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Background: Nicotine metabolism is a major factor in nicotine dependence, with approximately 70% to 80% of nicotine metabolized to cotinine in Caucasians. Cotinine formation is catalyzed primarily by CYP2A6, which also converts cotinine to trans-3′-hydroxycotinine (3HC). The goal of the present study was to examine the effects of CYP2A6 deficiency on nicotine metabolism profiles in vivo and the importance of genetic variants in nicotine-metabolizing enzyme genes on urinary nicotine metabolites levels. Methods: Urine samples from 722 smokers who participated in the Singapore Chinese Health Study were analyzed using UPLC-MS/MS to detect nicotine and eight of its urinary metabolites, and a total of 58 variants in 12 genes involved in nicotine metabolism were investigated in 475 of these subjects with informative genotyping data. Results: Urine samples stratified by the ratio of 3HC/cotinine exhibited a 7-fold increase in nicotine-N’-oxide, a 6-fold increase in nicotine-Glucuronide (Gluc), and a 5-fold decrease in 3HC-Gluc when comparing the lower versus upper 3HC/cotinine ventiles. Significant (P < 0.0001) associations were observed between functional metabolizing enzyme genotypes and levels of various urinary nicotine metabolites, including CYP2A6 genotype and levels of nicotine, nicotine-Gluc, nicotine-N’-oxide and 3HC, UGT2B10 genotype and levels of cotinine, nicotine-Gluc and cotinine-Gluc, UGT2B17 genotype and levels of 3HC-Gluc, FMO3 genotype and levels of nicotine-N’-oxide, and CYP2B6 genotype and levels of nicotine-N’-oxide and 4-hydroxy-4-(3-pyridyl)-butanoic acid. Conclusions: These data suggest that several pathways are important in nicotine metabolism. Impact: Genotype differences in several nicotine-metabolizing enzyme pathways may potentially lead to differences in nicotine dependence and smoking behavior and cessation.

Background: There is an urgent need to identify biomarkers for advanced adenoma, an important precursor of colorectal cancer (CRC). We aimed to determine alterations in ileal juice bile acids associated with colorectal advanced adenoma. Methods: We quantified a comprehensive panel of primary and secondary bile acids and their conjugates using an ultraperformance liquid chromatography triple-quadrupole mass spectrometric assay in ileal juice collected at colonoscopy from 46 study subjects (i.e., 14 biopsy-confirmed advanced adenomas and 32 controls free of adenoma or cancer). Using analysis of covariance (ANCOVA), we examined the differences in bile acid concentrations by disease status, adjusting for age, sex, body mass index, smoking status and type 2 diabetes. Results: The concentrations of hyodeoxycholic acid (HCA) species in ileal juice of the advanced adenoma patients (geometric mean = 4501.9 nM) were significantly higher than those of controls (geometric mean = 1292.3 nM, p = 0.001). The relative abundance of ursodeoxycholic acid (UDCA) in total bile acids was significantly reduced in cases than controls (0.73% in cases vs. 1.33% in controls; p = 0.046). No significant difference between cases and controls was observed for concentrations of total or specific primary bile acids (i.e., cholic acid (CA), chenodeoxycholic acid (CDCA) and their glycine- and taurine-conjugates) and total and specific major secondary bile acids (i.e., deoxycholic acid and lithocholic acid). Conclusions: Colorectal advanced adenoma was associated with altered bile acids in ileal juice. The HCA species may promote the development of colorectal advanced adenoma, whereas gut microbiota responsible for the conversion of CDCA to UDCA may protect against it. Our findings have important implications for the use of bile acids as biomarkers in early detection of colorectal cancer.

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.

Table S1. Number of [pyridine-D4]NNK cigarettes smoked per day by study participants by treatment sequence groups and treatment period, The PEITC Intervention Study 2008-2013 Table S2. Spearman correlation coefficients between number of cigarettes per day and urinary metabolites of nicotine, NNK, [pyridine-D4]NNK, and PGEM and 8-iso-PGF2α at the end of smoking adaption period (visit 2), The PEITC Intervention Study 2008-2013 Table S3. Urinary Levels of Total ITC and PEITC-NAC by treatment sequence groups and treatment period, The PEITC Intervention Study 2008-2013 Table S4. Geometric means of urinary [pyridine-D4]NNK metabolites by study period and treatment sequence assignment, The PEITC Intervention Study 2008-2013 Table S5. Mean level of urinary levels of PEITC-NAC and total ITC during PEITC treatment by glutathione-S-transferase (GST) genotypes, The PEITC Intervention Study 2008-2013 Table S6. Urinary levels of NNK and nicotine metabolites by PEITC treatment, The PEITC Intervention Study 2008-2013 Figure S1. Overview of the metabolism of [pyridine-D4]NNK and [pyridine-D4]NNAL leading to DNA adducts and urinary metabolites. Figure S2. Overview of the study design for the PEITC trial.

