Epidemiology Research Programs

Goals and objectives for research in the Department of Epidemiology include

• Fostering the highest quality epidemiologic research with the purpose of acquiring new knowledge related to the etiology and prevention of cancer
• Developing nationally and internationally recognized and well-funded multidisciplinary research programs in molecular, genetic and nutritional epidemiology
• Operating a molecular epidemiology research and resource laboratory that promotes collaborative and translational research
• Providing epidemiologic consultation, collaboration and service to support multidisciplinary basic science, clinical and translational research programs
• Developing institution-wide epidemiologic data resources and methodologies to support faculty research initiatives
• Generating community-based prospective research in high-risk and minority/ underserved populations

Molecular and Genetic epidemiology, in addition to Research in Minority Populations (Mexican-American Cohort Study) and the Population Sciences Laboratory comprise important areas of research in epidemiology. Following are descriptions of specific research programs.

A Major Gene for Lung Cancer on 6q23-25 Interacts with Tobacco Smoking to Greatly Increase Lung Cancer Risk

Chris Amos, Ph.D., Joan Bailey-Wilson, Ph.D., Susan Pinney, Ph.D., Mariza de Andrade, Ph.D., Qing Zhang, M.D., Dakai Zhu, M.S., John Minna, M.D.

Lung cancer risk is greatly increased by cigarette smoking and occupational exposures, but familial factors also play a major role. To identify genetic factors predisposing for lung cancer we collected extended pedigrees including multiple relatives with lung cancer. Multipoint linkage analysis of 38 pedigrees with four or more affecteds (lung or throat), under a dominant model with decreased penetrance, yielded a multipoint heterogeneity LOD (HLOD) score of 3.53 (p=1 x 10-4) on chromosome 6q at 156cM, near D6S2436. A subset of 23 multigenerational pedigrees with five or more affecteds yielded a multipoint homogeneity HLOD score of 4.67 (p=7 x 10-7). We then studied the impact that tobacco smoke has upon risk for lung cancer among carriers and noncarriers in the highest risk lung cancer families. To identify carriers in the highest risk families, we first haplotyped subjects for the three genetic loci flanking the susceptibility locus on chromosome 6q. We then performed Kaplan-Meier analysis to assess time to onset for carriers and noncarriers, according to their reported smoking behaviors. We found that tobacco smoking in carriers has a profound impact upon their lung cancer risk. For example, at age 65 for individuals who smoke 0, 1-19, 20-39 and 40+ pack years, the risks for lung cancer among carriers are respectively 29%, 53%, 58% and 47% indicating that among carriers of susceptibility any degree of smoking dramatically increases lung cancer risk. Among the noncarriers, the usual quantitative relationship between increasing smoking and increasing risk was observed. These results indicate the presence of a small subgroup of the population at extremely high lung cancer risks due to smoking.

Polymorphisms in the DNA Repair Gene XRCC1 and Hereditary Nonpolyposis Colorectal Cancer

Jinyun Chen, M.D., Pharm.D., Marsha Frazier, Ph.D.

Hereditary nonpolyposis colorectal cancer (HNPCC) is the predominant cause of familial colorectal cancer and is caused by germline mutations in DNA mismatch repair genes hMSH2, hMLH1, hPMS1, hPMS2 and hMSH6. Mutations causing loss of function in DNA mismatch repair protein genes results in the accumulation of DNA damage and eventual carcinogenesis. The DNA protein x-ray repair cross-complementing group 1 (XRCC1) plays a central role in the DNA base excision repair (BER) pathway, which is responsible for repairing damage caused by various kinds of insults, including oxidative stress, by interacting with DNA ligase III and with DNA polymerase ß. Three polymorphisms that induce amino acid changes have been found in XRCC1 (codon 194 Arg-->Trp, codon 280 Arg-->His and codon 399 Arg--Gln). It has been demonstrated that C- to -T substitution at codon 194 of exon 6 in the XRCC1 gene is associated with increased levels of markers of DNA damage, and has been linked to a variety of human cancers, such as esophageal squamous cell carcinoma (ESCC), breast cancer. The goal of the study is to discover the possible association between polymorphism in DNA repair genes XRCC1 (Arg194Trp) and susceptibility to colorectal cancer in HNPCC patients. We studied a series of 95 hMSH2 and hMLH1 mutation carriers (with 52 colorectal cancer and 43 unaffected carriers). Polymerase chain reaction, single-strand conformation polymorphism analysis and DNA sequencing were used to genotype the mutation carriers for the XRCC1 polymorphism. We examined the association between age of onset and XRCC1 genotype by comparing Kaplan-Meier survival curves. We found HNPCC patients with one mutant XRCC1 allele developed colorectal cancer an average of 4.4 years earlier than patients homozygous for the normal allele (P=0.045). The data suggest that the Arg194Trp XRCC1 polymorphism may affect DNA repair capacity and the age of onset of HNPCC. The findings may provide important information for identifying patients who will develop HNPCC at an earlier age.

Genetic Anticipation and Li-Fraumeni Syndrome

Tracy Costello, Ph.D., Chris Amos, Ph.D.

