Search

Cigarette smoke contains relatively large quantities of volatile organic toxicants or carcinogens such as benzene, acrolein, and crotonaldehyde. Among their detoxification products are mercapturic acids formed from glutathione conjugation, catalyzed in part by glutathione S-transferases (GST). A randomized phase II clinical trial with a crossover design was conducted to evaluate the effect of 2-phenethyl isothiocyanate (PEITC), a natural product formed from gluconasturtiin in certain cruciferous vegetables, on the detoxification of benzene, acrolein, and crotonaldehyde in 82 cigarette smokers. Urinary mercapturic acids of benzene, acrolein, and crotonaldehyde at baseline and during treatment were quantified. Overall, oral PEITC supplementation increased the mercapturic acid formed from benzene by 24.6% (P = 0.002) and acrolein by 15.1% (P = 0.005), but had no effect on crotonaldehyde. A remarkably stronger effect was observed among subjects with the null genotype of both GSTM1 and GSTT1: in these individuals, PEITC increased the detoxification metabolite of benzene by 95.4% (P < 0.001), of acrolein by 32.7% (P = 0.034), and of crotonaldehyde by 29.8% (P = 0.006). In contrast, PEITC had no effect on these mercapturic acids in smokers possessing both genes. PEITC had no effect on the urinary oxidative stress biomarker 8-iso-prostaglandin F2α or the inflammation biomarker prostaglandin E2 metabolite. This trial demonstrates an important role of PEITC in detoxification of environmental carcinogens and toxicants which also occur in cigarette smoke. The selective effect of PEITC on detoxification in subjects lacking both GSTM1 and GSTT1 genes supports the epidemiologic findings of stronger protection by dietary isothiocyanates against the development of lung cancer in such individuals. Cancer Prev Res; 9(7); 598-606. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: N-acetyltransferase 2 (NAT2) is involved in both carcinogen detoxification through hepatic N-acetylation and carcinogen activation through local O-acetylation. NAT2 slow acetylation status is significantly associated with increased bladder cancer risk among European populations, but its association in Asian populations is inconclusive., Methods: NAT2 acetylation status was determined by both single nucleotide polymorphisms (SNPs) and caffeine metabolic ratio (CMR), in a population-based study of 494 bladder cancer patients and 507 control subjects in Shanghai, China., Results: The CMR, a functional measure of hepatic N-acetylation, was significantly reduced in a dose-dependent manner among both cases and controls possessing the SNP-inferred NAT2 slow acetylation status (all P-values<5.0×10-10). The CMR-determined slow N-acetylation status (CMR<0.34) was significantly associated with a 50% increased risk of bladder cancer (odds ratio = 1.50, 95% confidence interval = 1.10-2.06) whereas the SNP-inferred slow acetylation statuses were significantly associated with an approximately 50% decreased risk of bladder cancer. The genotype-disease association was strengthened after the adjustment for CMR and was primarily observed among never smokers., Conclusions: The apparent differential associations for phenotypic and genetic measures of acetylation statuses with bladder cancer risk may reflect dual functions of NAT2 in bladder carcinogenesis because the former only measures the capacity of carcinogen detoxification pathway while the latter represents both carcinogen activation and detoxification pathways. Future studies are warranted to ascertain the specific role of N- and O-acetylation in bladder carcinogenesis, particularly in populations exposed to different types of bladder carcinogens., Competing Interests: The authors declare no competing interests.

Squamous cell carcinoma (SCC) of the skin is a malignancy arising from epithelial keratinocytes. Experimental and epidemiologic evidence raise the possibility that human polyomaviruses (PyV) may be associated with the occurrence of SCC. To investigate whether the risk for SCC was associated with PyV infection, seropositivity to 10 PyV types was assessed following diagnosis in a population-based case-control study conducted in the United States. A total of 253 SCC cases and 460 age group and gender-matched controls were included. Antibody response against each PyV was measured using a multiplex serology-based glutathione S-transferase capture assay of recombinantly expressed VP1 capsid proteins. Odds ratios (OR) for SCC associated with seropositivity to each PyV type were estimated using logistic regression, with adjustment for potentially confounding factors. SCC cases were seropositive for a greater number of PyVs than controls (P = 0.049). Those who were JC seropositive had increased odds of SCC when compared to those who were JC seronegative (OR = 1.37, 95% CI: 0.98-1.90), with an increasing trend in SCC risk with increasing quartiles of seroreactivity (P for trend = 0.04). There were no clear associations between SCC risk and serostatus for other PyV types. This study provides limited evidence that infection with certain PyVs may be related to the occurrence of SCC in the general population of the United States., (© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The role of the innate immune response in colorectal cancer is understudied. We examined the survival of colorectal cancer patients in relation to eosinophils, innate immune cells, infiltrating the tumor. Tissue microarrays were constructed from paraffin-embedded tumor tissues collected between 1986 and 2002 from 441 post-menopausal women diagnosed with colorectal cancer in the Iowa Women's Health Study. Tissue microarrays were stained with an eosinophil peroxidase antibody. Eosinophils in epithelial and stromal tissues within the tumor (called epithelial and stromal eosinophils, hereafter) were counted and scored into three and four categories, respectively. In addition, the degree of eosinophil degranulation (across epithelial and stromal tissues combined) was quantified and similarly categorized. We used Cox regression to estimate the hazard ratios and 95% confidence interval for all-cause and colorectal cancer death during 5-year follow-up after diagnosis and during follow-up through 2011 ('total follow-up'). The hazard ratios associated with eosinophil scores were adjusted for age of diagnosis, SEER (Surveillance, Epidemiology, and End Results) stage, tumor grade, body mass, and smoking history. High tumor stromal eosinophil score was inversely correlated with age and stage, and was associated with a decreased risk for all-cause and colorectal cancer death: hazard ratios (95% confidence intervals) were 0.61 (0.36-1.02; P-trend=0.02) and 0.48 (0.24-0.93; P-trend=0.01), respectively, during the 5-year follow-up for the highest vs lowest category. The inverse associations also existed for total follow-up for all-cause and colorectal cancer death for the highest vs lowest stromal eosinophil score: hazard ratios (95% confidence intervals) were 0.72 (0.48-1.08; P-trend=0.04) and 0.61 (0.34-1.12; P-trend=0.04), respectively. Further adjustment for treatment, comorbidities, additional lifestyle factors, tumor location, or molecular markers did not markedly change the associations, while adjustment for cytotoxic T cells slightly attenuated all associations. The infiltration of tumors with eosinophils, especially in stromal tissue, may be an important prognostic factor in colorectal cancer.

