Karl T. Kelsey

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.

TOXICITY TESTING IN THE 21ST CENTURY: A VISION AND A STRATEGY

With the release of the landmark report Toxicity Testing in the 21st Century: A Vision and a Strategy, the U.S. National Academy of Sciences, in 2007, precipitated a major change in the way toxicity testing is conducted. It envisions increased efficiency in toxicity testing and decreased animal usage by transitioning from current expensive and lengthy in vivo testing with qualitative endpoints to in vitro toxicity pathway assays on human cells or cell lines using robotic high-throughput screening with mechanistic quantitative parameters. Risk assessment in the exposed human population would focus on avoiding significant perturbations in these toxicity pathways. Computational systems biology models would be implemented to determine the dose-response models of perturbations of pathway function. Extrapolation of in vitro results to in vivo human blood and tissue concentrations would be based on pharmacokinetic models for the given exposure condition. This practice would enhance human relevance of test results, and would cover several test agents, compared to traditional toxicological testing strategies. As all the tools that are necessary to implement the vision are currently available or in an advanced stage of development, the key prerequisites to achieving this paradigm shift are a commitment to change in the scientific community, which could be facilitated by a broad discussion of the vision, and obtaining necessary resources to enhance current knowledge of pathway perturbations and pathway assays in humans and to implement computational systems biology models. Implementation of these strategies would result in a new toxicity testing paradigm firmly based on human biology.

Aging and environmental exposures alter tissue-specific DNA methylation dependent upon CPG island context

Epigenetic control of gene transcription is critical for normal human development and cellular differentiation. While alterations of epigenetic marks such as DNA methylation have been linked to cancers and many other human diseases, interindividual epigenetic variations in normal tissues due to aging, environmental factors, or innate susceptibility are poorly characterized. The plasticity, tissue-specific nature, and variability of gene expression are related to epigenomic states that vary across individuals. Thus, population-based investigations are needed to further our understanding of the fundamental dynamics of normal individual epigenomes. We analyzed 217 non-pathologic human tissues from 10 anatomic sites at 1,413 autosomal CpG loci associated with 773 genes to investigate tissue-specific differences in DNA methylation and to discern how aging and exposures contribute to normal variation in methylation. Methylation profile classes derived from unsupervised modeling were significantly associated with age (P<0.0001) and were significant predictors of tissue origin (P<0.0001). In solid tissues (n = 119) we found striking, highly significant CpG island–dependent correlations between age and methylation; loci in CpG islands gained methylation with age, loci not in CpG islands lost methylation with age (P<0.001), and this pattern was consistent across tissues and in an analysis of blood-derived DNA. Our data clearly demonstrate age- and exposure-related differences in tissue-specific methylation and significant age-associated methylation patterns which are CpG island context-dependent. This work provides novel insight into the role of aging and the environment in susceptibility to diseases such as cancer and critically informs the field of epigenomics by providing evidence of epigenetic dysregulation by age-related methylation alterations. Collectively we reveal key issues to consider both in the construction of reference and disease-related epigenomes and in the interpretation of potentially pathologically important alterations.

Interaction between tobacco and alcohol use and the risk of head and neck cancer: Pooled analysis in the international head and neck cancer Epidemiology consortium

Background: The magnitude of risk conferred by the interaction between tobacco and alcohol use on the risk of head and neck cancers is not clear because studies have used various methods to quantify the excess head and neck cancer burden. Methods: We analyzed individual-level pooled data from 17 European and American case-control studies (11,221 cases and 16,168 controls) participating in the International Head and Neck Cancer Epidemiology consortium. We estimated the multiplicative interaction parameter (ψ) and population attributable risks (PAR). Results: A greater than multiplicative joint effect between ever tobacco and alcohol use was observed for head and neck cancer risk (ψ = 2.15; 95% confidence interval, 1.53-3.04). The PAR for tobacco or alcohol was 72% (95% confidence interval, 61-79%) for head and neck cancer, of which 4% was due to alcohol alone, 33% was due to tobacco alone, and 35% was due to tobacco and alcohol combined. The total PAR differed by subsite (64% for oral cavity cancer, 72% for pharyngeal cancer, 89% for laryngeal cancer), by sex (74% for men, 57% for women), by age (33% for cases <45 years, 73% for cases >60 years), and by region (84% in Europe, 51% in North America, 83% in Latin America). Conclusions: Our results confirm that the joint effect between tobacco and alcohol use is greater than multiplicative on head and neck cancer risk. However, a substantial proportion of head and neck cancers cannot be attributed to tobacco or alcohol use, particularly for oral cavity cancer and for head and neck cancer among women and among young-onset cases. (Cancer Epidemiol Biomarkers Prev 2009;18(2):541–50)

MicroRNA Responses to Cellular Stress

Recent work has begun to explore the instrumental role that small noncoding RNA species, particularly microRNAs (miRNA), have both in classifying human tumors and in directing embryonic development. These studies suggest that developmental programs in essentially all organisms studied are set, in part, by varied expressions of miRNAs and that neoplasia is characterized by altered expression of miRNAs. Reasoning that these observations are linked, we examined whether cellular exposures that induce both developmental anomalies and cancer alter miRNAs. Using microarrays of 385 known human miRNAs, we studied human lymphoblastoid cells grown under various conditions or treatments. Folate deficiency induced a pronounced global increase in miRNA expression. We observed no significant alteration in miRNA expression in cells treated with gamma-irradiation, whereas exposure to sodium arsenite led to global increases in miRNA expression. The miRNA hsa-miR-222 was identified from these arrays as significantly overexpressed under folate-deficient conditions, and this finding was confirmed in vivo in human peripheral blood from individuals with low folate intake. Alterations to cellular miRNA expression profiles represent a novel mode of action of folate deprivation and arsenic exposure, and specific alterations in miRNA expression may be a powerful biomarker for these and other toxins with serious effects on human health.