Genetic anticipation is defined as a decrease in age of onset or increase in severity as the disorder is transmitted through subsequent generations. Anticipation effects have been observed in numerous complex diseases including mental disorders (e.g., schizophrenia, bipolar disorder) and cancers (Li-Fraumeni syndrome, leukemia). Specifically, anticipation in several diseases including Huntington's disease, myotonic dystrophy and Fragile X syndrome were shown to be caused by expansion of triplet repeats. We developed family-based likelihood modeling approaches to assess anticipation that appropriately model the underlying transmission of the disease gene and penetrance function. These methods can be applied in extended families that should increase the power to detect anticipation compared with existing methods based only upon parents and children. To evaluate the new methods, we performed extensive simulation studies and validated the method with data from an MD Anderson Li-Fraumeni cohort. Results of analyses of the LFS cohort with the likelihood based method showed evidence for both a generational effect a residual genetic effect after accounting for effects of the tumor suppressor gene, p53. Further analyses to verify a reported gender effect are ongoing.

Cell-Cycle Checkpoints, DNA Damage/Repair and Lung Cancer Risk

Jian Gu, Ph.D., Xifeng Wu, M.D., Ph.D., Charles Lu, M.D., Yunfei Wang, Ph.D., Hua Zhao, Ph.D., Sherry Luo, Waun Hong, M.D., Margaret R. Spitz, M.D., M.P.H.

Deregulated cell cycle checkpoints and deficient DNA repair capacity are among the hallmarks of cancer. We hypothesized that lung cancer patients would be more likely than healthy controls to exhibit deficiencies in cell-cycle checkpoints and/or DNA repair capacity, as gauged by cellular response to in vitro carcinogen exposure. In an ongoing case-control study of 808 patients with newly diagnosed lung cancer and 982 healthy controls, we used a fluorescence-activated cell sorting method to measure the accumulation of PBLs in the G1, S and G2 phases after exposure to g-radiation. The median g-radiation-induced percentages of cells in the S and G2 phases were significantly lower in cases (24.85% and 12.8%, respectively) than in controls (28.2% and 13.9%, respectively) (P<0.001). The shorter durations of the S and G2 phases resulted in 1.64- and 1.42-fold increased risks of lung cancer, respectively. In quartile analysis, significant trends of increasing lung cancer risk were observed as the accumulation of cells in either S or G2 phases decreased. We then used comet assay to measure the DNA repair capacity in a subset of these subjects (155 patients and 153 controls) upon carcinogen exposure. The median g-radiation-induced and BPDE-induced Olive tail moments, the comet assay parameter for measuring DNA damage, were significantly higher in the case group (5.31 and 4.22, respectively) than in the control group (4.42 and 2.83, respectively) (P<0.001). Higher tail moments of g-radiation and BPDE-induced comets were significantly associated with 2.32- and 4.49-fold elevated risks, respectively, of lung cancer. We also found that in general, shorter cell cycle delay was associated with higher levels of DNA damage, and joint effects existed between g-radiation-induced accumulation of S and G2 phase cells and mutagen-induced comets in determining lung cancer risk. This study provides the molecular epidemiologic evidence with the largest sample size linking phenotypic defects in cell-cycle checkpoints and DNA repair capacity to elevated lung cancer risk. We are currently genotyping a panel of genetic polymorphisms in critical cell cycle control genes, including cyclins, cyclin-dependant kinases (CDKs) and CDK inhibitors to evaluate genotype-phenotype correlations and determine the roles that genetic variations in cell cycle checkpoints may play in lung cancer susceptibility.

Chemotherapy Tailored to Fit: Genetic Variation in 5-FU Drug Action Pathway Modulates Therapeutic Response in Esophageal Cancer

Aditi Hazra, M.S., Jeffer Ajani, M.D., Tsung-The Wu, M.D., Stephen Swisher, M.D., Zhongxin Liao, M.D., Allene Correa, Ph.D., Jun Liu, M.S., Silvia Chiang, B.S., Xifeng Wu, M.D., Ph.D.

Further elucidation of genetic variation in critical drug action genes prior to the administration of chemotherapy may direct formative blueprints for optimizing individualized chemotherapy. We hypothesized that a combination of genetic variants in the 5-fluorouracil (5-FU) drug metabolism and action pathway will modulate response to 5-FU treatment in esophageal cancer (EC) patients. Genotyping of methylenetetrahydrofolate reductase (MTHFR) C677T, MTHFR A1298C and thymidylate synthase (TS) A277G was performed on 181 Caucasian patients treated with 5-FU. The patients had pre-therapy clinical stage II, III, or IVA EC. Epidemiologic and clinical data were ascertained through personal interviews and medical chart reviews, respectively. Overall, using Cox proportional hazards regression, we found that the MTHFR 1298 combined AC+CC genotypes were associated with significantly longer disease-free survival (OR=0.56 95% CI: 0.35 – 0.87). Disease-free survival curves, determined by the Kaplan-Meier method, demonstrated that patients with the AC+CC variants survived a median 51.30 months compared to patients with the AA genotype who survived a median 25.37 months (p=0.01). Next, we performed a joint effect analysis between the MTHFR 677 and MTHFR 1298 polymorphisms. The referent group was individuals with the MTHFR 677 CC and MTHFR 1298 AA genotypes. Individuals with the 1298 AC+CC genotypes and 677 CC genotypes had an OR of 0.54. Among individuals with the MTHFR 677 CT +TT genotypes, we found ORs of 0.92 and 0.17 for MTHFR 1298 AA and AC+CC genotypes, respectively. In addition, compared with patients with the TS 227 AA and MTHFR 1298 AA genotypes, individuals with the MHTFR 1298 AC+CC genotypes and TS 227 AA genotype had an OR of 0.19 (95% CI: 0.07 – 0.55). The joint effect of the MTHFR 1298, MTHFR 677, and TS 277 variants are associated with a considerably prolonged survival in EC patients. The magnitude of these findings suggests that MTHFR genotyping may predict patient outcome to 5-FU treatment and assist in developing improved individualized chemotherapeutic regimens for EC.