Background: Merkel cell polyomavirus (PyV) is causally related to Merkel cell carcinoma, a rare skin malignancy. Little is known about the serostability of other PyVs over time or associations with cutaneous squamous cell carcinoma (SCC)., Methods: As part of a U.S. nested case-control study, antibody response against the PyV VP1 capsid proteins of BK and John Cunningham virus (JC) was measured using multiplex serology on 113 SCC cases and 229 gender, age, and study center-matched controls who had a prior keratinocyte cancer. Repeated serum samples from controls and both pre and postdiagnosis samples from a subset of SCC cases were also tested. Odds ratios (OR) for SCC associated with seropositivity to each PyV type were estimated using conditional logistic regression., Results: Among controls, BK and JC seroreactivity was stable over time, with intraclass correlation coefficients of 0.86 for BK and 0.94 for JC. Among cases, there was little evidence of seroconversion following SCC diagnosis. JC seropositivity prior to diagnosis was associated with an elevated risk of SCC (OR = 2.54; 95% CI, 1.23-5.25), and SCC risk increased with increasing quartiles of JC (Ptrend = 0.004) and BK (Ptrend = 0.02) seroreactivity., Conclusions: PyV antibody levels were stable over time and following an SCC diagnosis. A history of PyV infection may be involved in the occurrence of SCC in a population at high risk for this malignancy., Impact: A single measure of PyV seroreactivity appears a reliable indicator of long-term antibody status, and PyV exposure may be a risk factor for subsequent SCC. Cancer Epidemiol Biomarkers Prev; 25(5); 736-44. ©2016 AACR., (©2016 American Association for Cancer Research.)

Cytochrome P450 2A6 (CYP2A6) catalyzes nicotine metabolism and contributes to the metabolism of the tobacco-specific lung carcinogen, NNK. Genetic variation in CYP2A6 may affect smoking behavior and contribute to lung cancer risk. A nested case-control study of 325 lung cancer cases and 356 controls was conducted within a prospective cohort of 18,244 Chinese men in Shanghai, China. Quantified were 4 allelic variants of CYP2A6 [*1(+51A), *4, *7, and *9] and urinary total nicotine, total cotinine, total trans-3'-hydroxycotinine (3HC) and total NNAL (an NNK metabolite). Calculated were total nicotine equivalents (TNE), the sum of total nicotine, total cotinine and total 3HC and the total 3HC:total cotinine ratio as a measure of CYP2A6 activity. The nicotine metabolizer status (normal, intermediate, slow and poor) was determined by CYP2A6 genotypes. The smoking-adjusted odds ratios (95% confidence intervals) of lung cancer for the highest vs lowest quartile of total nicotine, total cotinine, total 3HC, TNE and total NNAL were 3.03 (1.80-5.10), 4.70 (2.61-8.46), 4.26 (2.37-7.68), 4.71 (2.61-8.52), and 3.15 (1.86-5.33) (all Ptrend  < 0.001), respectively. Among controls CYP2A6 poor metabolizers had a 78% lower total 3HC:total cotinine ratio and 72% higher total nicotine (Ptrend ≤ 0.002). Poor metabolizers had an odds ratio of 0.64 (95% confidence interval = 0.43-0.97) for lung cancer, which was statistically nonsignificant (odds ratio = 0.74, 95% confidence interval = 0.48-1.15) after adjustment for urinary TNE and smoking intensity and duration. The lower lung cancer risk observed in CYP2A6 poor metabolizers is partially explained by the strong influence of CYP2A6 genetic polymorphisms on nicotine uptake and metabolism., (© 2015 UICC.)

2-Phenethyl isothiocyanate (PEITC), a natural product found as a conjugate in watercress and other cruciferous vegetables, is an inhibitor of the metabolic activation and lung carcinogenicity of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in F344 rats and A/J mice. We carried out a clinical trial to determine whether PEITC also inhibits the metabolic activation of NNK in smokers. Cigarette smokers were recruited and asked to smoke cigarettes containing deuterium-labeled [pyridine-D4]NNK for an acclimation period of at least 1 week. Then subjects were randomly assigned to one of two arms: PEITC followed by placebo, or placebo followed by PEITC. During the 1-week treatment period, each subject took PEITC (10 mg in 1 mL of olive oil, 4 times per day). There was a 1-week washout period between the PEITC and placebo periods. The NNK metabolic activation ratio [pyridine-D4]hydroxy acid/total [pyridine-D4]NNAL was measured in urine samples to test the hypothesis that PEITC treatment modified NNK metabolism. Eighty-two smokers completed the study and were included in the analysis. Overall, the NNK metabolic activation ratio was reduced by 7.7% with PEITC treatment (P = 0.023). The results of this trial, while modest in effect size, provide a basis for further investigation of PEITC as an inhibitor of lung carcinogenesis by NNK in smokers. Cancer Prev Res; 9(5); 396-405. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: Serum β-2 microglobulin (B2M), a major histocompatibility complex class I molecule that is a biomarker of kidney filtration and increased cell turnover, is elevated at the time of diagnosis in hematological and some solid cancers. However, serum B2M was not examined prospectively as a marker for cancer risk. We hypothesized that in a population without a prior cancer diagnosis, serum B2M is associated with risk of cancer (n = 2,436), including colorectal (n = 255), lung (n = 298), breast (n = 424), and prostate (n = 524) cancers, and hematological (n = 176) malignancies., Methods: The analytical cohort (n = 12,300) was followed for incident cancers from 1990 through 2006. B2M (range, 0.9-57.8 mg/L) was measured in stored serum collected in 1990-1992. Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals for cancer incidence and mortality in relation to quartiles of B2M., Results: Adjusting for age, sex, race, center, education, body mass index, smoking, aspirin, and hormone therapy (in women) and comparing highest to lowest B2M quartiles, HRs were 1.25 (1.06-1.47; Ptrend = 0.002) for total cancer risk and 2.21 (1.32-3.70; Ptrend=0.001) for colorectal cancer risk, with similar HRs for colon and rectal cancers. These associations remained after adjustment for an inflammatory biomarker, C-reactive protein, and after excluding the first three years of follow-up. Significant associations were also observed for mortality from total, lung, and hematological cancers., Conclusions: These findings provide the first evidence that higher serum B2M is associated with increased colorectal cancer risk., Impact: This study supports B2M as a potential biomarker for colorectal cancer risk. Cancer Epidemiol Biomarkers Prev; 25(4); 657-64. ©2016 AACR., (©2016 American Association for Cancer Research.)

