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1.  Key epigenetic changes associated with lung cancer development 
Epigenetics  2012;7(6):559-566.
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.
PMCID: PMC3398985  PMID: 22522909
DNA Methylation; goldengate; lung cancer; molecular epidemiology; pyrosequencing
2.  Urinary Levels of N-Nitroso Compounds in Relation to Risk of Gastric Cancer: Findings from the Shanghai Cohort Study 
PLoS ONE  2015;10(2):e0117326.
N-Nitroso compounds are thought to play a significant role in the development of gastric cancer. Epidemiological data, however, are sparse in examining the associations between biomarkers of exposure to N-nitroso compounds and the risk of gastric cancer.
A nested case-control study within a prospective cohort of 18,244 middle-aged and older men in Shanghai, China, was conducted to examine the association between urinary level of N-nitroso compounds and risk of gastric cancer. Information on demographics, usual dietary intake, and use of alcohol and tobacco was collected through in-person interviews at enrollment. Urinary levels of nitrate, nitrite, N-nitroso-2-methylthiazolidine-4-carboxylic acid (NMTCA), N-nitrosoproline (NPRO), N-nitrososarcosine (NSAR), N-nitrosothiazolidine-4-carboxylic acid (NTCA), as well as serum H. pylori antibodies were quantified in 191 gastric cancer cases and 569 individually matched controls. Logistic regression method was used to assess the association between urinary levels of N-nitroso compounds and risk of gastric cancer.
Compared with controls, gastric cancer patients had overall comparable levels of urinary nitrate, nitrite, and N-nitroso compounds. Among individuals seronegative for antibodies to H. pylori, elevated levels of urinary nitrate were associated with increased risk of gastric cancer. The multivariate-adjusted odds ratios for the second and third tertiles of nitrate were 3.27 (95% confidence interval = 0.76–14.04) and 4.82 (95% confidence interval = 1.05–22.17), respectively, compared with the lowest tertile (P for trend = 0.042). There was no statistically significant association between urinary levels of nitrite or N-nitroso compounds and risk of gastric cancer. Urinary NMTCA level was significantly associated with consumption of alcohol and preserved meat and fish food items.
The present study demonstrates that exposure to nitrate, a precursor of N-nitroso compounds, may increase the risk of gastric cancer among individuals without a history of H. pylori infection.
PMCID: PMC4319940  PMID: 25658333
3.  The relationship between tumor MSLN methylation and serum mesothelin (SMRP) in mesothelioma 
Epigenetics  2011;6(8):1029-1034.
Malignant pleural mesothelioma (MPM) remains a cancer of poor prognosis. It is hoped that implementation of effective screening biomarkers will lead to earlier diagnoses and improved outcomes. Serum-measured soluble mesothelin-related peptide (SMRP) has been demonstrated to have excellent specificity for MPM, but poor sensitivity precludes its use as a screening biomarker. Using a case series of MPM patients from the International Mesothelioma Program at the Brigham and Women's hospital, we sought to determine whether epigenetic change at the MSLN gene in patient tumors is responsible for the poor sensitivity of SMRP. We identified three potential target regions for CpG methylation silencing in the MSLN promoter, one of which was amenable to bisulfite pyrosequencing and located 214 bp upstream of the transcription start site. MSLN promoter methylation was significantly higher in normal pleura than tumor tissue (p < 6.0 × 10−9). Next, we compared cases according to serum SMRP status and observed that MSLN methylation was significantly higher among tumors from patients testing negative for SMRP (<1.5 nM) versus those that were SMRP positive (p < 0.03). These results demonstrate that MSLN is normally methylated in the pleura, and that methylation is lost in most tumors. However, in a subset of tumors methylation is retained, and this mechanism explains the poor sensitivity of the SMRP assay. These results may lead to additional biomarker targets that will resolve the poor sensitivity of the SMRP assay and allow implementation of screening among exposed populations.
PMCID: PMC3219084  PMID: 21775819
SMRP; MSLN; mesothelioma; methylation; screening
4.  Human papillomavirus serology and tobacco smoking in a community control group 
HPV infection is an established risk factor for oropharyngeal cancer, and it has been proposed that cigarette smoking may potentiate HPV infection in the oral epithelium. We sought to test the hypothesis that cigarette smoking increases HPV infection in an HPV16 serology study of cancer-free individuals.
Subjects were participants in a risk factor study for head and neck cancer, and were required to have no prior history of either HNSCC or any other cancer. Tobacco use and other risk factor data were gathered through interviewer-assisted questionnaires, while serology was conducted in a blinded fashion using a glutathione S-transferase capture enzyme-linked immunosorbent assay (ELISA) to detect antibodies against HPV16 L1, E1, E2, E4, E6 and E7 proteins. The differences in tobacco use by HPV serology were evaluated by ANOVA; and the reported odds ratios and 95% confidence intervals were determined by using unconditional logistic regression.