Organochlorine Pesticide (OCP) Exposure and Disease Prevalence in Mexican-American Migrant and Seasonal Farmworker (MSF) Adults and Children Homebased in Baytown, Texas

María A. Hernández-Valero, Dr.P.H.

The association between OCP exposure and disease prevalence among Mexican-American migrant MSFs is not known. A pilot study was conducted to measure 21 OCPS and correlate levels with epidemiological data (sociodemographics, anthropometrics, medical and occupational histories, children’s pathways of exposures) and obtain preliminary data among Mexican-American MSFs residing in Baytown, Texas. A modified version of bilingual Migrant Farmworker Questionnaire developed by NCI was administered and each participant donated two blood samples. Eight OCPs (DDT, DDE, mirex, ß-BHC, d-BHC, l-chlordane, oxychlordane, trans-nonachlor) were detected in the adults and two OCPs (DDE and mirex) in the children, even though these pesticides have been banned in the U.S. for decades. Adults measured higher levels for all detected OCPs than the comparison populations. Children measured higher levels of mirex [1.7 parts per billion (ppb)] than the non-migrant comparison populations (non-detectable), and similar levels as their parents (1.8 ppb). Seventy-two percent of the adults and 44% of the children reported suffering from different types of diseases or health problems (adults: diabetes, CVD, cancer, children: infections, gastric problems, asthma). Frequency of disease, country of birth, BMI and occupational exposures were significant indicators of OCP exposure in adult participants, and age, BMI, country of origin, breast-feeding, fieldwork, number of months working in the field and frequency of illness in the children. Even though, the number of participants in this study was small, results indicate an association between OCP exposure and disease prevalence among Mexican-American MSFs. Government should (1) enforce existing laws restricting children/adolescents from working in the fields and (2) larger studies need to be conducted to further investigate these preliminary findings.

Upregulation of PPARgmRNA and Protein Expression in PC-3 Prostate Cancer Cell Lines by Tocopherols and Tocotrienols

Koyamangalath Krishnan, M.D., F.R.C.P., Stephen Lee, B.S., Sharon Campbell, Ph.D., Sarah Whaley, B.S., Kyaw Tun, M.D., Min Qui, M.D., William Stone, Ph.D.

Prostate cancer is a common solid tumor in men worldwide with approximately 230,110 new cases of prostate carcinoma and 29,900 prostate carcinoma deaths in the United States. Peroxisome proliferators activator receptor g (PPARg) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPARg is expressed in the normal prostate, prostate cancer and human prostate cancer cell lines and contributes to prostate carcinogenesis. Heterozygous deletion of PPARg is seen in human prostatic cancer. PPARg ligands (troglitazone) inhibit proliferation of prostate carcinoma cells and suppresses growth of prostatic cancer cells in immunodeficient mice. Vitamin E has structural similarities to the PPARg ligand, troglitazone and hence we investigated the possibility that it may upregulate the expression of PPARg in PC-3 prostate cancer cells. PC-3 prostate cancer cell lines were purchased from American Type Culture Collection, ATCC (Manassas, VA) and maintained as a monolayer culture in RPMI 1680 media (Gibco BRL, Rockville, MD) supplemented with 10% FBS and 50 IU penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Cells were regularly split 1:3 when they had grown to 75% confluence. The effects of tocopherol and tocotrienol treatment on PPARg mRNA and protein expression in PC3 cells was evaluated by real-time PCR and Western blot. Total RNA was extracted from the cells using RNAzol B isolation kit and quantified. mRNA levels of PPARg were measured by RT-PCR using total RNA isolated from PC-3 cells. To accurately quantify the cDNA from the RNA a quantitative QPCR system was used. The PCR system used a fluorescent dye (SYBR green). QPCR optimization was performed using PPARg plasmid clones. cDNA standard curves were prepared using PPARg plasmids to calibrate PPARg. Our data suggest that all the vitamin E isoforms tested increased the relative copy number of PPARg mRNA and increase the PPARg protein levels. Tocotrienols were more efficient than tocopherols in its ability to upregulate PPARg. We have shown that tocotrienols are superior to tocopherols in cell growth arrest. Further work needs to be done to identify the mechanisms behind the superiority of tocotrienols over tocopherols.

