Effect of bead size and doxorubicin loading on tumor cellular injury after transarterial embolization and chemoembolization in a rat model of hepatocellular carcinoma.

Embolic agents used in transarterial embolization for intermediate stage hepatocellular carcinoma reduce blood flow into tumors and can deliver anticancer drugs. Tumor blood supply can be interrupted using doxorubicin-eluting beads (DEB-TACE) or non-loaded beads (TAE) of different calibers. In this preclinical study, we characterized the extent of remaining stressed tumor cells after treatment, hypoxia within the surviving tumor regions, and inflammatory immune cell infiltrates after embolization with 40-60 or 70-150 µm with non-loaded or doxorubicin-loaded beads at 3 and 7 days after treatment. TAE-treated tumors had more stressed and surviving tumor cells after 3 days, irrespective of bead size, compared with DEB-TACE-treated tumors. Hypoxic stress of residual cells increased after treatment with 70-150 µm beads without or with doxorubicin. Treatment with DEB-TACE of 70-150 µm resulted in increased inflammation and proliferation in the adjacent parenchyma. Inflammatory cell infiltrates were reduced at the periphery of tumors treated with 40-60 µm DEB-TACE.

Variants in folate pathway genes as modulators of genetic instability and lung cancer risk.

Genetic instability plays a crucial role in cancer development. The genetic stability of the cell as well as DNA methylation status could be modulated by folate levels. Several studies suggested associations between polymorphisms in folate genes and alterations in protein expression and variations in serum levels of the folate. The objective of this study was to investigate the effect of folate pathway polymorphisms on modulating genetic instability and lung cancer risk. Genotyping of 5 SNPs in folate pathway genes and cytokinesis-blocked micronucleus cytome assay analysis (to determine the genetic instability at baseline and following NNK treatment) was conducted on 180 lung cancer cases and 180 age-, gender-, and smoking-matched controls. Our results showed that individually, folate pathway SNPs were not associated with cytogenetic damage or lung cancer risk. However, in a polygenic disease such as lung cancer, gene-gene interactions are expected to play an important role in determining the phenotypic variability of the diseases. We observed that interactions between MTHFR677, MTHFR1298, and SHMT polymorphisms may have a significant impact on genetic instability in lung cancer patients. With regard to cytogenetic alterations, our results showed that lymphocytes from lung cancer patients exposed to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone [NNK] had considerably increased frequency of cytogenetic damage in presence of MTHFR 677, MTHFR 1298, and SHMT allelic variants. These findings support the notion that significant interactions may potentially modulate the lung cancer susceptibility and alter the overall the repair abilities of lung cancer patients when exposed to tobacco carcinogens such as NNK.

Hodgkin disease risk: role of genetic polymorphisms and gene-gene interactions in inflammation pathway genes.

Inflammation is a critical component of cancer development. The clinical and pathological features of Hodgkin disease (HD) reflect an abnormal immunity that results from cytokines secreted by Reed-Sternberg cells and the surrounding tumor. Numerous studies have reported the association between genetic polymorphisms in cytokine genes and the susceptibility to different hematologic cancers. However, the effects of such SNPs on modulating HD risk have not yet been investigated. We hypothesized that gene-gene interactions between candidate genes in the anti- and pro-inflammatory pathways carrying suspicious polymorphisms may contribute to susceptibility to HD. To test this hypothesis, we conducted a study on 200 HD cases and 220 controls to assess associations between HD risk and 38 functional SNPs in inflammatory genes. We evaluated potential gene-gene interactions using a multi-analytic strategy combining logistic regression, multi-factor dimensionality reduction, and classification and regression tree (CART) approaches. We observed that, in combination, allelic variants in the COX2, IL18, ILR4, and IL10 genes modify the risk for developing HD. Moreover, the cumulative genetic risk score (CGRS) revealed a significant trend where the risk for developing HD increases as the number of adverse alleles in the cytokine genes increase. These findings support the notion that epigenetic-interactions between these cytokines may influence pathogenesis of HD modulating the proliferation of regulatory T cells. In this way, the innate and adaptative immune responses may be altered and defy their usual functions in the host anti-tumor response. Our study is the first to report the association between polymorphisms in inflammation genes and HD susceptibility risk.