Importance: In the United States and Minnesota, melanoma incidence is rising more steeply among women than men younger than 50 years. To our knowledge, no study has examined age- and sex-specific associations between indoor tanning and melanoma to determine if these trends could be due to greater indoor tanning use among younger women., Objective: To examine associations between indoor tanning and melanoma among men and women younger than 50 years., Design, Setting, and Participants: Population-based case-control study conducted in Minnesota of 681 patients (465 [68.3%] women) diagnosed as having melanoma between 2004 and 2007, and 654 controls (446 [68.2%] women), ages 25 to 49 years., Exposure: Indoor tanning, defined as any use, first age of use, and total sessions., Main Outcomes and Measures: Crude and adjusted odds ratios (ORs) and 95% CIs were calculated for melanoma in relation to indoor tanning exposure for men and women by diagnosis or reference age (<30, 30-39, 40-49 years). Sex-specific associations for indoor tanning and melanoma by anatomic site were examined., Results: Compared with women aged 40 to 49 years, women younger than 40 years initiated indoor tanning at a younger age (16 vs 25 years, P < .001) and reported more frequent indoor tanning (median number of sessions, 100 vs 40, P < .001). Women younger than 30 years were 6 times more likely to be in the case than the control group if they tanned indoors (crude OR, 6.0; 95% CI, 1.3-28.5). Odds ratios were also significantly elevated among women, ages 30 to 49 years (adjusted OR, 3.5; 95% CI, 1.2-9.7 for women 30-39 years; adjusted OR, 2.3; 95% CI, 1.4-3.6 for women 40-49 years); a dose response was observed among women regardless of age. Among men, results by age were inconsistent. The strongest OR for indoor tanning by anatomic site was for melanomas arising on the trunk of women (adjusted OR, 3.7; 95% CI, 1.9-7.2)., Conclusions and Relevance: Indoor tanning is a likely factor for the steeper increase in melanoma rates in the United States among younger women compared with men, given the timing of when women initiated indoor tanning relative to diagnosis. The melanoma epidemic can be expected to continue unless indoor tanning is restricted and reduced.

Objectives: An excess of mesothelioma has been observed in iron ore miners in Northeastern Minnesota. Mining and processing of taconite iron ore generate exposures that include elongate mineral particles (EMPs) of amphibole and non-amphibole origin. We conducted a nested case-control study of mesothelioma in a cohort of 68,737 iron ore miners (haematite and taconite ore miners) to evaluate the association between mesothelioma, employment and EMP exposures from taconite mining., Methods: Mesothelioma cases (N=80) were identified through the Minnesota Cancer Surveillance System (MCSS) and death certificates. Four controls of similar age were selected for each case with 315 controls ultimately eligible for inclusion. Mesothelioma risk was evaluated by estimating rate ratios and 95% CIs with conditional logistic regression in relation to duration of taconite industry employment and cumulative EMP exposure [(EMP/cc)×years], defined by the National Institute for Occupational Safety and Health (NIOSH) 7400 method. Models were adjusted for employment in haematite mining and potential exposure to commercial asbestos products used in the industry., Results: All mesothelioma cases were male and 57 of the cases had work experience in the taconite industry. Mesothelioma was associated with the number of years employed in the taconite industry (RR=1.03, 95% CI 1.00 to 1.06) and cumulative EMP exposure (RR=1.10, 95% CI 0.97 to -1.24). No association was observed with employment in haematite mining., Conclusions: These results support an association between mesothelioma and employment duration and possibly EMP exposure in taconite mining and processing. The type of EMP was not determined. The potential role of commercial asbestos cannot be entirely ruled out., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Natural killer (NK)-cell phenotype is partially mediated through binding of killer-cell immunoglobulin-like receptors (KIR) with HLA class I ligands. The KIR gene family is highly polymorphic and not well captured by standard genome-wide association study approaches. Here, we tested the hypothesis that variations in KIR gene content combined with HLA class I ligand status is associated with keratinocyte skin cancers using a population-based study of basal cell carcinoma (BCC) and squamous cell carcinomas (SCC). We conducted an interaction analysis of KIR gene content variation and HLA-B (Bw4 vs. Bw6) and HLA-C (C1 vs. C2). KIR centromeric B haplotype was associated with significant risk of multiple BCC tumors (OR, 2.39; 95% confidence interval, 1.10-5.21), and there was a significant interaction between HLA-C and the activating gene KIR2DS3 for BCC (Pinteraction = 0.005). Furthermore, there was significant interaction between HLA-B and telomeric KIR B haplotype (containing the activating genes KIR3DS1 and KIR2DS1) as well as HLA-B and the activating KIR gene KIR2DS5 (Pinteraction 0.001 and 0.012, respectively). Similar but greatly attenuated associations were observed for SCC. Moreover, previous in vitro models demonstrated that p53 is required for upregulation of NK ligands, and accordingly, we observed there was a strong association between the KIR B haplotype and p53 alteration in BCC tumors, with a higher likelihood that KIR B carriers harbor abnormal p53 (P < 0.004). Taken together, our data suggest that functional interactions between KIR and HLA modify risks of BCC and SCC and that KIR encoded by the B genes provides selective pressure for altered p53 in BCC tumors., (©2016 American Association for Cancer Research.)