A p73 Exon 2 GC4/AT14 Polymorphism and Risk of Lung and Head and Neck Cancers

Guojun Li, M.D., Erich Sturgis, M.D., Li-E Wang, M.D., Robert Chamberlain, Ph.D., Margaret Spitz, M.D., M.P.H., Adel El-Naggar, M.D., Ph.D., Waun Hong, M.D., Qingyi Wei, M.D., Ph.D.

Genetic polymorphisms in genes controlling cell-cycle control, repair of DNA damage, and apoptosis may modulate cancer risk. One example is the p73 gene. A p73 exon 2 polymorphism was recently identified and may alter the efficiency of translation in the p73 protein. We hypothesize that this polymorphism plays a role in the etiology of lung and head and neck cancers. To test this hypothesis and evaluate the association between the p73 AT variant and risk of lung and head and neck cancers, two hospital-based case-control studies were conducted with 1054 patients newly diagnosed with lung cancer and 1139 cancer-free controls and 708 patients newly diagnosed with head and neck cancer and 1229 cancer-free controls, respectively. All controls were frequency-matched to the cases by age (±5 years), sex and smoking status, and all subjects were non-Hispanic whites. Our results showed that the AT variant and genotypes were more common in the cases than in the controls (P=0.0007 and P=0.0033 for lung cancer study and P=0.026 and P=0.009 for head and neck cancer study). Compared with the GC/GC genotype, the variant GC/AT and AT/AT genotypes were associated with a statistically significantly increased risk for lung cancer (adjusted OR =1.31, 95% CI= 1.10-1.57 and 1.56, 1.06-2.29, respectively) with a significant dose-effect relationship (trend test: P<0.001). The combined genotypes (GC/AT+AT/AT) were associated with a statistically significantly increased risk for head and neck cancer (adjusted OR =1.33, 95%CI =1.10-1.60). Further stratification analyses by age, sex, smoking and alcohol status, pack-years of smoking, and histologic types of both types of cancers indicated that the risk associated with the variant genotypes (GC/AT+AT/AT) were particularly evident in several subsets of both studies. Our results suggest that this p73 polymorphism may be a risk marker for genetic susceptibility to both lung and head and neck cancers.

Polymorphisms of Folate Metabolic Genes and Susceptibility to Bladder Cancer: A Case-Control Study

Jie Lin, Ph.D., Margaret Spitz, M.D., M.P.H., Yunfei Wang, Ph.D., Matthew Schabath, Ph.D., Ivan Gorlov, Ph.D., Ladia Hernandez, M.S., R.D., L.D., Patricia Pillow, M.S., R.D., L.D., Herbert Grossman, M.D., Xifeng Wu, M.D., Ph.D.

Epidemiological studies have shown an association between low folate intake and an increased cancer risk. Major genes involved in folate metabolism include methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase (MS). We investigated joint effects of polymorphisms of the MTHFR (677 C→T, 1298A→C) and MS (2756 A→G), dietary folate intake and cigarette smoking on the risk of bladder cancer in a case-control study. The study population consisted of 457 bladder cancer patients and 457 healthy controls, matched to cases in terms of age, gender and ethnicity. Genotype data were analyzed in a subset of 410 Caucasian cases and 410 controls. Compared with individuals carrying the MTHFR 677 wild type (CC) and reporting a high folate intake, those carrying the variant genotype (CT or TT) and reporting a low folate intake were at a significantly 3.51-fold increased risk of bladder cancer (95% CI: 1.59-6.52). In contrast, individuals carrying a variant genotype and reporting a high folate intake were at only 1.39-fold increased risk (95% CI: 0.71-2.70), and those carrying the wild type and reporting a low folate intake were at only 1.56-fold increased risk (95% CI: 0.82-2.97). The interaction between genetic polymorphism and folate intake was significant at the multiplicative scale (P=0.01). When analyzed in the context of smoking status, compared with never smokers with the MTHFR 677 wild type, the risk increased to 6.56-fold (95% CI: 3.28-13.12) in current smokers carrying the variant genotype. Analyses of the MTHFR 1298, MS 2756 genes revealed similar results. In addition, age at cancer onset in former smokers increased as the proportion of the heteromorphic haplotype in the individual increased (P=0.005). Our results strongly suggest that polymorphisms of the MTHFR and MS genes act together with low folate intake and smoking to increase the bladder cancer risk. These results have important implications for cancer prevention in susceptible populations.

The Identification of Prognostic Risk Factors for Second Primary Tumors of the Head and Neck

Charles Minard, M.S., Carol Etzel, Ph.D., Margaret Spitz, M.D., M.P.H.