We found no overall association of HPV16 serological markers with smoking. However, when the data were stratified by median age, smoking was positively associated with seropositivity for the HPV16 L1 capsid antigen in the younger controls while the older controls were less likely to be HPV16 L1 positive if they smoked (pinteraction < 0.002). There was no similar association of smoking and age with serological response to the early proteins (i.e E6, E7).
Exposure to HPV16 capsid protein (L1) is increased among relatively younger adults who smoke and diminished among older smokers. However, this pattern is not accompanied by a differential susceptibility for active infection (as determined by the early gene proteins such as E6 and E7) among young and older smokers.
PMCID: PMC4296688  PMID: 25572638
HPV; Smoking; Serology
5.  CTLA4 Variants, UV-Induced Tolerance, and Risk of Non-Melanoma Skin Cancer 
Cancer research  2009;69(15):6158-6163.
Although skin tumors are highly immunogenic, exposure to UV radiation is known to suppress immune responses via regulatory T cells. Specifically, the activity of cytotoxic lymphocyte-associated antigen-4 (CTLA-4) is integral in regulating the development of UV-induced tolerance and, concomitantly, skin cancers. Due to the inverse relationship between tumor surveillance and autoimmunity, we hypothesize that the same genetic variant in the CTLA4 locus that increases risk for autoimmune diseases is associated with decreased risk of non-melanoma skin cancer (NMSC). We analyzed whether the polymorphism CT60 or haplotypes of CTLA4 influence odds of developing the major types of NMSC, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), in a population-based case-control study of Caucasians in New Hampshire (849 controls, 930 BCC, and 713 SCC). The CTLA4 CT60 GG genotype was associated with decreased odds for BCC and SCC, controlling for age, sex, lifetime number of severe sunburns, and skin type [BCC: odds ratio (OR), 0.7; 95% confidence interval (95% CI), 0.5–0.9; SCC: OR, 0.7; 95% CI, 0.5–1.0]. For BCC, this decrease was apparent largely among those with a higher lifetime number of severe sunburns (Pinteraction = 0.0074). There were significantly decreased odds of disease associated with two haplotypes, which possess the CT60 G allele. Additionally, lifetime number of severe sunburns modestly altered the effects of the CTLA4 haplotypes in BCC, and the association seemed driven by the CT60 single nucleotide polymorphism. In conclusion, genetic variation at the CTLA4 locus may be etiologically important in NMSC, the most prevalent malignancy in the United States.
PMCID: PMC2928067  PMID: 19622768
6.  Cutaneous alpha, beta and gamma human papillomaviruses in relation to squamous cell carcinoma of the skin: a population-based study 
Human papillomavirus (HPV) infection is common worldwide and, in immunodeficient populations, may contribute to the pathogenesis of keratinocyte cancers, particularly squamous cell carcinomas (SCC). However, their role in SCC in the general population is less clear. We conducted a comprehensive analysis to investigate the independent effects of seropositivity for cutaneous alpha, beta and gamma HPV types on risk of SCC, and a meta-analysis of the available literature. In a population-based case-control study from New Hampshire, USA (n=1408), histologically-confirmed SCC cases and controls were tested for L1 antibodies to alpha, beta and gamma cutaneous HPV types 2–5, 7–10, 15, 17, 20, 23, 24, 27b, 36, 38, 48–50, 57, 65, 75–77, 88, 92, 95, 96, 101, 103, and 107 using multiplex serology. An increasing risk of SCC with number of beta HPVs to which an individual tested positive was observed even among those seronegative for gamma types (P for trend = 0.016) with an odds ratio of 1.95 (95% confidence interval (CI) = 1.07–3.56) for four or more beta types positive. In a meta-analysis of six case-control studies, increased SCC risks in relation to beta HPV seropositivity were found across studies, (meta odds ratio = 1.45, CI = 1.27–1.66). While the prevalence of gamma HPVs assayed was somewhat higher among SCC cases than controls, the association was only weakly evident among those seronegative for beta HPVs. Overall, the association between cutaneous HPVs and skin cancers appears to be specific to SCC and to genus beta HPVs in a general US population.
PMCID: PMC3713187  PMID: 23536363
human papillomavirus; squamous cell carcinoma; population-based; case-control; meta-analysis
7.  Exposure Profiles and Human Papillomavirus Infection in Skin Cancer: An Analysis of 25 Genus β-Types in a Population-Based Study 
An increasing number of studies report that genus β human papillomaviruses (HPVs) are associated with skin cancer, with suggestions of specificity for squamous cell carcinoma (SCC) of the skin. We have conducted a systematic examination of HPV DNA in tumors from immunocompetent hosts, including SCC and basal cell carcinoma (BCC), using a highly sensitive methodology and population-based samples to test the hypothesis that a differential prevalence of β-HPVs exists between SCC (n = 101) and BCC (n = 101) tumors. When testing for all known β-HPV types, we found no significant difference in HPV prevalence between the two histologies. However, SCC lesions were significantly more likely to be infected with HPV genus β-species 1 (includes types 5 and 8), than BCC samples (P = 0.01); this difference was not observed for any other species. A histologic difference was also observed for those HPV types previously reported to be important in skin cancer (P = 0.003). SCC samples showed a higher rate of infectivity (that is, were positive for multiple types) than BCC tumors (P = 0.02). These data highlight the potential importance of various genus β-HPV types, in particular genus β-species 1 in SCC, and support the hypothesis of a behavioral difference of the virus within the two major histological skin cancers.
