clinical outcomes of 48 children with gliomas. Eighty percent of high-grade gliomas (12/15) showed activation of the PI3K/Akt/mTOR pathway based on p-S6 and p-4EBP1 expression. The majority of high-grade gliomas were negative for PTEN expression (10/15), and 50% had PTEN promoter methylation (grade III: 2/4; grade IV: 3/6). Low-grade gliomas demonstrated PI3K/Akt/mTOR pathway activation in 14/32 (43.8%) by p-S6 and 16/32 (50%) by p-4EBP1. Over 50% of grade I (6/11) and almost all grade II tumors (6/7) showed PTEN promoter methylation. Tumor grade correlated negatively with PTEN expression and positively with expression of p-S6 and p-4EBP1 (PTEN: P =.0025; pS6: P =.0075; p-4EBP1: P =.0066). There was a trend toward inverse correlation of methylation of the PTEN promoter with expression of PTEN protein (P =.0990) and direct correlation of expression of p-S6 and p-4EBP1 with poorer clinical outcome, as measured by progression-free survival (p-S6: P =.0874; p-4EBP1: P =.0475). Tumors with no PTEN expression had a higher MIB labeling index (P =.007). The majority of pediatric gliomas show activation of the PI3K/Akt/mTOR pathway, with methylation of the PTEN promoter occurring commonly in these tumors. © The Author(s) 2012.

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.

BACKGROUND 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. METHODS 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. RESULTS 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). CONCLUSION 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.

There is increasing evidence that miRNA and transcription factors interact in an instructive fashion in normal and malignant hematopoiesis. We explored the impact of TEL-AML1 (ETV6-RUNX1), the most common fusion protein in childhood leukemia, on miRNA expression and the leukemic phenotype. Using RNA interference, miRNA expression arrays, and quantitative polymerase chain reaction, we identified miRNA-494 and miRNA-320a to be up-regulated upon TEL-AML1 silencing independently of TEL expression. Chromatin immunoprecipitation analysis identified miRNA-494 as a direct miRNA target of the fusion protein TEL-AML1. Using bioinformatic analysis as well as functional luciferase experiments, we demonstrate that survivin is a target of the 2 miRNAs. miRNA-494 and miRNA-320a were introduced to the cells by transfection and survivin expression determined by Western blot analysis. These miRNAs blocked survivin expression and resulted in apoptosis in a similar manner as TEL-AML1 silencing by itself; this silencing was also shown to be Dicer-dependent. miRNAs-494 and -320a are expressed at lower levels in TEL-AML1+ leukemias compared with immunophenotype-matched nonTEL-AML1 acute lymphoblastic leukemia subtypes, and within TEL-AML1+ leukemias their expression is correlated to survivin levels. In summary our data suggest that TEL-AML1 might exert its antiapoptotic action at least in part by suppressing miRNA-494 and miRNA-320a, lowering their expression causing enhanced survivin expression.

Mechanisms of action of nonmutagenic carcinogens such as asbestos remain poorly characterized. As pleural mesothelioma is known to have limited numbers of genetic mutations, we aimed to characterize the relationships among gene-locus–specific methylation alterations, disease status, asbestos burden, and survival in this rapidly fatal asbestos-associated tumor. Methylation of 1505 CpG loci associated with 803 cancer-related genes were studied in 158 pleural mesotheliomas and 18 normal pleura. After false-discovery rate correction, 969 CpG loci were independently associated with disease status (Q < 0.05). Classifying samples based on CpG methylation profile with a mixture model approach, methylation classes discriminated tumor from normal pleura (permutation P < 0.0001). In a random forests classification, the overall misclassification error rate was 3.4%, with