This study evaluated the effects of mutagen sensitivity and polymorphisms in select phase II genes on the risk of second primary tumors (SPT) in patients with early-stage head and neck squamous cell carcinoma (HNSCC). Mutagen sensitivity refers to an individual’s degree of sensitivity to genotoxicity and risk of cancer. In this study, the average number of bleomycin-induced chromatid breaks (BICB) per cell was used to quantify mutagen sensitivity. Data were collected for 1190 patients enrolled in a placebo-controlled chemoprevention trial of 13-cis-retinoic acid. The Cox proportional hazards model and Classification and Regression Tree (CART) analysis were used to explore the risk factors for development of SPTs. The complexity parameter and log-rank statistic were used to evaluate significant splits within the tree. After adjusting for the treatment group, age at diagnosis, smoking status and site of the primary cancer, the Cox proportional hazards model suggested an increased risk of SPT development for individuals with the GSTM1 null genotype (HR=1.86; 95% CI 1.04-3.32). The GSTT1 null genotype (HR=0.63; 95% CI 0.26-1.49) and the BICB (HR=0.79; 95% CI 0.45-1.37) were not associated with increased risk. However, CART analysis suggested that BICB is an important risk factor among specific subgroups of the population such as non-smokers from 56 to 68 years of age (chi-squared=4.6; p=0.0315). CART analysis did not implicate GSTM1 or GSTT1 as significant risk factors within subgroups. Important associations exist after treatment for HNSCC between SPT development and the GSTM1 null genotype as well as SPT development and mutagen sensitivity.

SULT1A1 Polymorphism and Age of Onset of Hereditary Non-Polyposis Colorectal Cancer

Mala Pande, M.P.H., Elizabeth Sablotne, B.S., Juliet Jones, Ph.D., Nancy Viscofsky, M.P.H., Marsha Frazier, Ph.D.

A series of 132 individuals from Hereditary Nonpolyposis Colorectal Cancer (HNPCC) families, at high risk for colorectal cancer due to germline mutations in the DNA mismatch repair (MMR) genes were evaluated for polymorphism in the sulphotransferase-SULT1A1 gene to determine if the polymorphic SULT1A1*2 allele, which causes an arginine-to-histidine amino acid change, was associated with an earlier age of onset of colorectal cancer (CRC) as compared to the wild type SULT1A1*1. SULT1A1 enzyme alters the biological activity of aromatic and heterocyclic amines through sulphation. Polymorphism in the gene may affect enzyme function resulting in variation in the rate of activation or detoxification of these substances. Individuals homozygous for the SULT1A1*2 (His) allele display only 10% of the enzyme activity of SULT1A1*1 (Arg) homozygotes. The average age of onset of colorectal cancer in this cohort is 45 years as compared to 70-75 years in the general population and 50% of the cohort presently has CRC. The increased risk is not explained by the MMR mutations alone, pointing to a role for other genetic and epigenetic factors. In this study there was no effect modification by the type of MMR mutation-hMLH1 or hMSH2 and no confounding of the exposure-disease association was observed by MMR mutation type, gender or ethnicity. There was no difference on comparison of the Kaplan-Meier survival curves by genotype. Hazard ratio from Cox’s proportional hazards regression analysis for age of onset of CRC for the variant genotype was not significantly different from the wild type (HR: 0.97; 95%CI: 0.58-1.61) therefore, in this study the SULT1A1*2 polymorphism is not linked to an earlier age of onset of CRC.

Colonoscopy Utilization Before and After HNPCC Genetic Testing

Susan Peterson, Ph.D., M.P.H., Ellen Gritz, M.D., Sally Vernon, Ph.D., Catherine Perz, Ph.D., Salma Marani, M.S., Beatty Watts, M.S., Chris Amos, Ph.D., Marsha Frazier, Ph.D., Patrick Lynch, M.D., J.D.

Genetic testing can identify HNPCC-predisposing mutations in persons with a cancer family history suggestive of this syndrome. Mutation carriers have up to an 85% lifetime risk of developing colorectal cancer (CRC), and are advised to have annual or biannual colonoscopy usually beginning at age 25 years. In the absence of genetic testing, persons who are at increased risk for HNPCC by virtue of their family history also are advised to follow the same cancer screening guidelines as mutation carriers. As part of a longitudinal study of psychosocial aspects of HNPCC genetic counseling and testing, we evaluated colonoscopy utilization before and after genetic testing. Subjects were 79 adults from families with a known HNPCC-predisposing mutation and with no prior history of CRC. Prior to genetic counseling, 53% (n=42) had ever had a colonoscopy. After genetic testing, 33% (n=26) were positive for an HNPCC-predisposing mutation. There was no difference in baseline prevalence of colonoscopy use between mutation carriers and non-carriers (50% and 54%, respectively). Within six months of receiving test results, 76% of mutation carriers reported that they had undergone colonoscopy since receiving their results, a significantly greater proportion compared with baseline utilization and with noncarriers (F=18.87, p<0.001). Compared with baseline responses, mutation carriers also expressed significantly greater self-confidence in their ability to follow through with colonoscopy, a greater level of commitment to having colonoscopy and fewer perceived barriers to CRC screening after test results disclosure. Undergoing HNPCC genetic counseling and testing may motivate mutation carriers to have a colonoscopy within a relatively short time period after disclosure of test results. HNPCC genetic counseling and testing also may positively influence mutation carriers’ attitudes toward CRC screening.

