Heather H. Nelson

DNA methylation arrays as surrogate measures of cell mixture distribution

BackgroundThere has been a long-standing need in biomedical research for a method that quantifies the normally mixed composition of leukocytes beyond what is possible by simple histological or flow cytometric assessments. The latter is restricted by the labile nature of protein epitopes, requirements for cell processing, and timely cell analysis. In a diverse array of diseases and following numerous immune-toxic exposures, leukocyte composition will critically inform the underlying immuno-biology to most chronic medical conditions. Emerging research demonstrates that DNA methylation is responsible for cellular differentiation, and when measured in whole peripheral blood, serves to distinguish cancer cases from controls.ResultsHere we present a method, similar to regression calibration, for inferring changes in the distribution of white blood cells between different subpopulations (e.g. cases and controls) using DNA methylation signatures, in combination with a previously obtained external validation set consisting of signatures from purified leukocyte samples. We validate the fundamental idea in a cell mixture reconstruction experiment, then demonstrate our method on DNA methylation data sets from several studies, including data from a Head and Neck Squamous Cell Carcinoma (HNSCC) study and an ovarian cancer study. Our method produces results consistent with prior biological findings, thereby validating the approach.ConclusionsOur method, in combination with an appropriate external validation set, promises new opportunities for large-scale immunological studies of both disease states and noxious exposures.

New common variants affecting susceptibility to basal cell carcinoma

In a follow-up to our previously reported genome-wide association study of cutaneous basal cell carcinoma (BCC), we describe here several new susceptibility variants. SNP rs11170164, encoding a G138E substitution in the keratin 5 (KRT5) gene, affects risk of BCC (OR = 1.35, P = 2.1 × 10−9). A variant at 9p21 near CDKN2A and CDKN2B also confers susceptibility to BCC (rs2151280[C]; OR = 1.19, P = 6.9 × 10−9), as does rs157935[T] at 7q32 near the imprinted gene KLF14 (OR = 1.23, P = 5.7 × 10−10). The effect of rs157935[T] is dependent on the parental origin of the risk allele. None of these variants were found to be associated with melanoma or fair-pigmentation traits. A melanoma- and pigmentation-associated variant in the SLC45A2 gene, L374F, is associated with risk of both BCC and squamous cell carcinoma. Finally, we report conclusive evidence that rs401681[C] in the TERT-CLPTM1L locus confers susceptibility to BCC but protects against melanoma.

Characterization of Phthalate Exposure among Pregnant Women Assessed by Repeat Air and Urine Samples

Background Although urinary concentrations of phthalate metabolites are frequently used as biomarkers in epidemiologic studies, variability during pregnancy has not been characterized. Methods We measured phthalate metabolite concentrations in spot urine samples collected from 246 pregnant Dominican and African-American women. Twenty-eight women had repeat urine samples collected over a 6-week period. We also analyzed 48-hr personal air samples (n = 96 women) and repeated indoor air samples (n = 32 homes) for five phthalate diesters. Mixed-effects models were fit to evaluate reproducibility via intraclass correlation coefficients (ICC). We evaluated the sensitivity and specificity of using a single specimen versus repeat samples to classify a woman’s exposure in the low or high category. Results Phthalates were detected in 85–100% of air and urine samples. ICCs for the unadjusted urinary metabolite concentrations ranged from 0.30 for mono-ethyl phthalate to 0.66 for monobenzyl phthalate. For indoor air, ICCs ranged from 0.48 [di-2-ethylhexyl phthalate (DEHP)] to 0.83 [butylbenzyl phthalate (BBzP)]. Air levels of phthalate diesters correlated with their respective urinary metabolite concentrations for BBzP (r = 0.71), di-isobutyl phthalate (r = 0.44), and diethyl phthalate (DEP; r = 0.39). In women sampled late in pregnancy, specific gravity appeared to be more effective than creatinine in adjusting for urine dilution. Conclusions Urinary concentrations of DEP and DEHP metabolites in pregnant women showed lower reproducibility than metabolites for di-n-butyl phthalate and BBzP. A single indoor air sample may be sufficient to characterize phthalate exposure in the home, whereas urinary phthalate biomarkers should be sampled longitudinally during pregnancy to minimize exposure misclassification.

Inactivation of the Fanconi anemia/BRCA pathway in lung and oral cancers: implications for treatment and survival.