Polyomaviruses (PyV) are potentially tumorigenic in humans. However, limited data exist on the population seroprevalence of PyVs and individual characteristics that relate to seropositivity. Using multiplex serology, we determined the seroprevalence of 10 human PyVs (BK, JC, KI, WU, MCV, HPyV6, HPyV7, TSV, HPyV9, and HPyV10) among controls from a population-based skin cancer case-control study (n = 460) conducted in New Hampshire between 1993 and 1995. On a subset of participants (n = 194), methylation at CpG dinucleotides across the genome was measured in peripheral blood using the Illumina Infinium HumanMethylation27 BeadChip array (Illumina Inc., San Diego, California), from which lymphocyte subtype proportions were inferred. All participants were seropositive for at least 1 PyV, with seroprevalences ranging from 17.6% (HPyV9) to 99.1% (HPyV10). Seropositivity to JC, MCV, and HPyV7 increased with age. JC and TSV seropositivity were more common among men than among women. Smokers were more likely to be HPyV9-seropositive but MCV-seronegative, and HPyV7 seropositivity was associated with prolonged glucocorticoid use. Based on DNA methylation profiles, differences were observed in CD8-positive T- and B-cell proportions by BK, JC, and HPyV9 seropositivity. Our findings suggest that PyV seropositivity is common in the United States and varies by sociodemographic and biological characteristics, including those related to immune function., (© The Author 2015. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Understanding the precise role of the immune system in cancer has been hindered by the complexity of the immune response and challenges in measuring immune cell types in health and disease in the context of large epidemiologic studies. In this review, we present the rationale to study immunity in cancer and highlight newly available tools to further elucidate the epidemiologic factors driving individual variation in the immune response in cancer. Here, we summarize key studies that have evaluated the role of immunologic status on risk of cancer, discuss tools that have been used in epidemiologic studies to measure immune status, as well as new evolving methodologies where application to epidemiology is becoming more feasible. We also encourage further development of novel emerging technologies that will continue to enable prospective assessment of the dynamic and complex role played by the immune system in cancer susceptibility. Finally, we summarize characteristics and environmental factors that affect the immune response, as these will need to be considered in epidemiologic settings. Overall, we consider the application of a systems biologic approach and highlight new opportunities to understand the immune response in cancer risk., (©2015 American Association for Cancer Research.)

Purpose: To evaluate cancer incidence among Minnesota taconite mining workers., Methods: We evaluated cancer incidence between 1988 and 2010 in a cohort of 40,720 Minnesota taconite mining workers used between 1937 and 1983. Standardized incidence ratios (SIRs) with 95% confidence intervals (CIs) were estimated by comparing numbers of incident cancers with frequencies in the Minnesota Cancer Surveillance System. SIRs for lung cancer by histologic subtypes were also estimated. We adjusted for out-of-state migration and conducted a probabilistic bias analysis for smoking-related cancers., Results: A total of 5700 cancers were identified, including 51 mesotheliomas and 973 lung cancers. The SIRs for lung cancer and mesothelioma were 1.3 (95% CI = 1.2-1.4) and 2.4 (95% CI = 1.8-3.2), respectively. Stomach, laryngeal, and bladder cancers were also elevated. However, adjusting for potential confounding by smoking attenuated the estimates for lung (SIR = 1.1, 95% CI = 1.0-1.3), laryngeal (SIR = 1.2, 95% CI = 0.8-1.6), oral (SIR = 0.9, 95% CI = 0.7-1.2), and bladder cancers (SIR = 1.0, 95% CI = 0.8-1.1)., Conclusions: Taconite workers may have an increased risk for certain cancers. Lifestyle and work-related factors may play a role in elevated morbidity. The extent to which mining-related exposures contribute to disease burden is being investigated., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Epidemiologic studies that evaluate the relationship between occupational asphalt exposure and head and neck cancer have had a limited ability to control for known risk factors such as smoking, alcohol and human papillomavirus (HPV)., Aims: To better elucidate this relationship by including known risk factors in a large case-control study of head and neck squamous cell carcinoma (HNSCC) from the greater Boston area., Methods: We analysed the relationship between occupational asphalt exposure and HNSCC among men in the Greater Boston area of Massachusetts. Analyses were conducted using unconditional multivariable logistic regression, performed with adjustments for age, race, education, smoking, alcohol consumption and HPV serology., Results: There were 753 cases and 913 controls. No associations between HNSCC and occupational asphalt exposure (neither among ever-exposed nor by occupational duration) were observed for exposures in any occupation or those restricted to the construction industry. We also observed no associations in subgroup analyses of never-smokers and ever-smokers. Adjusting for known risk factors further reduced the estimated effect of asphalt exposure on HNSCC risk., Conclusions: We found no evidence for an association between occupational asphalt exposure and HNSCC. The null findings from this well-controlled analysis could suggest that the risk estimates stemming from occupational cohort studies may be overestimated due to uncontrolled confounding and enhance the literature available for weighing cancer risk from occupational exposure to bitumen., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Occupational Medicine. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: To examine the association between employment duration, elongate mineral particle (EMP) exposure, silica exposure and the risk of lung cancer in the taconite mining industry., Methods: We conducted a nested case-control study of lung cancer within a cohort of Minnesota taconite iron mining workers employed by any of the mining companies in operation in 1983. Lung cancer cases were identified by vital records and cancer registry data through 2010. Two age-matched controls were selected from risk sets of cohort members alive and lung cancer free at the time of case diagnosis. Calendar time-specific exposure estimates were made for every job and were used to estimate workers' cumulative exposures. ORs and 95% CIs were estimated using conditional logistic regression. We evaluated total lung cancer risk and risk of histological subtype by total work duration and by cumulative EMP, and silica exposure by quartile of the exposure distribution., Results: A total of 1706 cases and 3381 controls were included in the analysis. After adjusting for work in haematite mining, asbestos exposure and sex, the OR for total duration of employment was 0.99 (95% CI 0.96 to 1.01). The ORs for quartile 4 versus 1 of EMP and silica exposure were 0.82 (95% CI 0.57 to 1.19) and 0.97 (95% CI 0.70 to 1.35), respectively. The risk of each histological subtype of lung cancer did not change with increasing exposure., Conclusions: This study suggests that the estimated taconite mining exposures do not increase the risk of developing lung cancer., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objectives/hypothesis: Head and neck squamous cell carcinoma (HNSCC) is strongly associated with tobacco use. We sought to examine the relationship between self-reported tobacco use and the level of urinary tobacco carcinogen metabolites in a cohort of patients with HNSCC., Study Design: Cross-sectional analysis., Methods: Eighty-four cigarette smokers with head and neck cancer completed tobacco and alcohol use questionnaires, and the following urinary tobacco metabolites were quantified: 1-hydroxypyrene (1-HOP), N'-nitrosonornicotine and its glucuronides (total NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), and cotinine. A cross-sectional analysis was performed with assessment of correlation coefficients., Results: When analyzed based on self-reported cigarettes per day (CPD), no significant correlation with any of the studied tobacco carcinogen metabolites was found. However, urinary cotinine showed significant correlation with total NNN, total NNAL, and 1-HOP. Total NNN, total NNAL, and 1-HOP showed significant correlation with each other suggesting exposure occurs to each proportionally., Conclusions: In smokers with HNSCC, self-reported tobacco use does not predict actual carcinogen exposure. In contrast, urinary cotinine levels significantly correlate with carcinogen levels. Therefore, urinary cotinine is the preferred value for estimating carcinogen dose in these patients. 1-HOP levels were significantly associated with total NNN and total NNAL suggesting that smokers are exposed to these carcinogens proportionally. These data indicate that utilizing conventional methods of estimating tobacco exposure (CPD) may not accurately approximate exposure to tobacco carcinogens in smokers with HNSCC. These data have implications for future studies focused on screening and epidemiology of smokers with HNSCC., Level of Evidence: NA, (© 2015 The American Laryngological, Rhinological and Otological Society, Inc.)