Dietary Phytoestrogens and Lung Cancer Risk

Matthew Schabath, Ph.D., Ladia Hernandez, M.S., R.D., L.D., Margaret Spitz, M.D., M.P.H.

Dietary phytoestrogens (PE) are plant-derived compounds that possess estrogen-like activity. They have gained much attention for their chemopreventive role in cancers of the breast, endometrium and prostate. In spite of lung-specific in vitro and in vivo studies supporting a chemopreventive role for PE, there is limited epidemiologic research focusing on PE intake and lung cancer. Using a case-control study, we examined self-reported dietary intake from a food frequency questionnaire for 12 individual PE, categorized into summary measures of lignans, isoflavones and total PE in 1,526 lung cancer cases and 1,483 healthy controls. Overall, cases compared to controls reported statistically significantly lower PE intake for 10 of the 12 individual PE and for summary measures of lignans, isoflavones and total PE. When total lignan intake was categorized by quartiles, high lignan intake was associated with a 32% reduced risk of lung cancer (OR = 0.68; 95% CI 0.55-0.86), after adjusting for age, gender, ethnicity, smoking and total energy. When the data were stratified by gender, the highest quartile of lignan intake was significantly protective for men (OR = 0.62) and borderline significant for women (OR = 0.82). When the lignans were categorized into their precursors and bioactive metabolites, the OR for the highest quartile of lignan metabolites was significantly protective both for women (OR = 0.49; 95% CI 0.36-0.67) and men (OR = 0.59; 95% CI 0.42-0.83). Similarly in the highest quartile of isoflavone intake, there was a 33% overall reduced risk (OR = 0.67; 95% CI 0.53-0.84) and reduced risks of 45% (OR = 0.55) and 21% (OR = 0.79) were evident for men and women, respectively. Finally, there was a 27% overall reduced risk (OR = 0.59; 95% CI 0.59-0.91) in the fourth quartile of total PE intake with reduced risks of 37% (OR = 0.63) and 21% (OR = 0.79) both for men and women, respectively. To our knowledge, this is the largest case-control study of PE intake and lung cancer risk. These results suggest that high intake of dietary PE is associated with an overall decrease in lung cancer risk and that high intake of specific PE may be more beneficial for men while other PE, specifically the bioactive lignan metabolites, may be more beneficial for women. These data may have important implications for cancer prevention.

Association Between Cigarette Smoking and Squamous Intraepithelial Lesions of the Cervix and Dealing with Residual Confounding by HPV Infection

Michael Scheurer, M.P.H., Guillermo Tortolero-Luna, M.D., Ph.D., Michele Follen, M.D., M.P.H.

A smoking and cervical carcinoma association has been supported by several studies showing increased levels of nicotine, cotinine and nitrosamines in cervical mucus; DNA damage in smokers’ cervical cells; and impaired mucosal immunity among smokers. In addition, lesion size reduction has been documented among women undergoing a smoking cessation program. Most epidemiologic evidence points to a two-fold increased risk among smokers and a dose-response relationship with duration and amount of cigarettes smoked. However, epidemiologists have debated the best way to control residual confounding by HPV infection. SIL cases and healthy controls were recruited from the MD Anderson Colposcopy Clinic and Harris County Health Department family planning and screening services, respectively. Detection of HPV DNA was performed using PCR with a consensus primer. Smoking history was obtained through face-to-face interview along with other risk factor information. Associations were estimated using ordinal logistic regression procedures, both controlling for HPV status and excluding HPV-negative subjects to assess any differences in the estimates. 323 cases and 270 controls were included in the complete analysis. Prevalence of HPV infection was 77% among cases compared to 20% among controls. In the HPV-positive analysis, 248 cases and 54 controls were included. Smoking was higher among cases (47 %) than controls (26%). Smokers had 2.5 (95% CI 1.78-3.57) times the odds of SIL than never smokers. After controlling for HPV positivity, age and race, women with LSIL had 1.38 (95% CI 0.83-2.28) times the odds of having ever smoked and women with HSIL had 1.80 (95% CI 1.02-3.17) the odds. When restricted to HPV-positive women, the odds of smoking increased among women with LSIL (OR=1.89; 95% CI 0.84-4.25) and HSIL (OR=2.53; 95% CI 1.13-5.65). These data support the role of smoking as a co-factor for SIL among women infected with HPV. Also, simply controlling for the effects of HPV positivity could lead to an underestimate of the association between smoking and cervical neoplasia.

Genetic Instability in Esophageal Cancer Assessed by Comet

Lina Shao, Ph.D., Jaffer Ajani, Maosheng Huang, Xifeng Wu, M.D., Ph.D.