PMCID: PMC2705138  PMID: 18548109
8.  HLA-C -35kb Expression SNP Is Associated with Differential Control of β-HPV Infection in Squamous Cell Carcinoma Cases and Controls 
PLoS ONE  2014;9(8):e103710.
A single nucleotide polymorphism (SNP) 35 kb upstream of the HLA-C gene is associated with HLA-C expression, and the high expressing genotype (CC) has been associated with HIV-I control. HLA-C is unique among the classical MHC class I molecules for its role in the control of viral infections and recognition of abnormal or missing self. This immunosurveillance is central to the pathogenesis of non-melanoma skin cancer (NMSC), and of squamous cell carcinoma (SCC) in particular. While sun exposure is a major risk factor for these cancers, cutaneous infections with genus β-HPV have been implicated in the development of SCC. We hypothesized that the high expression HLA-C genotype is associated with β-HPV infections. Therefore, we investigated the association between β-HPV serology and the −35 kb SNP (rs9264942) in a population-based case-control study of 510 SCC cases and 608 controls. Among controls, the high expression −35 kb SNP genotype (CC) reduced the likelihood of positive serology for multiple (≥2) β-HPV infections (OR = 0.49, 95% CI: 0.25–0.97), and β-HPV species 2 infection (OR = 0.43, 95% CI: 0.23–0.79). However, no association with β-HPV status was observed among SCC cases. Our findings suggest that underlying immunogenotype plays an important role in differential control of β-HPV in SCC cases and controls.
PMCID: PMC4118903  PMID: 25083782
9.  Gastric reflux is an independent risk factor for laryngopharyngeal carcinoma 
Gastric reflux can reach into the upper airway, inducing cellular damage in the epithelial lining. This condition is believed to be a risk factor for development of laryngopharyngeal squamous cell carcinoma (LPSCC), although the literature is conflicting.
To better clarify this relationship, we assessed the association of self-reported heartburn history and medication use among 631 LPSCC patients and 1234 control subjects (frequency-matched on age, gender and town of residence) enrolled as part of a population-based case-control study of head and neck squamous cell carcinoma in the greater Boston area.
After adjusting for age, gender, race, smoking, alcohol consumption, HPV16 seropositivity, education and body mass index, subjects reporting a history of frequent heartburn and who were neither a heavy smoker nor heavy drinker had a significantly elevated risk of LPSCC (OR = 1.78, 95% CI: 1.00–3.16). Among those with a history of heartburn, there was an inverse association between antacid use and LPSCC relative to those never taking heartburn medication (OR = 0.59, 95% CI: 0.38–0.93) that remained consistent when analyzed by smoking/drinking status, HPV16 status, or by primary tumor site.
Our data show that gastric reflux is an independent risk factor for squamous cancers of the pharynx and larynx. Further studies are needed to clarify the possible chemopreventive role of antacid use for patients with gastric reflux.
Elucidation of additional risk factors for head and neck cancer can allow for risk stratification and inform surveillance of high-risk patients.
PMCID: PMC3681904  PMID: 23703970
Heartburn; antacids; proton pump inhibitor; histamine H2 receptor antagonist; head and neck cancer
10.  RNASEL and MIR146A SNP-SNP Interaction as a Susceptibility Factor for Non-Melanoma Skin Cancer 
PLoS ONE  2014;9(4):e93602.
Immunity and inflammatory pathways are important in the genesis of non-melanoma skin cancers (NMSC). Functional genetic variation in immune modulators has the potential to affect disease etiology. We investigated associations between common variants in two key regulators, MIR146A and RNASEL, and their relation to NMSCs. Using a large population-based case-control study of basal cell (BCC) and squamous cell carcinoma (SCC), we investigated the impact of MIR146A SNP rs2910164 on cancer risk, and interaction with a SNP in one of its putative targets (RNASEL, rs486907). To examine associations between genotype and BCC and SCC, occurrence odds ratios (OR) and 95% confidence intervals (95%CI) were calculated using unconditional logistic regression, accounting for multiple confounding factors. We did not observe an overall change in the odds ratios for SCC or BCC among individuals carrying either of the RNASEL or MIR146A variants compared with those who were wild type at these loci. However, there was a sex-specific association between BCC and MIR146A in women (ORGC = 0.73, [95%CI = 0.52–1.03]; ORCC = 0.29, [95% CI = 0.14–0.61], p-trend<0.001), and a reduction in risk, albeit not statistically significant, associated with RNASEL and SCC in men (ORAG = 0.88, [95%CI = 0.65–1.19]; ORAA = 0.68, [95%CI = 0.43–1.08], p-trend = 0.10). Most striking was the strong interaction between the two genes. Among individuals carrying variant alleles of both rs2910164 and rs486907, we observed inverse relationships with SCC (ORSCC = 0.56, [95%CI = 0.38–0.81], p-interaction = 0.012) and BCC (ORBCC = 0.57, [95%CI = 0.40–0.80], p-interaction = 0.005). Our results suggest that genetic variation in immune and inflammatory regulators may influence susceptibility to NMSC, and novel SNP-SNP interaction for a microRNA and its target. These data suggest that RNASEL, an enzyme involved in RNA turnover, is controlled by miR-146a and may be important in NMSC etiology.