HPV infection is a causal agent in many epithelial cancers, yet our understanding of genetic susceptibility to HPV infection and resultant cancer risk is limited. Epidermodysplasia Verruciformis is a rare condition of extreme susceptibility to cutaneous HPV infection primarily attributable to mutations in TMC6 and TMC8. Genetic variation in the TMC6/TMC8 region has been linked to beta-type HPV infection and squamous cell carcinoma of the skin, cervical cancer, HPV persistence and progression to cervical cancer. Here, we have tested the hypothesis that the common TMC8 SNP rs7208422 is associated with high-risk HPV infection and risk of head and neck squamous cell carcinoma (HNSCC). Seropositivity to the HPV L1 protein (HPV16, 18, 11, 31, 33, 35, 45, 52, 58) was measured in 514 cases and 452 population-based controls. Genotype was significantly associated with seropositivity to HPV18 L1 (OR TT vs AA = 0.48, 95% CI = 0.22-0.99) and borderline significantly associated with HPV16 L1 (OR TT vs AA = 0.58, 95% CI = 0.22-1.17). There was a consistent inverse association between TMC8 genotype and infection with other HPV types, including statistically significant associations for HPV31 and HPV52. Consistent with these results, the variant T genotype was associated with a reduced risk of HNSCC (ORAT: 0.63, 95% CI 0.45-0.89, ORTT: 0.54, 95% CI 0.36-0.81), even among subjects seronegative for all HPV types (ORAT: 0.71, 95% CI 0.45-1.11, ORTT: 0.54, 95% CI 0.31-0.93). Our data indicate that common genetic variation in TMC8 is associated with high-risk HPV infection and HNSCC etiology.

Background: 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., Methods: 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., Results: 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., Conclusion: 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.

Background: 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., Methods: 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., Results: 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)., Conclusions: 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.

Purpose: Previous studies examining the association of body mass index (BMI) with risk of and survival from head and neck squamous cell carcinoma (HNSCC) have been inconsistent, although an inverse association has been noted for obesity and risk of HNSCC in several studies. Previous studies have not examined whether these associations differ by human papillomavirus (HPV) status., Methods: We utilized the resources of a population-based case-control study of HNSCC from the greater Boston area (959 cases and 1,208 controls were eligible for this analysis). Anthropometric history was collected through personal interviews, and HPV status was assessed using serology. We analyzed the association between BMI (assessed 5 years prior to disease incidence) and disease risk and survival using logistic regression and Cox proportional hazards regression, respectively., Results: After adjusting for known risk factors, the association between obesity and overall risk of HNSCC was not significant (OR 0.79, 95 % CI 0.60-1.04). However, obesity (BMI ≥30 kg/m(2)) was inversely associated with HNSCC risk among HPV-seronegative cases (OR 0.48, 95 % CI 0.32-0.70), but not among HPV-seropositive cases (OR 0.91, 95 % CI 0.68-1.21). BMI was not associated with survival overall or by HPV status. However, being overweight (BMI 25-29.9 kg/m(2)) was associated with longer survival among HPV-seropositive smokers (HR 0.48, 95 % CI 0.31-0.74)., Conclusions: Our findings are consistent with previous observations that obesity is inversely associated with the risk of HNSCC; however, this association appears to be confined to HPV-seronegative cases. Overall, obesity was not associated with HNSCC survival overall or by HPV status., Impact: Obesity is associated with risk of non-HPV HNSCC, but not HPV HNSCC.