Interactions between genetic instability and environmental exposures have been implicated in esophageal carcinogenesis. To assess genetic instability and its association with cigarette smoking as an environmental exposure in esophageal carcinogenesis, we used the comet assay to measure baseline, benzo[α]pyrene diol epoxide (BPDE)-induced, and g-radiation-induced genetic instability in peripheral lymphocytes of 82 esophageal cancer patients and 79 healthy controls. We found that DNA damage was significantly higher in case patients than in control subjects at baseline (case vs. control: 2.59 vs. 1.57, P <0.001) and after mutagen BPDE (case vs. control: 3.81 vs. 2.45, P <0.001) and g-radiation (case vs. control: 5.01 vs. 3.58, P <0.001) treatments. After deduction of the baseline DNA damage, the net BPDE-induced (case vs. control: 1.24 vs. 0.91, P <0.05) and g-radiation-induced (case vs. control: 2.43 vs. 2.02, P <0.05) DNA damage was still significantly higher in case patients than in control subjects. When data were dichotomized with the median value of control subjects and then adjusted by age, sex, ethnicity and smoking status, an increased estimated relative risk of esophageal cancer was statistically significantly associated with DNA damage at baseline (odds ratio [OR] = 12.48, 95% confidence interval [CI] = 4.72 to 33.02), after BPDE treatment (OR 11.20, 95% CI = 1.97 to 63.7), and after g-radiation treatment (OR 8.59, 95% CI = 1.77 to 41.82). We further analyzed the data by stratifying smoking status and found that DNA damage was heavier in never-smokers than ever-smokers in case patients at baseline and after treatment with mutagens, but lower in never-smokers than ever-smokers in control subjects at baseline and after treatment with mutagens. Thus, we conclude that genetic instability may be associated with esophageal cancer and that smoking may decrease the threshold of DNA damage for the development of esophageal cancer. Further studies utilizing a larger sample size are necessary to validate our findings and to determine if this comet assay-based approach could be used in clinical screenings to determine individual susceptibility to esophageal cancer.

Case-Control Analysis of Methylene-Tetrahydrofolate Reductase Polymorphisms and Risk of Lung Cancer

Qiuling Shi, Zhengdong Zhang, Ana Neumann, Guojun Li, Ph.D., Margaret Spitz, M.D., M.P.H., Qingyi Wei, M.D., Ph.D.

Methylene-tetrahydrofolate reductase (MTHFR) is involved in the metabolism of folate and nucleotides needed for DNA synthesis and repair, and variations in MTHFR functions likely play a role in lung cancer etiology. The MTHFR gene have three major single nucleotide polymorphisms (SNPs): C677T, A1298C and G1793A. We investigated the frequencies of these polymorphisms in a hospital-based case-control study of 1051 lung cancer patients and 1141 cancer-free controls in a non-Hispanic white population. We genotyped these polymorphisms and found that, compared with subjects homozygous for the MTHFR 1298AA genotype, the 1298CC genotype were associated with significantly increased risk of lung cancer (OR =1.36, 95% CI = 1.01-1.85), which was predominant in females (OR =2.09, 95% CI = 1.32-3.29). Also in females, MTHFR 677TT showed a decreased risk of lung cancer (OR =0.60, 95% CI = 0.40-0.92). Similar pattern was not found in males. Significant gene-dietary interactions of MTHFR C677T and vitamin B6, vitamin B12 and methionine intake were found in females, whereas gene-environmental interactions of MTHFR C677T, A1298C with smoking were found in males. The haplotype analysis showed that subjects with four or more risk alleles (677C, 1298C and 1793A) of the three MTHFR polymorphisms had higher lung cancer risk when compared with those with zero or one risk allele (OR =1.34, 95% CI = 1.01-1.78), which was more pronounced in females (OR =1.93, 95% CI = 1.27-2.94) and subjects reporting low folate intake (OR =1.95, 95% CI = 1.08-3.52). In conclusion, the polymorphisms of MTHFR contribute to the risk of lung cancer in non-Hispanic whites and this contribution is modified by the dietary and environmental exposure with gender specific.

Prostate Cancer Progression: Role of Obesity and Diet After Prostatectomy

Sara Strom, Ph.D., Richard Babaian, M.D., Curtis Pettaway, M.D., Patricia Troncoso, M.D., Christopher Logothetis, M.D., Yuko Yamamura, B.A.

Several lines of evidence suggest that diet and weight gain may be important factors in prostate carcinogenesis, especially in tumor progression. The purpose of this study was to evaluate measures of obesity at different ages in a well-characterized cohort of prostate cancer (PC) patients treated with radical prostatectomy and to develop a prognostic model that incorporates measures of obesity. We carried out a prospective study of 526 patients registered at MD Anderson Cancer Center. Kaplan-Meier, Cox proportional hazard and receiver operating characteristic curve analyses were performed. During an average follow-up of 54 months, 97 (18%) post-prostatectomy patients experienced biochemical failure (BF). Patients who were obese (body mass index (BMI) >30 kg/m2) at diagnosis had a higher rate of BF than non-obese men (p=0.07) and those obese at 40 years had an even greater rate of BF (p=0.001). Obesity at diagnosis (hazard ratio (HR)=1.79, p=0.01), pathological stage (HR=2.00, p=0.006) and Gleason score (p trend< 0.001) remained significant predictors of BF in multivariate analysis. Men who gained weight at the greatest rate (>1.5 kg/yr) between 25 years and diagnosis progressed significantly sooner (mean time= 17 months) than those who exhibited a slower weight gain (mean time= 39 months, p trend=0.005). The inclusion of obesity to the clinical nomogram improved its performance. Patients who experienced disease progression were significantly more likely to consume more calories (2498 vs. 2224, p=0.01), fats (111.2 g vs. 92.4 g, p=0.01), protein (91.4 g vs. 81.9g, p=0.04) and vitamin E (12.6 a-TE vs. 11.1 a-TE, p=0.03) as compared to men whose disease did not progress. We did not find significant differences in reported consumption of calcium, zinc, lycopene, genistein and selenium. Our findings validate the importance for a role of energy balance in prostate cancer progression and suggest a link to the biological basis of prostate cancer progression that can be therapeutically exploited.