PMCID: PMC3974770  PMID: 24699816
11.  LINE-1 DNA Methylation, Smoking and Risk of Parkinson’s Disease 
Journal of Parkinson's disease  2012;2(4):303-308.
Long interspersed nucleotide element-1 (LINE-1) retrotransposons are located throughout the human genome. Those retaining an intact 5′ promoter can copy and insert themselves into the DNA of neural progenitor cells that express tyrosine hydroxylase, which may influence differentiation and survival of these cells. Because LINE-1 promoter methylation is associated with decreased LINE-1 propagation, we compared LINE-1 methylation profiles in blood mononuclear cells between 292 newly diagnosed Parkinson’s disease (PD) cases and 401 unrelated, neurologically normal controls. Overall, PD was not associated with percent methylation of the LINE-1 promoter. However, the predictable inverse association between PD and ever smoking tobacco was strongest for men and women with the lowest LINE-1 promoter methylation, and less apparent as LINE-1 methylation increased. Underlying this possible interaction, ever regularly smoking tobacco was associated with decreased LINE-1 methylation in controls (age- and sex-adjusted linear regression β = −0.24, 95% confidence interval [CI] −0.43, −0.04), but not in cases (β = 0.06, 95% CI −0.17, 0.28, interaction p = 0.06). PD cases may have innate differences in their ability to respond to tobacco smoke.
PMCID: PMC3962286  PMID: 23938260
12.  Quantitative reconstruction of leukocyte subsets using DNA methylation 
Genome Biology  2014;15(3):R50.
Cell lineage-specific DNA methylation patterns distinguish normal human leukocyte subsets and can be used to detect and quantify these subsets in peripheral blood. We have developed an approach that uses DNA methylation to simultaneously quantify multiple leukocyte subsets, enabling investigation of immune modulations in virtually any blood sample including archived samples previously precluded from such analysis. Here we assess the performance characteristics and validity of this approach.
Using Illumina Infinium HumanMethylation27 and VeraCode GoldenGate Methylation Assay microarrays, we measure DNA methylation in leukocyte subsets purified from human whole blood and identify cell lineage-specific DNA methylation signatures that distinguish human T cells, B cells, NK cells, monocytes, eosinophils, basophils and neutrophils. We employ a bioinformatics-based approach to quantify these cell types in complex mixtures, including whole blood, using DNA methylation at as few as 20 CpG loci. A reconstruction experiment confirms that the approach could accurately measure the composition of mixtures of human blood leukocyte subsets. Applying the DNA methylation-based approach to quantify the cellular components of human whole blood, we verify its accuracy by direct comparison to gold standard immune quantification methods that utilize physical, optical and proteomic characteristics of the cells. We also demonstrate that the approach is not affected by storage of blood samples, even under conditions prohibiting the use of gold standard methods.
Cell mixture distributions within peripheral blood can be assessed accurately and reliably using DNA methylation. Thus, precise immune cell differential estimates can be reconstructed using only DNA rather than whole cells.
PMCID: PMC4053693  PMID: 24598480
13.  Tobacco Smoke Biomarkers and Cancer Risk Among Male Smokers in the Shanghai Cohort Study 
Cancer letters  2012;334(1):34-38.
Metabolites of tobacco smoke constituents can be quantified in urine and other body fluids providing a realistic measure of carcinogen and toxicant dose in a smoker. Many previous studies have demonstrated that these metabolites – referred to as biomarkers in this paper – are related to tobacco smoke exposure. The studies reviewed here were designed to answer another question: are these substances also biomarkers of cancer risk? Using a prospective study design comparing biomarker levels in cancer cases and controls, all of whom were smokers, the results demonstrate that several of these biomarkers – total cotinine, total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), r-1-,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), and total N’-nitrosonornicotine (NNN) - are biomarkers of cancer risk. Therefore, these biomarkers have the potential to become part of a cancer risk prediction algorithm for smokers.
PMCID: PMC3648613  PMID: 22824243
tobacco smoke; biomarkers; cotinine; NNAL; PheT; NNN
14.  Epigenetic biomarkers of T-cells in human glioma 
Epigenetics  2012;7(12):1391-1402.