With 2.3 million breast cancer survivors in the US today, identification of modifiable factors associated with breast cancer recurrence and survival is increasingly important. Only recently new studies have been designed to examine the impact of lifestyle factors on prognosis, including Pathways, a prospective study of women with breast cancer in Kaiser Permanente Northern California (KPNC). Pathways aims to examine the effect on recurrence and survival of (1) lifestyle factors such as diet, physical activity, quality of life, and use of alternative therapies and (2) molecular factors such as genetic polymorphisms involved in metabolism of chemotherapeutic agents. Eligibility includes any woman diagnosed with invasive breast cancer within KPNC, no previous diagnosis of other invasive cancer, age 21 years or older, and ability to speak English, Spanish, Cantonese, or Mandarin. Newly diagnosed patients are identified daily from electronic pathology records and are enrolled within two months of diagnosis. An extensive baseline interview is conducted, blood and saliva samples are collected, and body measurements are taken. Women are followed for lifestyle updates, treatment, and outcomes by self-report and query of KPNC databases. Recruitment began in 9 January, 2006, and as of 16 January, 2008, 1,539 women have been enrolled along with collection of 1,323 blood samples (86%) and 1,398 saliva samples (91%). The Pathways Study will become a rich resource to examine behavioral and molecular factors and breast cancer prognosis.

Histone H3 lysine 9 trimethylation (H3K9Me3) has been associated with transcriptional repression, but recent findings implicate this chromatin modification in transcriptional activation and mRNA elongation by RNA polymerase II. Here, we applied immunoprecipitation (IP) with a custom DNA tiling microarray containing many transcription factors important in development and cancer (for example homeotic genes; N=683 total genes) to explore the relationship between H3K9Me3 and other histone modifications with the differential expression of genes. Cancer cell lines derived from different tissues (2 leukemia, 2 medulloblastoma) were characterized with IP antibodies to H3K9Me3, H3K4 dimethylation (H3K4Me2) and H3K9 acetylation (H3K9Ac). MV4-11 is known to overexpress the HOXA9 and MEIS1 genes, whereas D283 overexpresses the OTX2 homeobox gene. Gene expression was assessed by Affymetrix U133 array. Mapping the number and size of histone markings demonstrated significant colocalization of H3K9Ac and H3K4Me2 with H3K9Me3, indicating a pattern of putative 'activating' and 'repressive' markings. The median site size was 600-821 bp and 72-95% or 53-80% of chromatin signal sites were located within 1 kb or 500 bp of transcription start sites (TSS), respectively. A relatively small number of genes displayed additional H3K9Me3 sites in the 5'-region distant from the TSS. Comparing genes with modification sites to those without sites in their promoters confirmed the positive associations of H3K9Ac and H3K4Me2 with gene expression and revealed that H3K9Me3 is associated with active genes rather than being a repressive marking as previously thought. The positive regulatory effect of all three types of modifications were quantitatively correlated with site size, and applied to absolute gene expression within a single cell line as well as relative expression among pairs of cell lines. Extended patterns of H3K9Me3 upstream of some genes (for example HOXA9 and OTX2) may result from the action of multiple promoter elements. We found an inverse relationship between promoter DNA hypermethylation and H3K9Me3 in three studied genes (HOXA9, TMS1, RASSF1A). The localization of H3K9Me3 downstream of the TSSs of expressed genes and not within promoter regions of hypermethylated and silenced genes is consistent with the proposed coupling of H3K9Me3 with RNA polymerase II. Our results indicate a need for revising aspects of the histone code involving H3 lysine methylation. Awareness of H3K9Me3 as a mark of gene activity, not repression, is especially important for the classification of human cancer using chromatin and histone profiles.