Mutation in the LKB1 and p53 Genes Cooperate in Tumorgenesis

Chongjuan Wei, Ph.D., Richard Behringe, Ph.D., Christopher Amos, Ph.D., Marsha Frazier, Ph.D.

Peutz-Jeghers syndrome (PJS) is a rare inherited disease in which patients develop gastrointestinal hamartomatous polyps and exhibit mucocutaneous pigmentation on the skin and oral mucosa. The main PJS locus has been mapped to chromosome 19p13.3, and the gene responsible was identified as LKB1, encoding a serine/threonine kinase. PJS also is a cancer predisposition syndrome, with patients exhibiting increased risk of several cancers, including small bowel, colon, pancreas, stomach, breast and ovarian cancers. The mechanisms involved in the tumor suppressor function of LKB1 remain largely uncharacterized. To evaluate the role of LKB1 in tumorgenesis, we created a conditional knockout gene targeting mouse model employing Cre-loxp technology. We cloned the mouse lkb1 gene piece by piece using Long-Range PCR, then determined the whole sequence, which is 14081 bp in length containing a 7894-bp intron 1. The targeted LKB1 fragment covers exons 2-8 that are from 8172 bp to 11892 bp. A PGK-neo fragment flanked by two loxP sites was inserted inside of the intron 8. One more loxP site was added at the 3’ end of intron 1. All of these three loxP sites were in the same direction encompassing the exons 2-8. A TK fragment (a negative selection marker) was introduced in intron 1 just 5’ of the loxP site. The LKB1 3loxp allele resulted form homologous recombination in mouse ES cells. The chimeric mice derived from recombinant ES clones transmitted the LKB1 3loxp allele to the offspring. The lkb1 3loxp/+ mice were mated with CMV-Cre transgenic mice to generate the lkb1 null alleles. We found that mice heterozygous for LKB1 knockout develop severe gastrointestinal polyps that are hamartomas. Pathological diagnosis indicated all the polyps detected in lkb1 knockout mice are hamartomatous and indistinguishable from the polyps in PJS patients, suggesting that LKB1+/- models human PJS polyposis. It has been reported that LKB1 is the mediator of the p53-dependant apoptosis. To analyze possible molecular mechanisms between LKB1 and p53 genes, crosses were made between LKB1 knockout mice and p53 knockout mice. Hamartomatous polyps were found in the gastrointestinal tract of mice that are heterozygous for LKB1 and p53 gene. Also, heterozygous knockout mice for LKB1 and p53 genes have shown a reduced life span and dramatically increased tumor incidence than single gene knockout mice for either LKB1 or p53 gene. These results indicate that p53 and LKB1 cooperate in accelerating tumorgenesis.

COX-2-Specific NSAID, Celecoxib, Affects Cyclin D1 and Cell Cycle in Human Colorectal Cancer Cells

Jing Zhu, Ph.D., Marsha Frazier, Ph.D.

Cyclin D1 is a cell cycle G1/S transition checkpoint protein. It’s involved in both normal regulation of the cell cycle and neoplasia. A polymorphism (G to A) in the cyclin D1 gene produces an alternate splice product, cyclin D1 b, which lacks the carboxy-terminal amino acid sequences found in the normal cyclin D1 a splice product. Cyclin D1 a is important for cyclin D1 degradation, but the two isoforms share the same amino acid sequence that is responsible for the cyclin D1 cell cycle regulation function. Celecoxib, a COX-2-specific non-steroidal anti-inflammatory drug, was reported to reduce the number and size of colorectal adenomas. We treated COX-2-expressing and COX-2-deficient colorectal cancer cell lines HT-29 and HCT-15 with 0, 20, 50 and 100 mM of celecoxib for 24 or 48 hours. We found that the percentage of cells in G0/G1 phase increased with the increasing concentration of celecoxib. Real time PCR results showed that steady state levels of cyclin D1 transcript a and b decrease with increasing drug concentration and the decrease was greater for transcript b. Western blot results showed that cyclin D1 a levels decreased after treatment with celecoxib, but protein b did not. The results suggest that the stability of cyclin D1 protein b does not parallel the abundance of its mRNA with celecoxib treatment. A decrease in cyclin D1 arrests cells in G0/ G1 phase, thus inhibiting cell proliferation. Both the COX-2-expressing cell line HT-29 and the COX-2-deficient cell line HCT-15 responded to celecoxib treatment. These findings suggest that, in addition to a COX-2-dependent pathway, COX-2-independent pathways may also play a role in the response to celecoxib.