Factors impacting technical success rate of image-guided intra-arterial therapy in rat orthotopic liver tumor model.

Transcatheter hepatic arterial chemoembolization (TACE) is the current standard of care for intermediate stage hepatocellular carcinoma (HCC) patients. To study the effects of TACE in the tumor immune microenvironment, an immunocompetent rat model is required. The purpose of this study was to determine factors influencing technical success during hepatic arterial catheterization in immunocompetent orthotopic rat liver models. To this end, 91 Sprague-Dawley and eighty-three F344 rats underwent transcatheter hepatic arterial embolization using a transcarotid approach and were divided into a non-tumor-bearing (n = 41) and tumor-bearing (n = 133) groups. Vascular diameters of the hepatic arterial branches were evaluated from angiographic images. Catheterization of the proper hepatic artery (PHA) was achieved in 92% of the tumor-bearing and 68.3% of the non-tumor-bearing rats. We found a strong positive association between the diameter of the PHA and animals' body weight in both groups (P < 0.005), independently of the rat's strain. Results of the logistic regression model predicting a successful catheter placement into the PHA according to the animal's weight indicate that successful PHA catheterization is likely to be achieved in tumor-bearing animals weighing ≥ 250 g and > 308 g in non-tumor-bearing rats, with a sensitivity and specificity of 91.3% and 100.0% and 96.4% and 92.3%, respectively. In conclusion, animal's body weight at the time of catheterization is the principal determinant of technical success for transcatheter arterial embolization. Familiarity with these technical factors during animal selection will improve TACE technical success rates.

Cytokinesis-blocked micronucleus cytome assay biomarkers identify lung cancer cases amongst smokers.

The multi-endpoint cytokinesis-blocked micronucleus assay is used for assessing chromosome aberrations. We have recently reported that this assay is extremely sensitive to genetic damage caused by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and that the binucleated cells with micronuclei, nucleoplasmic bridges, and nuclear buds in lymphocytes (chromosome damage endpoints measured by the assay) are strong predictors of lung cancer risk. In the current study, we refined our analysis to include toxicity endpoints (micronuclei in mononucleated cells, apoptosis, necrosis, and nuclear division index) to investigate the benefit of including these variables on improving the predictive value of the assay. Baseline and NNK-induced micronuclei in mononucleated cells were significantly higher in patients (n = 139) than controls (n = 130; P < 0.001). Baseline apoptosis was higher among cases; however, the controls showed a significant higher fold increase in NNK-induced apoptosis compared with baseline (P < 0.001). Principal components analysis was used to derive a summary measure for all endpoints and calculate the positive predictive value (PPV) and negative predictive value (NPV) for disease status. First principal component for NNK-induced chromosome damage endpoints (binucleated cells with micronuclei, nucleoplasmic bridges, and nuclear buds) had an area under the curve = 97.9 (95% confidence interval, 95.9-99.0), PPV = 94.8, and NPV = 92.6. The discriminatory power improved when micronuclei in mononucleated cells were included: area under the curve = 99.1 (95% confidence interval, 97.9-100.0), PPV = 98.7 and NPV = 95.6. The simplicity, rapidity, and sensitivity of the assay together with potential for automation make it a valuable tool for screening and prioritizing potential cases for intensive screening.

Cytokinesis-blocked micronucleus assay as a novel biomarker for lung cancer risk.

In this case-control study, we modified the cytokinesis-block micronucleus (CBMN) assay, an established biomarker for genomic instability, to evaluate susceptibility to the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by measuring the frequency of NNK-induced chromosomal damage endpoints (micronuclei, nucleoplasmic bridges, and nuclear buds) per 1,000 binucleated lymphocytes. Spontaneous and NNK-induced chromosomal damage were significantly higher in lung cancer patients compared with controls. Forty-seven percent of cases (versus 12% of controls) had >or=4 spontaneous micronuclei, 66% of cases (and no controls) had >or=4 spontaneous nucleoplasmic bridges, and 25% of cases (versus 5% of controls) had >or=1 spontaneous nuclear bud (P < 0.001). Similarly, 40% of cases (versus 6% of the controls) had >or=5 NNK-induced micronuclei, 89% of cases (and no controls) had >or=6 induced nucleoplasmic bridges, and 23% of cases (versus 2% of controls) had >or=2 induced nuclear buds (P < 0.001). When analyzed on a continuous scale, spontaneous micronuclei, nucleoplasmic bridges, and nuclear buds were associated with 2-, 29-, and 6-fold increases in cancer risk, respectively. Similarly, NNK-induced risks were 2.3-, 45.5-, and 10-fold, respectively. We evaluated the use of CBMN assay to predict cancer risk based on the numbers of micronuclei, nucleoplasmic bridges, and nuclear buds defined by percentile cut points in controls. Probabilities of being a cancer patient were 96%, 98%, and 100% when using the 95th percentiles of spontaneous and NNK-induced micronuclei, nucleoplasmic bridges, and nuclear buds, respectively. Our study indicates that the CBMN assay is extremely sensitive to NNK-induced genetic damage and may serve as a strong predictor of lung cancer risk.