Detection of EBV-specific IgG in sera from incident NPC cases and healthy controls in the SCHS cohort. Heatmap depicting IgG heavy and light chain-specific (IgGH+L) detection values from 20 incident NPC case-control pairs and the data is represented similarly to Fig 2 with Fisher’s exact test identifying proteins (bolded) that discriminate case from controls and denoted *p

Table S1. Baseline demographic and lifestyle characteristics of study participants who developed nasopharyngeal carcinoma (cases) and those who remained cancer free (controls), the Singapore Chinse Health Study and the Shanghai Cohort Study; Table S2. EBV ORF Expression Library; Table S3. Anti-EBV IgA prediction or protection of NPC by EBV ORF class; Table S4. Anti-EBV IgG prediction or protection of NPC by EBV ORF class; Table S5. Detection of EBNA1 IgA in NPC cases sorted from the shortest to the longest time interval from blood collection to diagnosis of NPC, along with matched controls in the Singapore Chinese Health Study (SCHS) and the Shanghai Cohort Study (SCS).

EBV Expression Library; Multiplex Immunoblot Assay; Conservation Plots

Purpose:The favorable prognosis of stage I and II nasopharyngeal carcinoma (NPC) has motivated a search for biomarkers for the early detection and risk assessment of Epstein-Barr virus (EBV)–associated NPC. Although EBV seropositivity is ubiquitous among adults, a spike in antibodies against select EBV proteins is a harbinger of NPC. A serologic survey would likely reveal which EBV antibodies could discriminate those at risk of developing NPC.Experimental Design:Lysates from a new EBV mammalian expression library were used in a denaturing multiplex immunoblot assay to survey antibodies against EBV in sera collected from healthy individuals who later developed NPC (incident cases) in a prospective cohort from Singapore and validated in an independent cohort from Shanghai, P.R. China.Results:We show that IgA against EBV nuclear antigen 1 (EBNA1) discriminated incident NPC cases from matched controls with 100% sensitivity and 100% specificity up to 4 years before diagnosis in both Singapore and Shanghai cohorts. Incident NPC cases had a greater IgG repertoire against lytic-classified EBV proteins, and the assortment of IgA against EBV proteins detected by the immunoblot assay increased closer to diagnosis.Conclusions:Although NPC tumors consistently harbor latent EBV, the observed heightened systemic and mucosal immunity against lytic-classified antigens years prior to clinical diagnosis is consistent with enhanced lytic transcription. We conclude that an expanding EBV mucosal reservoir (which can be latent and/or lytic) is a risk factor for NPC. This presents an opportunity to identify those at risk of developing NPC using IgA against EBNA1 as a biomarker.

Purpose: The favorable prognosis of stage I and II nasopharyngeal carcinoma (NPC) has motivated a search for biomarkers for the early detection and risk assessment of Epstein-Barr virus (EBV)–associated NPC. Although EBV seropositivity is ubiquitous among adults, a spike in antibodies against select EBV proteins is a harbinger of NPC. A serologic survey would likely reveal which EBV antibodies could discriminate those at risk of developing NPC. Experimental Design: Lysates from a new EBV mammalian expression library were used in a denaturing multiplex immunoblot assay to survey antibodies against EBV in sera collected from healthy individuals who later developed NPC (incident cases) in a prospective cohort from Singapore and validated in an independent cohort from Shanghai, P.R. China. Results: We show that IgA against EBV nuclear antigen 1 (EBNA1) discriminated incident NPC cases from matched controls with 100% sensitivity and 100% specificity up to 4 years before diagnosis in both Singapore and Shanghai cohorts. Incident NPC cases had a greater IgG repertoire against lytic-classified EBV proteins, and the assortment of IgA against EBV proteins detected by the immunoblot assay increased closer to diagnosis. Conclusions: Although NPC tumors consistently harbor latent EBV, the observed heightened systemic and mucosal immunity against lytic-classified antigens years prior to clinical diagnosis is consistent with enhanced lytic transcription. We conclude that an expanding EBV mucosal reservoir (which can be latent and/or lytic) is a risk factor for NPC. This presents an opportunity to identify those at risk of developing NPC using IgA against EBNA1 as a biomarker.