The tumor suppressor PTEN encodes a lipid phosphatase that negatively regulates the phosphatidylinositol 3-kinase/AKT cell survival pathway. Mutations of this gene are common in brain, prostate, endometrial, and gastric cancers but occur rarely in non-small-cell lung cancer (NSCLC), although the PTEN protein is often lost in lung tumors. We have studied hypermethylation of the PTEN promoter, loss of heterozygosity (LOH) at microsatellites in chromosome 10q23 (surrounding and intragenic to the PTEN locus), and hypermethylation of PTEN's highly homologous pseudogene, PTENP1, and their association with PTEN protein loss in a surgical case series study of primary NSCLC. PTEN protein expression was reduced or lost in 74% (86/117) of tumors, with loss occurring more often in well to moderately differentiated tumors. In squamous cell carcinomas, PTEN loss occurred significantly more often in early-stage (stage I or II) disease. PTEN protein loss also occurred more frequently in tumors with low to no aberrant TP53 staining. Methylation of PTEN occurred in 26% (39/151) of tumors, and LOH at 10q23 was rare, occurring in only 19% (17/90) of informative tumors. Neither methylation nor LOH was a significant predictor of PTEN protein expression, although LOH occurred exclusively in early-stage disease. In NSCLC, loss of PTEN protein expression occurs frequently, although the mechanism responsible for loss is not clearly attributable to deletion or epigenetic silencing. PTEN loss may also be a favorable prognostic marker, although further studies are needed to confirm this finding.

We investigated the role of glutathione S-transferase (GST) enzymes (M1, T1), methylenetetrahydrofolate (MTHFR) 677 and 1298, and the NAD(P)H:quinone oxidoreductase (NQO1) polymorphisms in a population-based bladder cancer case – control study in Argentina. Buccal cell DNA was obtained from 106 cases and 109 controls. The strongest evidence was for an interaction between NQO1 genotype and smoking. For ever smoking vs. never smoking the odds ratio was 8.6 (95% confidence interval (CI) 2.7 – 27), in the CC genotype, and 1.3 (95% CI 0.5– 3.5) in the CT and TT genotypes combined. Also, elevated bladder cancer risks associated with GSTM1 and GSTT1 null genotypes were found in smokers. Having both null polymorphisms conferred the highest risks. The MTHFR 677 CT and TT polymorphisms appeared protective against bladder cancer.

The human FHIT (fragile histidine triad) gene is a putative tumor suppressor gene located at chromosome region 3p14.2. Previous studies have shown that loss of heterozygosity, homozygous deletions, and abnormal expression of the FHIT gene are involved in several types of human malignancies. A CpG island is present in the 5′ promoter region of the FHIT gene, and methylation in this region correlates with loss of FHIT expression. To test whether aberrant methylation of the FHIT gene may play a role in pediatric leukemia, we assessed the FHIT methylation status of 10 leukemia cell lines and 190 incident population-based cases of childhood acute lymphocytic and myeloid leukemias using methylation-specific PCR. Conventional and fluorescence in situ hybridization cytogenetic data were also collected to examine aneuploidy, t(12, 21), and other chromosomal rearrangements. Four of 10 leukemia cell lines (40%) and 52 of 190 (27.4%) bone marrows from childhood leukemia patients demonstrated hypermethylation of the promoter region of FHIT. Gene expression analyses and 5-aza-2′-deoxycytidine treatment showed that promoter hypermethylation correlated with FHIT inactivation. Among primary leukemias, hypermethylation of FHIT was strongly correlated with acute lymphoblastic leukemia (ALL) histology (P = 0.008), high hyperdiploid (P < 0.0001), and translocation-negative (P < 0.0001) categories. Hyperdiploid B-cell ALLs were 23-fold more likely to be FHIT methylated compared with B-cell ALL harboring TEL-AML translocations. FHIT methylation was associated with high WBC counts at diagnosis, a known prognostic indicator. These results suggest that hypermethylation of the promoter region CpG island of the FHIT gene is a common event and may play an important role in the etiology and pathophysiology of specific cytogenetic subtypes of childhood ALL.

Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.