Immune factors are thought to influence glioma risk and outcomes, but immune profiling studies to further our understanding of the immune response are limited by current immunodiagnostic methods. We developed a new assay to capture glioma immune biology based on quantitative methylation specific PCR (qMSP) of two T-cell genes (CD3Z: T-cells, and FOXP3: Tregs). Flow cytometry of T-cells correlated well with the CD3Z demethylation assay (r = 0.93; p < 2.2 × 10−16), demonstrating the validity of the assay. Furthermore, there was a high correlation between qMSP and immunohistochemistry (IHC) in quantifying tumor infiltrating T-cells (r = 0.85; p = 3.4 × 10−11). Applying our qMSP methods to archival whole blood from 65 glioblastoma multiforme (GBM) cases and 94 non-diseased controls, GBM cases had highly statistically significantly lower T-cells (p = 1.7 × 10−9) as well as Tregs (p = 5.2 × 10−11) and a modestly lower ratio of Tregs/T-cells (p = 0.024). Applying the methods to 120 excised glioma tumors, we observed that tumor infiltrating CD3+ T-cells were positively correlated with glioma tumor grade (p = 5.7 × 10−7), and that Tregs were enriched in tumors compared with peripheral blood indicating active chemoattraction of suppressive Tregs into the tumor compartment. Poorer patient survival was correlated with higher levels of tumor infiltrating T-cells (p = 0.01) and Tregs (p = 0.04). DNA methylation based immunodiagnostics represent a new generation of powerful laboratory tools offering many advantages over conventional methods that will facilitate large clinical epidemiologic studies and capitalize on stored archival blood and tissue banks.
PMCID: PMC3528694  PMID: 23108258
DNA methylation; glioma; Tregs; T-cells; biomarkers
15.  Occupational dust exposure and head and neck squamous cell carcinoma risk in a population-based case–control study conducted in the greater Boston area 
Cancer Medicine  2013;2(6):978-986.
Head and neck cancers account for an estimated 549,000 global cancer diagnoses each year. While tobacco use, alcohol consumption, and HPV16 infection are considered to be the major risk factors for this disease, occupational risk factors, including exposure to asbestos, have also been described, although dust exposures other than asbestos have been historically understudied. We have investigated the relationship between occupational exposures to five types of dusts, including sawdust, concrete dust, leather dust, metal dust, and chimney soot, and head and neck squamous cell carcinomas (HNSCC) in the greater Boston area. We report findings from a population-based case–control study involving 951 incident HNSCC cases and 1193 controls, frequency matched on age (±3 years), sex, and town/neighborhood of residence. Multivariable logistic regression was used to assess the association between occupational exposure to each type of dust and HNSCC, overall and by primary tumor site. After adjusting for age, sex, race, smoking, alcohol consumption, education, and HPV16 serology, laryngeal carcinoma risk increased for each decade of occupational exposure to sawdust (OR = 1.2, 95% CI: 1.0, 1.3) and metal dust (OR = 1.2, 95% CI: 1.0, 1.4); and HNSCC risk increased for each decade of occupational leather dust exposure (OR = 1.5, 95% CI: 1.2, 1.9). We have provided evidence for an association between occupational sawdust and metal dust and laryngeal squamous cell carcinoma, and leather dust and HNSCC, with increasing risk with longer duration at the exposed occupation.
PMCID: PMC3892403  PMID: 24403272
Concrete dust; epidemiology; HNSCC; leather dust; metal dust; sawdust; soot
16.  Decreased NK cells in patients with head and neck cancer determined in archival DNA 
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.
Pyrosequencing and qMSP confirmed that a demethylated DNA region in NKp46 distinguishes NK cells from other leukocytes, and serves as a quantitative NK cell marker. Demethylation of NKp46 was significantly lower in HNSCC patient bloods compared with controls (p < 0.001). Individuals in the lowest NK tertile had over 5-fold risk of being a HNSCC case, controlling for age, gender, HPV16 status, cigarette smoking, alcohol consumption, and BMI (OR = 5.6, 95% CI: 2.0, 17.4). Cases did not show differences in NKp46 demethylation based on tumor site or stage.
The results of this study indicate a significant depression in NK cells in HNSCC patients that is unrelated to exposures associated with the disease. DNA methylation biomarkers of NK cells represent an alternative to conventional flow cytometry that can be applied in a wide variety of clinical and epidemiologic settings including archival blood specimens.
PMCID: PMC3500449  PMID: 23014525
natural killer cells; NK cells; head and neck cancer; HNSCC; DNA methylation
17.  Biomarkers of HPV in Head and Neck Squamous Cell Carcinoma 
Cancer research  2012;72(19):5004-5013.