Inactivation of the FANC–BRCA pathway via promoter methylation of the FANCF gene renders cells sensitive to DNA crosslinking agents, and has been identified in ovarian cancer cell lines and sporadic primary tumor tissues. We investigated epigenetic alterations in the FANC–BRCA pathway in head and neck squamous cell carcinomas (HNSCC) and non-small-cell lung cancers (NSCLC) using methylation-specific PCR. Promoter methylation of FANCF occurred in 15% (13/89) of HNSCCs and 14% (22/158) of NSCLCs. Methylation of BRCA1 occurred only in 6/158 NSCLC, and was limited to adenocarcinomas and large-cell carcinomas of the lung. No methylation of BRCA2 was detected. FANCF methylation was associated with a shorter duration of tobacco use (P=0.03) and a younger age of starting smoking (P=0.06) in NSCLC, and with a greater number of years of alcohol drinking (P=0.02) in HNSCC. In adenocarcinomas of the lung, FANCF promoter methylation was a significant predictor of poor survival with a hazard ratio of 3.1 (95% CI 1.2–7.9). This study demonstrates that inactivation of the FANC–BRCA pathway is relatively common in solid tumors and may be related to tobacco and alcohol exposure and survival of these patients.

Patterns of gene promoter methylation in squamous cell cancer of the head and neck.

Promoter methylation is an important pathway in transcriptional silencing of known and candidate tumor suppressor genes in Head and Neck Squamous Cell Carcinoma (HNSCC). In order to study the association of tumor suppressor gene promoter methylation in HNSCC with patient clinical characteristics, especially alcohol consumption and tobacco smoking, we examined promoter methylation of the p16INK4a, DAP-kinase, E-Cadherin, and RASSF1A genes using methylation-specific PCR (MSP) in 80 patients. The prevalence of p16INK4a, DAP-kinase, E-Cadherin, and RASSF1A promoter methylation was 26/80 (32.5%), 19/80 (23.8%), 29/80 (36.3%), 6/80 (7.5%) respectively. In 48 cases (60%), at least one of these promoters was methylated. There was a significant association of methylation of any of these genes and ever smoking (P=0.006). p16INK4a gene promoter methylation was associated with a younger age of smoking initiation (P<0.03); E-Cadherin promoter methylation was associated with an increased number of pack years smoked (P<0.03). We also found an association of methylation of any gene and T status (OR=2.7, P<0.05). Tumors with p16INK4a methylation were significantly less likely to show lymph node metastasis (P<0.001). DAP-kinase promoter methylation was significantly associated with lymph node metastasis and this relationship was dependent upon p16INK4a promoter methylation status. Our results suggest that, in HNSCC, promoter methylation of these four genes accumulates with increasing tumor size. This may reflect distinct pathways of somatic inactivation leading to cancer; additional larger studies are needed to further investigate this possibility. Tobacco smoking may play an important role in both the occurrence of promoter methylation as well as delineating the precise pathway that eventually results in a tumorigenic phenotype.

DNA Methylation, Isocitrate Dehydrogenase Mutation, and Survival in Glioma

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.

Model-based clustering of DNA methylation array data: a recursive-partitioning algorithm for high-dimensional data arising as a mixture of beta distributions.

BackgroundEpigenetics is the study of heritable changes in gene function that cannot be explained by changes in DNA sequence. One of the most commonly studied epigenetic alterations is cytosine methylation, which is a well recognized mechanism of epigenetic gene silencing and often occurs at tumor suppressor gene loci in human cancer. Arrays are now being used to study DNA methylation at a large number of loci; for example, the Illumina GoldenGate platform assesses DNA methylation at 1505 loci associated with over 800 cancer-related genes. Model-based cluster analysis is often used to identify DNA methylation subgroups in data, but it is unclear how to cluster DNA methylation data from arrays in a scalable and reliable manner.ResultsWe propose a novel model-based recursive-partitioning algorithm to navigate clusters in a beta mixture model. We present simulations that show that the method is more reliable than competing nonparametric clustering approaches, and is at least as reliable as conventional mixture model methods. We also show that our proposed method is more computationally efficient than conventional mixture model approaches. We demonstrate our method on the normal tissue samples and show that the clusters are associated with tissue type as well as age.ConclusionOur proposed recursively-partitioned mixture model is an effective and computationally efficient method for clustering DNA methylation data.