BACKGROUND: In 2012, using a genome-wide association study (GWAS) of UCSF Adult Glioma Study patients and replication in patients from the Mayo Clinic, TCGA and GliomaSE, we showed that a SNP in SSBP2 on chromosome 5 was associated with survival of glioblastoma (GBM) patients who received standard of care (SOC). Since then we have examined 41 SNPs that were potentially associated with survival after glioma diagnosis based on our preliminary results or published results of others. METHODS: The data set consisted of 401 patients with grade 2 or grade 3 astrocytic, oligodendroglial, or mixed oligoastrocytic tumors and 339 GBM patients not included in the previous GWAS. RESULTS: The SSBP2 minor allele was again associated with poorer survival among GBM patients who received SOC, but interestingly with improved survival among Grade 2 and Grade 3 patients. SNPs in another region of chromosome 5 were associated with GBM survival irrespective of whether or not the patients had SOC. Several other SNPs were significantly (p < 0.05) associated with survival of GBM or lower grade glioma. The results are currently being replicated in a similar sized group of patients from the Mayo Clinic. CONCLUSIONS: Inherited germline variation may influence glioma survival. SECONDARY CATEGORY: n/a.

E2A-PBX1 is expressed as a result of the t(1;19) chromosomal translocation in nearly 5% of cases of childhood acute lymphoblastic leukemia. The E2A-PBX1 chimeric transcription factor contains the N-terminal transactivation domain of E2A (TCF3) fused to the C-terminal DNA-binding homeodomain of PBX1. While there is no doubt of its oncogenic potential, the mechanisms of E2A-PBX1-mediated pre-B cell transformation and the nature of direct E2A-PBX1 target genes and pathways remain largely unknown. Herein we used chromatin immunoprecipitation assays (ChIP-chip) to identify direct targets of E2A-PBX1, and we used gene expression arrays of siRNA E2A-PBX1-silenced cells to evaluate changes in expression induced by the fusion protein. Combined ChIP-chip and expression data analysis gave rise to direct and functional targets of E2A-PBX1. Further we observe that the set of ChIP-chip identified E2A-PBX1 targets show a collective down-regulation trend in the E2A-PBX1 silenced samples compared to controls suggesting an activating role of this fusion transcription factor. Our data suggest that the expression of the E2A-PBX1 fusion gene interferes with key regulatory pathways and functions of hematopoietic biology. Among these are members of the WNT and apoptosis/cell cycle control pathways, and thus may comprise an essential driving force for the propagation and maintenance of the leukemic phenotype. These findings may also provide evidence of potentially attractive therapeutic targets.

Buccal cells are increasingly used as a source of quality DNA to improve participation rates in molecular studies. Here, three buccal cell collection protocols were compared to determine factors affecting the yield of cells, total DNA per sample, and DNA yield per cell. In addition, kinetic quantitative polymerase chain reaction (PCR) (TaqMan™) was used to quantify human DNA available for PCR. The method of collection used influenced the overall DNA yield per sample. The collection buffer used influenced the number of cells but not the overall DNA yield per sample. Repeated freezing and thawing did not affect overall DNA yield per sample, DNA yield per cell, or the total number of cells collected. Mouthwashes had the highest DNA yield per sample (20.8 μg) compared with cytobrush samples (1.9 μg from three cytobrushes) and tongue depressors (0.8 μg from three tongue depressors). However, mouthwash samples may contain significant non-human DNA and other contaminants that could interfere with some molecular studies. Spectrometry grossly overestimated the total DNA recovered from mouthwash samples compared with fluorometry or quantitative PCR.