High-risk populations exhibit early transformation of localized prostate cancer (CaP) disease to metastasis which results in the mortality of such patients. The paucity of knowledge about the molecular mechanism involved in acquiring of metastatic behavior by primary tumor cells and non-availability of reliable phenotype-discriminating biomarkers are stumbling blocks in the management of CaP disease. Here, we determine the role and translational relevance of ROBO1 (an organogenesis-associated gene) in human CaP. Employing CaP-progression models and prostatic tissues of Caucasian and African-American patients, we show that ROBO1 expression is localized to cell-membrane and significantly lost in primary and metastatic tumors. While Caucasians exhibited similar ROBO1 levels in primary and metastatic phenotype, a significant difference was observed between tumor phenotypes in African-Americans. Epigenetic assays identified promoter methylation of ROBO1 specific to African-American metastatic CaP cells. Using African-American CaP models for further studies, we show that ROBO1 negatively regulates motility and invasiveness of primary CaP cells, and its loss causes these cells to acquire invasive trait. To understand the underlying mechanism, we employed ROBO1-expressing/ROBO1-C2C3-mutant constructs, immunoprecipitation, confocal-microscopy and luciferase-reporter techniques. We show that ROBO1 through its interaction with DOCK1 (at SH3-SH2-domain) controls the Rac-activation. However, loss of ROBO1 results in Rac1-activation which in turn causes E-Cadherin/β-catenin cytoskeleton destabilization and induction of cell migration. We suggest that ROBO1 is a predictive biomarker that has potential to discriminate among CaP types, and could be exploited as a molecular target to inhibit the progression of disease as well as treat metastasis in high-risk populations such as African-Americans., (© 2014 UICC.)

The Notch signaling pathway plays a substantial role in human NK cell development. However, the role of Notch on killer Ig-like receptor (KIR) upregulation and acquisition of effector function has not been explored. To evaluate how Notch influences terminal differentiation, cord blood-derived NK cells or sorted peripheral blood NK cells were cultured with IL-15 for 7 d with inhibitory or activating Notch signals. Inhibition of Notch signaling significantly decreased KIR expression, whereas activation enhanced it. Overexpression of activated Notch on cord blood-derived NK cells resulted in a 2-fold increase in KIR expression, indicating that Notch signaling plays a direct, cell-intrinsic role in KIR regulation. Moreover, Notch-mediated KIR expression on NK cells is regulated through cis inhibition by delta-like ligand 1. Notch signaling also enhances CD16 upregulation that precedes KIR expression. Concomitant with the upregulation of KIR and CD16, Notch signaling induces increased cytolytic effector capacity and cytokine secretion, even in posttransplant samples in which NK cell function is inherently defective. Given these attributes of Notch signaling, we propose that Notch agonists may enhance NK cell maturation and tumor killing in a posttransplant setting., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Although HPV16 has been strongly implicated in oropharyngeal carcinogenesis, the role of other high-risk HPV types in the etiology of head and neck cancer remains unclear. To date, few data exist addressing the nature of the association between antibodies to oncogenic proteins of non-HPV16 HPVs in relation to head and neck cancer. We examined the relationship between multiple HPV types (HPV6, 11, 16, 18, 31, 33, 45, 52, 58) and head and neck squamous cell carcinoma (HNSCC) in a large population-based case-control study (1069 cases and 1107 controls). Serological measures for HPV types included antibodies to L1, E6 and/or E7. In a secondary analysis, we excluded HPV16 seropositive subjects to examine independent associations with other high-risk HPVs. All analyses were adjusted for age, race, sex, education, smoking and alcohol consumption. Statistically significant associations were observed for HPV16, 18, 33 and 52 and risk of HNSCC after mutually adjusting for HPV types. Among HPV16 seronegative subjects, elevated risks of HNSCC were observed for HPV18 E6 (OR = 4.19, 95% CI = 1.26-14.0), HPV33 E6 (OR = 7.96, 95% CI = 1.56-40.5) and HPV52 E7 (OR = 3.40, 95% CI = 1.16-9.99). When examined by tumor type, associations with HPV18 and HPV33 remained statistically significant for oropharyngeal cancer, and HPV52 was associated with oral cancer. In addition, magnitude of associations for HNSCC increased markedly with increasing number of seropositive high-risk HPV infections. High-risk HPV types, other than HPV16, are likely to be involved in the etiology of HNSCC., (© 2014 UICC.)

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.

Indoor tanning is carcinogenic to humans. Individuals report that they tan indoors before planning to be in the sun to prevent sunburns, but whether skin cancer is subsequently reduced is unknown. Using a population-based case-control study, we calculated the association between melanoma and indoor tanning after excluding exposed participants reporting indoor tanning-related burns, stratified by their number of lifetime sunburns (0, 1-2, 3-5, >5). Confounding was addressed using propensity score analysis methods. All statistical tests were two-sided. We observed increased risk of melanoma across all sunburn categories for participants who had tanned indoors without burning compared with those who never tanned indoors, including those who reported zero lifetime sunburns (odds ratio = 3.87; 95% confidence interval = 1.68 to 8.91; P = .002). These data provide evidence that indoor tanning is a risk factor for melanoma even among persons who reported never experiencing burns from indoor tanning or outdoor sun exposure., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objective: Indoor tanning with UV radiation-emitting lamps is common among adolescents and young adults. Rising incidence rates of basal cell carcinoma (BCC) have been reported for the United States and elsewhere, particularly among those diagnosed at younger ages. Recent epidemiologic studies have raised concerns that indoor tanning may be contributing to early occurrence of BCC, and younger people may be especially vulnerable to cancer risk associated with this exposure. Therefore, we sought to address these issues in a population-based case-control study from New Hampshire., Methods: Data on indoor tanning were obtained on 657 cases of BCC and 452 controls ≤50 years of age., Results: Early-onset BCC was related to indoor tanning, with an adjusted odds ratio (OR) of 1.6 (95% confidence interval, 1.3-2.1). The strongest association was observed for first exposure as an adolescent or young adult, with a 10% increase in the OR with each age younger at first exposure (OR per year of age ≤23 = 1.1; 95% confidence interval, 1.0-1.2). Associations were present for each type of device examined (ie, sunlamps, tanning beds, and tanning booths)., Conclusions: Our findings suggest early exposure to indoor tanning increases the risk of early development of BCC. They also underscore the importance of counseling adolescents and young adults about the risks of indoor tanning and for discouraging parents from consenting minors to this practice., (Copyright © 2014 by the American Academy of Pediatrics.)