Human papillomavirus (HPV) is an accepted cause of head and neck squamous cell carcinoma (HNSCC) and patients with HPV-associated HNSCC have a favorable prognosis. Currently there is no general guidance on the most appropriate biomarkers for clinical assessment of HPV in these malignancies. We compared PCR-based and serological HPV assays, as well as p16 immunohistochemistry, individually and in combination in a single population-based study to assess their associations with overall survival among HNSCC patients, and thus their potential value as biomarkers. HPV16 serology was determined for 488 patients, immunohistochemical detection of p16 expression in tumors was performed in a subset of 233 cases, and PCR-based methods to assess the presence of HPV16 DNA in a subset of 179 cases’ tumors. Considering each biomarker individually in the subset of patients studied for all endpoints, seropositivity for the E6 and E7 proteins was significantly associated with enhanced all-cause survival in oropharyngeal disease (HRE6/E7+ =0.1, 95%CI=0.02–0.3). Neither the presence of HPV16 DNA or p16 immunostaining was associated with significant enhanced overall survival in oropharyngeal disease ( HRDNA=0.9, 95% CI-0.3–2.9; HRp16=0.3, 95%CI=0.1–1.1). However, the combination of HPV positive DNA and E6 or E7 serology was associated with enhanced overall survival in oropharyngeal disease (HRDNA +/E6/E7+=0.1, 95%CI=0.02–1.0), while E6/E7 seronegative patients with evidence of HPV in tumor DNA did not show any evidence of favorable survival (HRDNA+/E6−/E7−=3.4, 95%CI = 0.6–18.1). Further, patients with p16 staining and E6 or E7 seropositivity had favorable survival from oropharyngeal disease (HRp16+/E6/E7+=0.1, 95%CI=0.02–0.4), while patients who were p16 positive and E6/E7 seronegative had significantly increased hazard of all causes of death (HRp16+/E6−/E7−=3.1, 95%CI=1.2–7.7). A stronger association of HPV presence with prognosis (assessed by all-cause survival) is observed when "HPV-associated" HNSCC is defined using tumor status (HPV DNA status or P16) and HPV E6/E7 serology in combination rather using tumor HPV status alone.
PMCID: PMC3463756  PMID: 22991304
human papillomavirus; head and neck cancer; p16 immunostaining
18.  Plasma S-adenosylmethionine, DNMT polymorphisms, and peripheral blood LINE-1 methylation among healthy Chinese adults in Singapore 
BMC Cancer  2013;13:389.
Global hypomethylation of repetitive DNA sequences is believed to occur early in tumorigenesis. There is a great interest in identifying factors that contribute to global DNA hypomethylation and associated cancer risk. We tested the hypothesis that plasma S-adenosylmethionine (SAM) level alone or in combination with genetic variation in DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was associated with global DNA methylation extent at long interspersed nucleotide element-1 (LINE-1) sequences.
Plasma SAM level and LINE-1 DNA methylation index were measured using stored blood samples collected from 440 healthy Singaporean Chinese adults during 1994-1999. Genetic polymorphisms of 13 loci in DNMT1, DNMT3A and DNMT3B were determined.
LINE-1 methylation index was significantly higher in men than in women (p = 0.001). LINE-1 methylation index was positively associated with plasma SAM levels (p ≤ 0.01), with a plateau at approximately 78% of LINE-1 methylation index (55 nmol/L plasma SAM) in men and 77% methylation index (50 nmol/L plasma SAM) in women. In men only, the T allele of DNMT1 rs21124724 was associated with a statistically significantly higher LINE-1 methylation index (ptrend = 0.001). The DNMT1 rs2114724 genotype modified the association between plasma SAM and LINE-1 methylation index at low levels of plasma SAM in men.
Circulating SAM level was associated with LINE-1 methylation status among healthy Chinese adults. The DNMT1 genetic polymorphism may exert a modifying effect on the association between SAM and LINE-1 methylation status in men, especially when plasma SAM level is low. Our findings support a link between plasma SAM and global DNA methylation status at LINE-1 sequences.
PMCID: PMC3765398  PMID: 23957506
19.  Peripheral blood immune cell methylation profiles are associated with non-hematopoietic cancers 
Blood leukocytes from patients with solid tumors exhibit complex and distinct cancer-associated patterns of DNA methylation. However, the biological mechanisms underlying these patterns remain poorly understood. Since epigenetic biomarkers offer significant clinical potential for cancer detection, we sought to address a mechanistic gap in recently published works, hypothesizing that blood-based epigenetic variation may be due to shifts in leukocyte populations.
We identified differentially methylated regions (DMRs) among leukocyte subtypes using epigenome-wide DNA methylation profiling of purified peripheral blood leukocyte subtypes from healthy donors. These leukocyte-tagging DMRs were then evaluated using epigenome-wide blood methylation data from three independent case-control studies of different cancers.
A substantial proportion of the top 50 leukocyte DMRs were significantly differentially methylated among head and neck squamous cell carcinoma (HNSCC) cases and ovarian cancer cases compared to cancer-free controls (48 and 47 out of 50, respectively). Methylation classes derived from leukocyte DMRs were significantly associated cancer case status (p < 0.001, p < 0.03, and p < 0.001) for all three cancer types: HNSCC, bladder cancer, and ovarian cancer, respectively and predicted cancer status with a high degree of accuracy (AUC = 0.82, 0.83, and 0.67).