p14(ARF) is a putative tumor suppressor gene thought to modify the levels of p53. CpG sites within the 5'-flanking region and exon 1beta of p14(ARF) are targets of aberrant methylation and transcriptional silencing in human colorectal cancer (CRC). Here we have developed methylation-specific polymerase chain reaction (MSPCR) methods to detect methylation of CpG sites in p14(ARF) in CRC cell lines and primary CRC tumors, and correlated p14(ARF) mRNA expression with methylation in CRC cell lines using competitive quantitative reverse transcription-polymerase chain reaction methods. Ten CRC cell lines were studied; three (DLD-1, HCT15 and SW48) showed extensive methylation and six (Colo320, SW480, HT29, Caco2, SW837 and WiDr) were unmethylated; the other cell line, LoVo, showed partial methylation that affected exon 1beta but not the immediate upstream CpG sites. p14(ARF) mRNA was expressed at extremely low levels in fully methylated cell lines and at 10(4)- to 10(5)-fold higher levels in unmethylated cell lines. p14(ARF) expression in the partially methylated LoVo cell line was intermediate. Treatment of LoVo cells with 2 microM 5-aza-2'-deoxycytidine for 72 h was associated with marked (100-fold) induction of mRNA levels. Of 119 primary CRCs, 18% contained p14(ARF) methylation, although partial methylation was the most common pattern observed (in 67% of methylated tumors). Methylation of p14(ARF) was often accompanied by p16(INK4a) methylation; however, 50% of p14(ARF) methylated tumors contained unmethylated p16(INK4a). Methylation at p14(ARF) was associated with female gender, greater age, proximal anatomic location and poor differentiation, but not stage at diagnosis. A two-step MSPCR method for assaying p14(ARF) methylation in human tumors is described.

Normal tissues express the M1 isoform of pyruvate kinase (PK) that helps generate and funnel pyruvate into the mitochondria for ATP production. Tumors, in contrast, express the less active PKM2 isoform, which limits pyruvate production and spares glycolytic intermediates for the generation of macromolecules needed for proliferation. Although high PKM2 expression and low PK activity are considered defining features of tumors, very little is known about how PKM expression and PK activity change along the continuum from low grade to high grade tumors, and how these changes relate to tumor growth. To address this issue, we measured PKM isoform expression and PK activity in normal brain, neural progenitor cells, and in a series of over 100 astrocytomas ranging from benign grade I pilocytic astrocytomas to highly aggressive grade IV glioblastoma multiforme (GBM). All glioma exhibited comparably reduced levels of PKM1 expression and PK activity relative to normal brain. In contrast, while grade I-III gliomas all had modestly increased levels of PKM2 RNA and protein expression relative to normal brain, GBM, regardless of whether they arose de novo or progressed from lower grade tumors, showed a 3-5 fold further increase in PKM2 RNA and protein expression. Low levels of PKM1 expression and PK activity were important for cell growth as PKM1 over-expression and the accompanying increases in PK activity slowed the growth of GBM cells. The increased expression of PKM2, however, was also important, because shRNA-mediated PKM2 knockdown decreased total PKM2 and the already low levels of PK activity, but paradoxically also limited cell growth in vitro and in vivo. These results show that pyruvate kinase M expression, but not pyruvate kinase activity, is regulated in a grade-specific manner in glioma, but that changes in both PK activity and PKM2 expression contribute to growth of GBM.

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.