Epigenome-wide studies of DNA methylation using blood-derived DNA from cancer patients are complicated by the heterogeneity of cell types within blood and the associated cell lineage specification of DNA methylation signatures. Here, we applied a novel set of analytic approaches to assess the association between cancer case-status and DNA methylation adjusted for leukocyte variation using blood specimens from three case-control cancer studies (bladder: 223 cases, 205 controls; head and neck: 92 cases, 92 controls; and ovarian: 131 cases, 274 controls). Using previously published data on leukocyte-specific CpG loci and a recently described approach to deconvolute subject-specific blood composition, we performed an epigenome-wide analysis to examine the association between blood-based DNA methylation patterns and each of the three aforementioned solid tumor types adjusted for cellular heterogeneity in blood. After adjusting for leukocyte profile in our epigenome-wide analysis, the omnibus association between case-status and methylation was significant for all three studies (bladder cancer: P = 0.047; HNSCC: P = 0.013; ovarian cancer: P = 0.0002). Subsequent analyses revealed that CpG sites associated with cancer were enriched for transcription factor binding motifs involved with cancer-associated pathways. These results support the existence of cancer-associated DNA methylation profiles in the blood of solid tumor patients that are independent of alterations in normal leukocyte distributions. Adoption of the methods developed here will make it feasible to rigorously assess the influence of variability of normal leukocyte profiles when investigating cancer related changes in blood-based epigenome-wide association studies.

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.

Background: 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., Results: 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., Conclusions: 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.

Exposures to polycyclic aromatic hydrocarbons (PAHs) from various environmental and occupational sources are considered a primary risk factor for lung cancer among lifelong never smokers, based largely on results from epidemiologic studies utilizing self-reported exposure information. Prospective, biomarker-based human studies on the role of PAH and other airborne carcinogens in the development of lung cancer among lifelong non-smokers have been lacking. We prospectively investigated levels of urinary metabolites of a PAH and volatile organic compounds in relation to lung cancer risk in a nested case-control study of 82 cases and 83 controls among lifelong never smokers of the Shanghai Cohort Study, a prospective cohort of 18 244 Chinese men aged 45-64 years at enrollment. We quantified three PAH metabolites: r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), 3-hydroxyphenanthrene (3-OH-Phe) and total hydroxyphenanthrenes (total OH-Phe, the sum of 1-, 2-, 3- and 4-OH-Phe), as well as metabolites of the volatile organic compounds acrolein (3-hydroxypropyl mercapturic acid), benzene (S-phenyl mercapturic acid), crotonaldehyde (3-hydroxy-1-methylpropylmercapturic acid) and ethylene oxide (2-hydroxyethyl mercapturic acid). Urinary cotinine was also quantified to confirm non-smoking status. Compared with the lowest quartile, odds ratios (95% confidence intervals) for lung cancer risk for the highest quartile levels of PheT, 3-OH-Phe and total OH-Phe were 2.98 (1.13-7.87), 3.10 (1.12-7.75) and 2.59 (1.01-6.65) (all P trend < 0.05), respectively. None of the metabolites of the volatile organic compounds were associated with overall lung cancer risk. This study demonstrates a potentially important role of exposure to PAH in the development of lung cancer among lifelong never smokers.

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., (© 2013 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

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 = 1,408), 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., (© 2013 UICC.)

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

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., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: 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., Methods: To better clarify this relationship, we assessed the association of self-reported heartburn history and medication use among 631 patients with LPSCCs 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., Results: 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 LPSCCs [OR, 1.78; 95% confidence interval (CI), 1.00-3.16]. Among those with a history of heartburn, there was an inverse association between antacid use and LPSCCs 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., Conclusions: 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., Impact: Elucidation of additional risk factors for head and neck cancer can allow for risk stratification and inform surveillance of high-risk patients.

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.

L1 retrotransposons comprise 17% of the human genome and are its only autonomous mobile elements. Although L1-induced insertional mutagenesis causes Mendelian disease, their mutagenic load in cancer has been elusive. Using L1-targeted resequencing of 16 colorectal tumor and matched normal DNAs, we found that certain cancers were excessively mutagenized by human-specific L1s, while no verifiable insertions were present in normal tissues. We confirmed de novo L1 insertions in malignancy by both validating and sequencing 69/107 tumor-specific insertions and retrieving both 5' and 3' junctions for 35. In contrast to germline polymorphic L1s, all insertions were severely 5' truncated. Validated insertion numbers varied from up to 17 in some tumors to none in three others, and correlated with the age of the patients. Numerous genes with a role in tumorigenesis were targeted, including ODZ3, ROBO2, PTPRM, PCM1, and CDH11. Thus, somatic retrotransposition may play an etiologic role in colorectal cancer.

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., (©2012 AACR.)