Fundamental Differences in Cell Cycle Deregulation in Human Papillomavirus–Positive and Human Papillomavirus–Negative Head/Neck and Cervical Cancers

Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, cervical cancers, and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV(+) HNCs and cervical cancers differed in their patterns of gene expression yet shared many changes compared with HPV(-) HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV(+) HNCs and cervical cancers were found to be up-regulated in their expression of a distinct and larger subset of cell cycle genes than that observed in HPV(-) HNC. Moreover, HPV(+) cancers overexpressed testis-specific genes that are normally expressed only in meiotic cells. Many, although not all, of the hallmark differences between HPV(+) HNC and HPV(-) HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testis-specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV(+) and HPV(-) cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV(+) cancers.

Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is commonly associated with tobacco and alcohol exposures, although dietary factors, particularly folate, and human papillomavirus, are also risk factors. Epigenetic alterations are increasingly implicated in the initiation and progression of cancer. Genome-wide (global) hypomethylation seems to occur in early neoplasia and is a feature of genomic DNA derived from solid tumor tissues, including HNSCC. This study aimed to determine whether global methylation in DNA derived from whole blood, a proxy tissue, is associated with HNSCC and to assess potential modification of this property by environmental or behavioral risk factors. Methods: Global DNA methylation levels were assessed using a modified version of the combined bisulfite restriction analysis of the LRE1 sequence in a population-based case-control study of HNSCC from the Boston area. Results: Hypomethylation lead to a significant 1.6-fold increased risk for disease (95% confidence interval, 1.1-2.4), in models controlled for other HNSCC risk factors. Smoking showed a significant differential effect (P < 0.03) on blood relative methylation between cases and controls. Furthermore, in cases, variant genotype in the MTHFR gene and low folate intake showed relationships with decreased global methylation, whereas in controls, antibody response to human papillomavirus 16 was associated with an increased global methylation level. Discussion: DNA hypomethylation in nontarget tissue was independently associated with HNSCC and had a complex relationship with the known risk factors associated with the genesis of HNSCC. (Cancer Epidemiol Biomarkers Prev 2007;16(1):108–14)

Sexual behaviours and the risk of head and neck cancers: a pooled analysis in the International Head and Neck Cancer Epidemiology (INHANCE) consortium

BACKGROUND Sexual contact may be the means by which head and neck cancer patients are exposed to human papillomavirus (HPV). METHODS We undertook a pooled analysis of four population-based and four hospital-based case-control studies from the International Head and Neck Cancer Epidemiology (INHANCE) consortium, with participants from Argentina, Australia, Brazil, Canada, Cuba, India, Italy, Spain, Poland, Puerto Rico, Russia and the USA. The study included 5642 head and neck cancer cases and 6069 controls. We calculated odds ratios (ORs) of associations between cancer and specific sexual behaviours, including practice of oral sex, number of lifetime sexual partners and oral sex partners, age at sexual debut, a history of same-sex contact and a history of oral-anal contact. Findings were stratified by sex and disease subsite. RESULTS Cancer of the oropharynx was associated with having a history of six or more lifetime sexual partners [OR = 1.25, 95% confidence interval (CI) 1.01, 1.54] and four or more lifetime oral sex partners (OR = 2.25, 95% CI 1.42, 3.58). Cancer of the tonsil was associated with four or more lifetime oral sex partners (OR = 3.36, 95 % CI 1.32, 8.53), and, among men, with ever having oral sex (OR = 1.59, 95% CI 1.09, 2.33) and with an earlier age at sexual debut (OR = 2.36, 95% CI 1.37, 5.05). Cancer of the base of the tongue was associated with ever having oral sex among women (OR = 4.32, 95% CI 1.06, 17.6), having two sexual partners in comparison with only one (OR = 2.02, 95% CI 1.19, 3.46) and, among men, with a history of same-sex sexual contact (OR = 8.89, 95% CI 2.14, 36.8). CONCLUSIONS Sexual behaviours are associated with cancer risk at the head and neck cancer subsites that have previously been associated with HPV infection.

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.

Recommendations for the design and analysis of epigenome-wide association studies

Epigenome-wide association studies (EWAS) hold promise for the detection of new regulatory mechanisms that may be susceptible to modification by environmental and lifestyle factors affecting susceptibility to disease. Epigenome-wide screening methods cover an increasing number of CpG sites, but the complexity of the data poses a challenge to separating robust signals from noise. Appropriate study design, a detailed a priori analysis plan and validation of results are essential to minimize the danger of false positive results and contribute to a unified approach. Epigenome-wide mapping studies in homogenous cell populations will inform our understanding of normal variation in the methylome that is not associated with disease or aging. Here we review concepts for conducting a stringent and powerful EWAS, including the choice of analyzed tissue, sources of variability and systematic biases, outline analytical solutions to EWAS-specific problems and highlight caveats in interpretation of data generated from samples with cellular heterogeneity.