These results suggest that shifts in leukocyte sub-populations may account for a considerable proportion of variability in peripheral-blood DNA methylation patterns of solid tumors.
This illustrates the potential utility of DNA methylation profiles for identifying shifts in leukocyte populations representative of disease, and that such profiles may represent powerful new diagnostic tools, applicable to a range of solid tumors.
PMCID: PMC3415587  PMID: 22714737
DNA methylation; cancer; leukocytes; immune system; biomarkers
20.  Allergies and risk of head and neck cancer 
Cancer causes & control : CCC  2012;23(8):1317-1322.
Individuals with allergies have a heightened Th2 (T helper 2) immunity which may provide advantages in controlling tumor growth. Inverse associations have been reported among individuals with allergies and risk of brain and pancreatic cancers.
We examined the relationship between allergies and risk of head and neck squamous cell carcinoma (HNSCC) in a population-based case-control study with 1014 cases and 1193 frequency-matched controls. Logistic regression models were used to estimate odds ratio (OR) and 95% confidence intervals (95% CI) controlling for age, sex, race, smoking history, alcohol consumption, and education. In addition, in a subset of the population, models were adjusted for HPV16 status.
Individuals with allergies had a 19% lower risk of HNSCC (OR = 0.81, 95% CI = 0.67-0.98). Associations with allergies were stronger for laryngeal (OR = 0.66, 95% CI = 0.45-0.97) and oropharyngeal (OR =0.73, 95% CI=0.57-0.92) cancers, while no association was observed for oral cavity cancers (OR = 0.98, 95% CI = 0.76-1.26). History of asthma was not associated with overall HNSCC, but the association was statistically significant for oropharyngeal cancer (OR = 0.67, 95% CI = 0.44-0.99). HPV16 status did not confound or modify the associations with allergies.
Elevated Th2 immunity in individuals with history of allergies and asthma may reduce the risk of HNSCC. Additional research into related mechanisms may provide new insights into how to treat HNSCC.
These findings may provide new insight into biological pathways that could lead to a better understanding of the etiology of this disease.
PMCID: PMC3598569  PMID: 22710748
allergies; atopy; head and neck cancer
21.  Aberrant promoter methylation of CDH13 and MGMT genes is associated with clinicopathological characteristics of primary non small cell lung carcinoma 
Clinical Lung Cancer  2011;13(4):297-303.
Systemic methylation changes may be a diagnostic marker for tumor development or prognosis. Here, we investigate the relationship between gene methylation in lung tumors relative to normal lung tissue, and whether DNA methylation changes can be detected in paired blood samples.
Material and methods
Sixty five patients were enrolled in a surgical case series of non-small cell lung cancer (NSCLC) at a single institution. Using bisulfite pyrosequencing, CpG methylation was quantified at five genes (RASSF1A, CDH13, MGMT, ESR1 and DAPK) in lung tumor, pathologically normal lung tissue, and circulating blood from enrolled cases.
The analyses of methylation in tumors compared to normal lung tissue identified higher methylation of CDH13, RASSF1A, and DAPK genes, while ESR1 and MGMT methylation did not differ significantly between these tissue types. We then examined whether the three aberrantly methylated genes could be detected in blood. The difference in methylation observed in tumors was not reflected in methylation status of matching blood samples, indicating a low feasibility of detecting lung cancer by analyzing these genes in a blood-based test. Lastly we probed whether tumor methylation was associatied with clinical and demographic characteristics. Histology and gender were associated with methylation at the CDH13 gene, while stage was associated with methylation at MGMT.
Our results show higher methylation of RASSF1A, CDH13, and DAPK genes in lung tumors compared to normal lung. The lack of reflection of these methylation changes in blood samples from patients with NSCLC indicate their poorly suitability for a screening test.
PMCID: PMC3346856  PMID: 22169480
methylation; non-small cell lung cancer; CDH13; MGMT; clinicopathological characteristics
22.  Gene-Environment Interactions of Novel Variants Associated with Head and Neck Cancer 
Head & neck  2011;34(8):1111-1118.
A genome-wide association study for upper aerodigestive tract cancers identified 19 candidate single-nucleotide polymorphisms (SNPs). We used these SNPs to investigate the potential gene-gene and gene-environment interactions in head and neck squamous cell carcinoma (HNSCC) risk.
The 19 variants were genotyped using Taqman (Applied Biosystems) assays among 575 cases and 676 controls in our population-based case-control study.
A restricted cubic spline model suggested both ADH1B and HEL308 modified the association between smoking pack-years and HNSCC. Classification and regression tree analysis demonstrated a higher order interaction between smoking status, ADH1B, FLJ13089 and FLJ35784 in HNSCC risk. Compared with ever smokers carrying ADH1B T/C+T/T genotypes, smokers carrying ADH1B C/C genotype and FLJ13089 A/G+A/A genotypes had a highest risk of HNSCC (OR=1.84).