Implications of LINE1 Methylation for Bladder Cancer Risk in Women

Purpose: Epigenetic alterations including changes to cellular DNA methylation levels contribute to carcinogenesis and may serve as powerful biomarkers of the disease. This investigation sought to determine whether hypomethylation at the long interspersed nuclear elements (LINE1), reflective of the level of global DNA methylation, in peripheral blood–derived DNA is associated with increased risk of bladder cancer. Experimental Design: LINE1 methylation was measured from blood-derived DNA obtained from participants of a population-based incident case-control study of bladder cancer in New Hampshire. Bisulfite-modified DNA was pyrosequenced to determine LINE1 methylation status; a total of 285 cases and 465 controls were evaluated for methylation. Results: Being in the lowest LINE1 methylation decile was associated with a 1.8-fold increased risk of bladder cancer [95% confidence interval (95% CI), 1.12-2.90] in models controlling for gender, age, and smoking, and the association was stronger in women than in men (odds ratio, 2.48; 95% CI, 1.19-5.17 in women; and odds ratio, 1.47; 95% CI, 0.79-2.74 in men). Among controls, women were more likely to have lower LINE1 methylation than men (P = 0.04), and levels of arsenic in the 90th percentile were associated with reduced LINE1 methylation (P = 0.04). Conclusions: LINE1 hypomethylation may be an important biomarker of bladder cancer risk, especially among women. Clin Cancer Res; 16(5); 1682–9

Promoter methylation of DAP-kinase: association with advanced stage in non-small cell lung cancer.

Death associated protein (DAP)-kinase is a 16 kDa calmodulin-dependent serine/threonine kinase that carries a death domain at its C-terminus. DAP-kinase functions as a positive mediator of apoptosis that is induced by interferon-γ. Recent studies suggest that DAP-kinase is involved in tumor metastasis and that it can be inactivated by methylation of CpG islands in the promoter region of the gene in some human tumors. However, little is known about the factors that are associated with the occurrence of DAP-kinase promoter methylation. We investigated both the possible associations of tobacco carcinogen and asbestos exposure with DAP-kinase promoter methylation, and the demographic and clinical factors associated with DAP-kinase promoter methylation in non-small cell lung cancer (NSCLC). One hundred and eighty-five patients diagnosed with NSCLC undergoing surgical resection from June, 1992 through December, 1996 at Massachusetts General Hospital participated in this study. Methylation-Specific PCR (MSP), performed using fresh-frozen tissue, was used to determine the methylation status of the promoter region of the DAP-kinase gene. Forty-seven (25%) of 185 tumors showed DAP-kinase promoter methylation. There was a significant association between methylation and an advanced pathologic stage (P=0.003, Fishers exact test). Methylation of the DAP-kinase promoter was also associated with an increase in tumor size (P=0.009, Fishers exact test) and lymph node involvement (P=0.04). No association was found between promoter methylation of DAP-kinase and k-ras or p53 mutation. In addition there was no association with a history of exposure to tobacco or asbestos. Controlling for age, sex, and histology, the odds ratios describing the association of DAP-kinase hypermethylation with stage were 2.70 (1.13–6.45), 3.11 (1.37–7.08) and 7.77 (1.21–50.03) in stages II, III and IV, respectively. Stage I cases with DAP-kinase promoter methylation had worse overall survival, but with the small sample size and limited follow-up this did not reach statistical significance. Our findings suggest that methylation of the promoter region of the DAP-kinase gene is not associated with exposure to tobacco or asbestos. However, they strongly suggest that DAP-kinase may be important in the progression of non-small cell lung cancer from early to late stage disease.

Human papillomavirus 16 and head and neck squamous cell carcinoma

Evidence suggests that human papillomavirus (HPV)16 seropositivity reflects past HPV16 exposure and is associated with risk for head and neck squamous cell carcinoma (HNSCC). Our objectives were to test the hypothesis that HPV16 seropositivity is associated with risk for HNSCC, to correlate HPV16 seropositivity with HPV16 tumor DNA, and to correlate HPV16 seropositivity and HPV16 DNA with sexual history and patient survival. In a case–control study of approximately 1,000 individuals, we assessed serology to the HPV16 L1 protein and in cases only, assayed tumors for HPV16 DNA. HPV16 seropositivity was associated with 1.5‐ and 6‐fold risks for tumors of the oral cavity and pharynx, respectively. There was a dose response trend for HPV16 titer and increasing risk of HNSCC (p < 0.0001) and HPV16 tumor DNA (p < 0.0001). In cases, HPV16 DNA and seropositivity were significantly associated with sexual activity; odds ratios (ORs) of 12.8 and 3.7 were observed for more than 10 oral sexual partners and ORs of 4.5 and 3.2 were associated with a high number of lifetime sexual partners, respectively. Finally, HPV16 seropositivity and HPV16 tumor DNA were associated with hazard ratios of 0.4 and 0.5, respectively, indicating better survival for HPV positive individuals. HPV16 seropositivity was associated with risk for HNSCC, with greatest risk for pharyngeal cancer. We observed dose response relationships between serology titer and both risk for HNSCC and HPV16 tumor DNA. In cases, HPV16 tumor DNA and positive serology were associated with sexual history and improved disease free survival.