Leishmania major are intramacrophage parasites whose eradication requires the induction of T helper 1 (Th1) effector cells capable of activating macrophages to a microbicidal state. Interleukin 12 (IL-12) has been recently identified as a macrophage-derived cytokine capable of mediating Th1 effector cell development, and of markedly enhancing interferon gamma (IFN-gamma) production by T cells and natural killer cells. Infection of macrophages in vitro by promastigotes of L. major caused no induction of IL-12 p40 transcripts, whereas stimulation using heat-killed Listeria or bacterial lipopolysaccharide induced readily detectable IL-12 mRNA. Using a competitor construct to quantitate a number of transcripts, a kinetic analysis of cytokine induction during the first few days of infection by L. major was performed. All strains of mice examined, including susceptible BALB/c and resistant C57BL/6, B10.D2, and C3H/HeN, had the appearance of a CD4+ population in the draining lymph nodes that contained transcripts for IL-2, IL-4, and IFN-gamma (and in some cases, IL-10) that peaked 4 d after infection. In resistant mice, the transcripts for IL-2, IL-4, and IL-10 were subsequently downregulated, whereas in susceptible BALB/c mice, these transcripts were only slightly decreased, and IL-4 continued to be reexpressed at high levels. IL-12 transcripts were first detected in vivo by 7 d after infection, consistent with induction by intracellular amastigotes. Challenge of macrophages in vitro confirmed that amastigotes, in contrast to promastigotes, induced IL-12 p40 mRNA. Reexamination of the cytokine mRNA at 4 d revealed expression of IL-13 in all strains analyzed, suggesting that IL-2 and IL-13 may mediate the IL-12-independent production of IFN-gamma during the first days after infection. Leishmania have evolved to avoid inducing IL-12 from host macrophages during transmission from the insect vector, and cause a striking induction of mRNAs for IL-2, IL-4, IL-10, and IL-13 in CD4+ T cells. Each of these activities may favor survival of the organism.

Acute lymphoblastic leukemia (ALL) likely has a multistep etiology, with initial genetic aberrations occurring early in life. An abnormal immune response to common infections has emerged as a plausible candidate for triggering the proliferation of pre-leukemic clones and the fixation of secondary genetic mutations and epigenetic alterations. We investigated whether evidence of infection with a specific common myelotropic childhood virus, parvovirus B19 (PVB19), relates to patterns of gene promoter DNA methylation in ALL patients. We serologically tested bone marrow samples at diagnosis of B-cell ALL for PVB19 infection and DNA methylation using a high-throughput bead array and found that 4.2% and 36.7% of samples were seroreactive to PVB19 IgM and IgG, respectively. Leukemia samples were grouped by DNA methylation pattern. Controlling for age and immunophenotype, unsupervised modeling confirmed that the DNA methylation pattern was associated with history of PVB19 (assessed by IgG, p = 0.02), but not recent infection (assessed by IgM). Replication assays on single genes were consistent with the association. The data indicate that a common viral illness may drive specific DNA methylation patterns in susceptible B-precursor cells, contributing to the leukemogenic potential of such cells. Infections may impact childhood leukemia by altering DNA methylation patterns and specific key genes in susceptible cells; these changes may be retained even after the clearance of infection.

Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively). We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245) through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment.

Recent evidence suggests the Akt-mTOR pathway may play a role in development of low-grade gliomas (LGG). We sought to evaluate whether activation of this pathway correlates with survival in LGG by examining expression patterns of proteins within this pathway. Forty-five LGG tumor specimens from newly diagnosed patients were analyzed for methylation of the putative 5′-promoter region of PTEN using methylation-specific PCR as well as phosphorylation of S6 and PRAS40 and expression of PTEN protein using immunohistochemistry. Relationships between molecular markers and overall survival (OS) were assessed using Kaplan-Meier methods and exact log-rank test. Correlation between molecular markers was determined using the Mann-Whitney U and Spearman Rank Correlation tests. Eight of the 26 patients with methylated PTEN died, as compared to 1 of 19 without methylation. There was a trend towards statistical significance, with PTEN methylated patients having decreased survival (P = 0.128). Eight of 29 patients that expressed phospho-S6 died, whereas all 9 patients lacking p-S6 expression were alive at last follow-up. There was an inverse relationship between expression of phospho-S6 and survival (P = 0.029). There was a trend towards decreased survival in patients expressing phospho-PRAS40 (P = 0.077). Analyses of relationships between molecular markers demonstrated a statistically significant positive correlation between expression of p-S6(235) and p-PRAS40 (P = 0.04); expression of p-S6(240) correlated positively with PTEN methylation (P = 0.04) and negatively with PTEN expression (P = 0.03). Survival of LGG patients correlates with phosphorylation of S6 protein. This relationship supports the use of selective mTOR inhibitors in the treatment of low grade glioma.