Our results suggest that the risk associated with these variants may be specifically important amongst specific exposure groups.
PMCID: PMC3662053  PMID: 22052802
post-genome wide association study; head and neck cancer; gene and environment interaction
23.  Identification of an Epigenetic Profile Classifier That Is Associated with Survival in Head and Neck Cancer 
Cancer research  2012;72(11):2728-2737.
Panels of prognostic biomarkers selected using candidate approaches often do not validate in independent populations, so additional strategies are needed to identify reliable classifiers. In this study, we used an array-based approach to measure DNA methylation and applied a novel method for grouping CpG dinucleotides according to well-characterized genomic sequence features. A hypermethylation profile among 13 CpG loci characterized by polycomb group target genes, mammalian interspersed repeats, and transcription factor binding sites (PcG/MIR/TFBS), was associated with reduced survival (hazard ratio: 3.98, p=0.001) in head and neck squamous cell carcinoma patients. This association was driven by CpGs associated with the TAP1 and ALDH3A1 genes, findings that were validated in an independent patient group (hazard ratio: 2.86, p=0.04). Together, the data not only elucidate new potential targets for therapeutic intervention in head and neck cancer, but also may aid in the identification of poor prognosis patients who may require more aggressive treatment regimens.
PMCID: PMC3650639  PMID: 22507853
Epigenetics; DNA Sequence; Head and Neck Carcinoma; Survival; Microarray
24.  LINE-1 hypomethylation is associated with bladder cancer risk among non-smoking Chinese 
Reduced levels of global DNA methylation, assessed in peripheral blood, have been associated with bladder cancer risk in European and American populations. Similar data are lacking in Asian populations where genetic differences, lifestyle factors, and different environmental exposures may affect DNA methylation and its risk relationship with bladder cancer. The association between global DNA methylation measured at long interspersed nuclear element (LINE-1) repeat regions through bisulfite pyrosequencing in lymphocyte DNA and bladder cancer risk was examined in a case-control study of 510 bladder cancer patients and 528 healthy control subjects in Shanghai, China. In an initial analysis restricted to control subjects, LINE-1 methylation was elevated among men, those who frequently consumed cruciferous vegetables, and those with a null genotype for either glutathione S-transferase M1 (GSTM1) or GSTT1. In contrast, reduced LINE-1 methylation was found in current smokers with a high cytochrome P450 1A2 (CYP1A2) phenotype index. In a case-control analysis, there was no significant association of LINE-1 methylation with case status, although reduced LINE-1 methylation was associated with increased risk of bladder cancer among never smokers (P for trend = 0.03); analysis by tertile revealed odds ratios (ORs) of 1.91 (lowest tertile; 95% CI = 1.17–3.13) and 1.34 (middle tertile; 95% CI = 0.79–2.28) when compared to the highest tertile. This association was strongest among nonsmokers null for either the GSTM1 or GSTT1 genotype (P for trend = 0.006). Further research is needed to understand the relationships between methyl group availability and LINE-1 methylation in relation to bladder cancer risk.
PMCID: PMC3208798  PMID: 21445976
25.  Peripheral blood DNA methylation profiles are indicative of head and neck squamous cell carcinoma 
Epigenetics  2012;7(3):291-299.
Head and neck cancer accounts for an estimated 47,560 new cases and 11,480 deaths annually in the United States, the majority of which are squamous cell carcinomas (HNSCC). The overall 5 year survival is approximately 60% and declines with increasing stage at diagnosis, indicating a need for non-invasive tests that facilitate the detection of early disease. DNA methylation is a stable epigenetic modification that is amenable to measurement and readily available in peripheral blood. We used a semi-supervised recursively partitioned mixture model (SS-RPMM) approach to identify novel blood DNA methylation markers of HNSCC using genome-wide methylation array data for peripheral blood samples from 92 HNSCC cases and 92 cancer-free control subjects. To assess the performance of the resultant markers, we constructed receiver operating characteristic (RJC) curves and calculated the corresponding area under the curve (AUC). Cases and controls were best differentiated by a methylation profile of six CpG loci (associated with FGD4, SERPINF1, WDR39, IL27, HYAL2 and PLEKHA6), with an AUC of 0.73 (95% CI: 0.62–0.82). After adjustment for subject age, gender, smoking, alcohol consumption and HPV16 serostatus, the AUC increased to 0.85 (95% CI: 0.76–0.92). We have identified a novel blood-based methylation profile that is indicative of HNSCC with a high degree of accuracy. This profile demonstrates the potential of DNA methylation measured in blood for development of non-invasive applications for detection of head and neck cancer.
PMCID: PMC3335952  PMID: 22430805
semi-supervised RPMM; HNSCC; epigenetics; biomarkers; Infinium; methylation array

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