Background: Chronic inflammation plays a significant role in hepatocellular carcinoma (HCC). With rising incidence and poor prognosis of HCC, identifying relatively less invasive biomarkers, especially inflammatory ones, may improve the assessment and stratification of HCC risk. The objective of our studies was to assess if CD8+ T cell cytokines in pre-diagnostic serum are associated with risk of HCC development. Methods: We conducted two parallel case-control studies of HCC nested within the Shanghai Cohort Study (SCS) and the Singapore Chinese Health Study (SCHS), two prospective cohorts of 81,000 individuals with 25+ years of follow-up. The serum concentrations of five CD8+ T cell cytokines ─ soluble CD137 (sCD137), soluble Fas (sFas), perforin, macrophage inflammatory protein 1-beta (MIP-1β), and tumor necrosis factor alpha (TNF-α) ─ were determined using Luminex bead-based immunoassay on 315 HCC cases and 315 individually matched controls in the SCS, and on 197 HCC case-control pairs in the SCHS. Conditional logistic regression was used to estimate odds ratio (OR) and 95% confidence interval (CI) for HCC with elevated levels of cytokines with adjustment for body mass index, alcohol drinking, cigarette smoking, seropositivity of hepatitis surface antigen (HBsAg), and history of diabetes. Results: sCD137 levels were statistically significantly higher in HCC cases than controls in both cohorts. Compared with the 1st quartile (Q1) of sCD137, multivariable-adjusted ORs (95% CI) of HCC for the 4th quartile (Q4) were 3.8 (1.7-8.3) in the SCS and 3.5 (1.4 -8.5) in the SCHS (both Ptrend’s < 0.001). Among HBsAg-negative individuals, ORs (95% CIs) for Q2, Q3, & Q4 of sCD137 were 2.7 (1.5-4.9), 2.7 (1.4-5.0), and 4.5 (2.4-8.5), respectively, compared with Q1 (Ptrend < 0.001) in both SCS and SCHS combined. The corresponding ORs (95% CIs) among HBsAg- positive individuals were 21.0 (9.3-47.6), 34.1 (13.8-84.2), and 56.7 (23.3-137.9), respectively, with a P=0.095 for multiplicative interaction. The sCD137-HCC risk association remained constant over different time periods from blood draw to HCC diagnosis: ORs (95% CIs) for Q4 vs. Q1 were 4.7 (1.2-17.5) for Conclusion: These novel and validated findings demonstrated that serum sCD137 levels were significantly elevated many years prior to HCC diagnosis, and had a potential synergistic effect with chronic viral infection on the HCC risk. sCD137 may be developed as a immune monitoring biomarker for risk stratification and assessment, which can lead to early diagnosis and improve prognosis of HCC patients. Citation Format: Claire E. Thomas, Jennifer J. Adibi, Allison L. Kuipers, Brenda Diergaarde, Hung N. Luu, Aizhen Jin, Woon-Puay Koh, Yu-Tang Gao, Jennifer Adams-Haduch, Renwei Wang, Anna Lokshin, Jaideep Behari, Jian-Min Yuan. Soluble CD137 and risk of hepatocellular carcinoma: nested case-control studies in cohorts in Shanghai and Singapore [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3015.

Background: Serine and glycine play an important role in the folate-dependent one-carbon metabolism. The metabolism of serine and glycine has been shown to be associated with cancer cell proliferation. No prior epidemiologic study has investigated the associations for serum levels of serine and glycine with pancreatic cancer risk. Methods: We conducted a nested case-control study involved 129 incident pancreatic cancer cases and 258 individually matched controls within a prospective cohort study of 18,244 male residents in Shanghai, China. Glycine and serine and related metabolites in pre-diagnostic serum were quantified using gas chromatography-tandem mass spectrometry. A conditional logistic regression method was used to evaluate the associations for serine, glycine, and related metabolites with pancreatic cancer risk with adjustment for potential confounders. Results: Odds ratios (95% confidence intervals) of pancreatic cancer for the highest quartile of serine and glycine were 0.33 (0.14–0.75) and 0.25 (0.11–0.58), respectively, compared with their respective lowest quartiles (both p’s < 0.01). No significant association with risk of pancreatic cancer was observed for other serine- or glycine related metabolites including cystathionine, cysteine, and sarcosine. Conclusion: The risk of pancreatic cancer was reduced by more than 70% in individuals with elevated levels of glycine and serine in serum collected, on average, more than 10 years prior to cancer diagnosis in a prospectively designed case-control study. These novel findings support a protective role of serine and glycine against the development of pancreatic cancer in humans that might have an implication for cancer prevention. publishedVersion