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 serologic 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 patients with HNSCC, and thus their potential value as biomarkers. HPV16 serology was determined for 488 patients; immunohistochemical detection of p16 expression in tumors was conducted in a subset of 233 cases, and PCR-based methods to assess the presence of HPV16 DNA in a subset of 179 cases of 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 [HR(E6/E7+) = 0.1, 95% confidence interval (CI) = 0.02-0.3]. Neither the presence of HPV16 DNA nor p16 immunostaining was associated with significant enhanced overall survival in oropharyngeal disease (HR(DNA) = 0.9, 95% CI = 0.3-2.9; HR(p16) = 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 (HR(DNA+/E6/E7+) = 0.1, 95% CI = 0.02-1.0), whereas E6/E7 seronegative patients with evidence of HPV in tumor DNA did not show any evidence of favorable survival (HR(DNA+/E6-/E7-) = 3.4, 95% CI = 0.6-18.1). Furthermore, patients with p16 staining and E6 or E7 seropositivity had favorable survival from oropharyngeal disease (HR(p16+/E6/E7+) = 0.1, 95% CI = 0.02-0.4), whereas patients who were p16 positive and E6/E7 seronegative had significantly increased hazard of all causes of death (HR(p16+/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., (©2012 AACR.)

Background: 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., Methods: We examined the relationship between allergies and risk of head and neck squamous cell carcinoma (HNSCC) in a population-based case-control study with 1,014 cases and 1,193 frequency-matched controls. Logistic regression models were used to estimate odds ratios (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., Results: 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., Conclusions: 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., Impact: These findings may provide new insight into biological pathways that could lead to a better understanding of the etiology of this disease.

Background: 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., Methods: The 19 variants were genotyped using Taqman assays among 575 cases and 676 controls in our population-based case-control study., Results: 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 the highest risk of HNSCC (odds ratio = 1.84)., Conclusions: Our results suggest that the risk associated with these variants may be specifically important among specific exposure groups., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: Blood leukocytes from patients with solid tumors exhibit complex and distinct cancer-associated patterns of DNA methylation. However, the biologic mechanisms underlying these patterns remain poorly understood. Because 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., Methods: We identified differentially methylated regions (DMR) 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., Results: 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 with cancer-free controls (48 and 47 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 (area under the curve [AUC] = 0.82, 0.83, and 0.67)., Conclusions: These results suggest that shifts in leukocyte subpopulations may account for a considerable proportion of variability in peripheral blood DNA methylation patterns of solid tumors., Impact: This illustrates the potential use 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., (©2012 AACR.)

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 (HR, 3.98; P = 0.001) in patients with head and neck squamous cell carcinoma. This association was driven by CpGs associated with the TAP1 and ALDH3A1 genes, findings that were validated in an independent patient group (HR, 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., (©2012 AACR)

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.

Background: There 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., Results: Here 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., Conclusions: Our method, in combination with an appropriate external validation set, promises new opportunities for large-scale immunological studies of both disease states and noxious exposures.

Background: While a possible link between assisted reproductive technology (ART) and rare imprinting disorders has been found, it is not clear if this is indicative of subtler disruptions of epigenetic mechanisms. Results from previous studies have been mixed, but some methylation differences have been observed., Methods: Children conceived through ART and children conceived spontaneously were recruited for this cross-sectional study. Information about reproductive history, demographic factors, birth characteristics, and infertility treatment was obtained from maternal interview and medical records. Peripheral blood lymphocytes and buccal cell samples were collected from participating children. Methylation analysis was performed on five loci using pyrosequencing. Statistical analysis of methylation differences was performed using linear regression with generalized estimating equations. Results are reported as differences with 95% confidence intervals (CI)., Results: A total of 67 ART children and 31 spontaneously conceived (SC) children participated. No significant difference in methylation in lymphocyte samples was observed between groups for any loci. Possible differences were found in buccal cell samples for IGF2 DMR0 (Difference: 2.07; 95% confidence interval (CI): -0.28, 4.42; p = 0.08) and IGF2R (Difference: -2.79; 95% CI: -5.74, 0.16; p = 0.06). Subgroup analysis indicated potential lower methylation in those whose parents used ART for unexplained infertility., Conclusions: Observed differences in methylation between the ART and SC groups were small for all loci in the two sample types examined and no statistical differences were observed. It is still unclear whether or not small differences observed in several studies represent a real difference between groups and if this difference is biologically meaningful. Larger studies with long term follow-up are needed to fully answer these questions.

Arsenic is a carcinogen that contaminates drinking water worldwide. Accumulating evidence suggests that both exposure and genetic factors may influence susceptibility to arsenic-induced malignancies. We sought to identify novel susceptibility loci for arsenic-related bladder cancer in a US population with low to moderate drinking water levels of arsenic. We first screened a subset of bladder cancer cases using a panel of approximately 10,000 non-synonymous single nucleotide polymorphisms (SNPs). Top ranking hits on the SNP array then were considered for further analysis in our population-based case-control study (n = 832 cases and 1,191 controls). SNPs in the fibrous sheath interacting protein 1 (FSIP1) gene (rs10152640) and the solute carrier family 39, member 2 (SLC39A2) in the ZIP gene family of metal transporters (rs2234636) were detected as potential hits in the initial scan and validated in the full case-control study. The adjusted odds ratio (OR) for the FSIP1 polymorphism was 2.57 [95% confidence interval (CI) 1.13, 5.85] for heterozygote variants (AG) and 12.20 (95% CI 2.51, 59.30) for homozygote variants (GG) compared to homozygote wild types (AA) in the high arsenic group (greater than the 90th percentile), and unrelated in the low arsenic group (equal to or below the 90th percentile) (P for interaction = 0.002). For the SLC39A2 polymorphism, the adjusted ORs were 2.96 (95% CI 1.23, 7.15) and 2.91 (95% CI 1.00, 8.52) for heterozygote (TC) and homozygote (CC) variants compared to homozygote wild types (TT), respectively, and close to one in the low arsenic group (P for interaction = 0.03). Our findings suggest novel variants that may influence risk of arsenic-associated bladder cancer and those who may be at greatest risk from this widespread exposure.