Incidence rates of non-Hodgkin lymphoma (NHL) and distributions of certain viruses differ between East Asian and Western populations. There are limited data on associations between serologic markers of multiple viral infections in pre-diagnostic blood and NHL risk in East Asians. We conducted a nested case-control study of 214 NHL cases and 214 matched controls from three population-based prospective cohorts in Shanghai and Singapore. Antibodies against antigens from herpesviruses, Hepatitis B (HBV) and C (HCV) virus and polyomaviruses were measured in plasma or serum using fluorescent bead-based multiplex assays. Conditional logistic regression was used to evaluate associations between antibody levels and NHL risk. An increased risk of NHL was observed for higher compared to lower EA-D (Odds Ratio (OR) = 2.04, 95% Confidence Interval (CI) = 1.10-3.81; p

Background: Organochlorines (OCs) are environmentally persistent compounds that have been extensively used as pesticides and for other industrial applications. Residues of OCs have been detected at hazardous waste sites and in various environmental media worldwide and serum levels of OCs continue to be detectable in the general population. Specific OCs have been associated with non-Hodgkin lymphoma (NHL) risk with varying degrees of evidence. These associations have not been evaluated in Asia, where the exposure patterns of substantially high levels of certain OC pesticides and lower levels of polychlorinated biphenyls (PCBs) are different from Western populations. China accounted for nearly 20% of the worldwide production of dichlorodiphenyltrichloroethane (DDT) through 1983 when it was restricted, and historical usage of hexachlorocyclohexane (HCH) in China has been among the highest in the world. Methods: We evaluated the risk of NHL in relation to pre-diagnostic blood levels of five OC pesticides/metabolites (hexachlorobenzene, β-HCH, oxychlordane, trans-nonachlor, and p,p’-DDE, the primary metabolite of DDT) and four PCB congeners (118, 138-158, 153, 180) in a case-control study of 167 NHL cases and 167 controls matched on age, sex, and blood collection date. The study was nested within three population-based cohorts of Chinese men and women in Shanghai and Singapore. Associations between lipid-adjusted OC concentrations and NHL risk were analyzed using conditional logistic regression. Results: Median levels of p,p’-DDE and β-HCH were up to 12 and 65 times higher, respectively, in Shanghai (blood collected between 1986-2000) compared to reported levels in population-based cohorts in the United States (CLUE; Nurse’s Health Study) and Norway (Janus) with blood collected between 1972-1990. Median levels of p,p’-DDE and β-HCH were more comparable in Singapore (blood collected between 2000-2004) relative to the Western cohorts (1-2 fold concentration differences). Levels of β-HCH were associated with increased risk of overall NHL (3rd vs. 1st tertile OR=1.78, 95%CI=0.98-3.23; ptrend =0.049) in the pooled analysis of three cohorts. No significant associations were observed for other OCs and NHL risk, including for p,p’-DDE. Results for β-HCH and p,p’-DDE were consistent across cohorts. Discussion: Associations between β-HCH and NHL risk have not been consistent in studies of Western populations. Our findings provide the first evidence suggesting associations between blood levels of β-HCH and NHL risk in a population in Asia that experienced far higher exposures. Although there is limited evidence that DDT (IARC Group 2A) is associated with NHL based on studies in Western populations, our findings among Asians, who had higher p,p’-DDE levels than reported in the general population in the West, do not support an association with environmental exposure. Citation Format: Bryan A. Bassig, Xiao-Ou Shu, Andreas Sjodin, Woon-Puay Koh, Yu-Tang Gao, Jennifer Adams-Haduch, Mark Davis, Renwei Wang, Yong-Bing Xiang, Mark Purdue, Bu-Tian Ji, Gong Yang, Richard Jones, H. Dean Hosgood, Wei Jie Seow, Wei Hu, Wei Zheng, Jian-Min Yuan, Qing Lan, Nathaniel Rothman. Pre-diagnostic blood levels of organochlorines and risk of non-Hodgkin lymphoma in three population-based cohorts in China and Singapore [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5043.

Background: Chronic inflammation causes persistent liver injury and consecutive regeneration, potentially leading to fibrosis and cirrhosis, and consequently, to the development of hepatocellular carcinoma (HCC). Prostaglandin E2 (PGE2) is one of the major end-products of the cyclooxygenase-2 (COX-2) pathway, an enzyme that is an important mediator of inflammation. Oxidative stress, which results from the generation of reactive oxygen species (ROS) by environmental risk factors or cellular mitochondrial dysfunction, may be involved in the progression of chronic liver disease to the development of HCC. 8-epi-prostaglandin F2α (8-epi-PGF2α) is a product of lipid peroxidation, which has been recognized as a specific, chemically stable, quantitative marker of systemic and integrated measure of oxidative stress. Method: We evaluated the associations for urinary levels of 8-epi-PGF2α and PGE-M, a metabolite of PGE2, with HCC risk in a prospective cohort of 18,244 men in Shanghai, China. After 25 years of follow-up, 347 participants developed HCC. For each case, two control subjects were chosen and matched on age (±2 years), date of sample collection (±1 months), and neighborhood of residence. Urinary 8-epi-PGF2α and PGE-M were quantified using validated liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assays. The logistic regression method was used to calculate the odds ratio (OR) and its 95% confidence interval (CI) associated with higher levels of both urinary 8-epi-PGF2α and PGE-M. Results: 8-epi-PGF2α levels were significantly higher in HCC cases than control subjects (geometric means 0.92 vs 0.80 pmol/mg creatinine, P 1 B surface antigen. This association remained significant after excluding all HCC cases diagnosed within the first 10 years of follow-up. The present study did not find any significant association between urinary PEG-M and HCC risk. Discussion: 8-Isoprostanes have been found to be significantly elevated in patients with non-alcoholic steatohepatitis (NASH). Our findings support a significant role of oxidative stress in the development of HCC, especially in those without traditional risk factors. The null finding on PGE-M and HCC risk is consistent with results of epidemiological studies that did not show a statistically significant association for HCC risk with use of COX-2 inhibitors. Citation Format: Jian-Min Yuan, Menno Grouls, Steven G. Carmella, Renwei Wang, Alicia Heskin, Yang Jiang, Yuting Tan, Jennifer Adams-Haduch, Yu-Tang Gao, Stephen S. Hecht. Prediagnostic levels of urinary 8-epi-prostaglandin F2α and prostaglandin E2 metabolite, biomarkers of oxidative damage and inflammation, and risk of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2681.

Background. Lean smokers have increased oxidative DNA damage relative to their non-lean counterparts, indicating their heightened susceptibility to tobacco carcinogens-induced DNA damage. Epidemiological association between body mass index (BMI) and lung cancer is inconclusive. We have reported that BMI is a protective factor for smoking-related lung cancer and so did a recent meta-analysis. Knowledge about the association between BMI and non-smoking related lung cancer is limited as this is rare in Western population. We therefore sought to investigate the association between BMI and the risk of never-smoking related lung cancer in an on-going prospective cohort study, the Singapore Chinese Health Study. Methods. The current analysis used data from the Singapore Chinese Health Study, a cohort of 63,257 middle-aged and older Chinese men and women enrolled between 1993 and 1998. As of December 31, 2015, 2,001 study participants who were free of lung cancer at baseline developed lung cancer of whom 621 were never smokers. Cox proportional hazard regression method was used to calculate hazard ratio (HR) and the corresponding 95% confidence interval (CI) for lung cancer associated with BMI in never-smokers. Results. Our results shown that increasing BMI was a protective factor for never-smoking related lung cancer and there is no interaction by gender. The respective HRs and 95% CIs were 0.66 (0.45-0.95) for overall and 0.60 (0.39-0.93) in female for those with BMI>27.5 compared to those with BMI Conclusion. There was an inverse association between BMI and the risk of never-smoking related lung cancer and there is no interaction by gender for this association. This association was also not different by histologic subtypes. Citation Format: Hung N. Luu, Renwei Wang, Jennifer Adams-Haduch, Aizhen Jin, Woon-Puay Koh, Jian-Min Yuan. High BMI in relation to low risk of lung cancer among never smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 631.