Lack of Breastfeeding History in Parous Women with Inflammatory Breast Cancer Predicts Poor Disease-Free Survival.

Purpose: Breastfeeding alters the breast microenvironment, and several lines of evidence suggest the breast microenvironment contributes to the clinical phenotype of inflammatory breast cancer. We investigated breastfeeding history as a modifier of locoregional recurrence (LRR), distant metastasis (DM), disease-free survival (DFS), and overall survival (OS) in parous women with inflammatory breast cancer. Methods: Parous women with inflammatory breast cancer were identified from a prospective registry at The University of Texas MD Anderson Cancer Center. We compared patient and tumor characteristics, LRR, DM, DFS, and OS patients with (BF+) and without (BF-) a history of breastfeeding. Results: Eighty-two patients were included. At a median follow-up of 50 months, BF+ patients had significantly lower risk of LRR (9.0% vs. 23.6%; p=0.01), a lower risk of DM (26.8% vs. 53.8%; p=0.008), and better DFS (73.1% vs. 48.1%; p=0.006) than BF- patients. On multivariate analysis, BF+ history was associated with significantly lower risk of DM (hazard ratio 0.38, 95% confidence interval 0.15-0.97; p=0.04) and better DFS (hazard ratio 0.37, 95% confidence interval 0.15-0.93; p=0.04) after adjusting for established predictive and prognostic variables. The prognostic significance of breastfeeding may be most pronounced in women with triple-negative IBC. Conclusion: A lack of breastfeeding history in parous women with inflammatory breast cancer may predict worse prognosis. We speculate that breastfeeding-induced alterations in the breast microenvironment may alter the aggressiveness of inflammatory breast cancer.

Human sensitivity to PhIP: a novel marker for prostate cancer risk.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been implicated in the development of colon, prostate and mammary gland tumors in rats. In this study, we developed a modified in vitro mutagen sensitivity assay, with activated PhIP (N-OH-PhIP) as the challenge mutagen and chromosome aberrations as the endpoint, and applied it in a pilot prostate cancer case-control study of 81 cases and 84 age and ethnicity-matched controls. Our results showed significantly higher baseline breaks among the cases, mean+/-S.E.=1.86+/-0.23 versus 0.96+/-0.14 in controls; P=0.006. Individuals with high baseline breaks (dichotomized at the control median) had a 36% increased risk for PC (OR=1.36; 95% CI=1.08-1.72). In stratified analysis, high baseline breaks was associated in younger participants (< or = 60 years) with an OR of 1.71 (1.14-2.57) and in those with a positive family history of PC, an OR of 1.43 (0.97-2.11). PhIP treatment induced significantly higher breaks in cases, mean+/-S.E.=5.07+/-0.39 versus 3.83+/-0.24 in controls; P=0.05. Higher PhIP-induced breaks was associated with an overall 17% increased risk for PC (OR=1.17; 95% CI=1.03-1.33), a significantly increased risks (OR=1.19; 95% CI=1.00-1.41) among younger participants, non-smokers (OR=1.39, 1.03-1.88) and 1.20 (1.00-1.45) among those with no family history of PC. Results from this pilot study demonstrate differential sensitivity to PhIP among subgroups and therefore, this assay have potential as a susceptibility marker for prostate cancer risk.

Cytogenetic effects in children treated with methylphenidate.

In recent years there has been a surge in methylphenidate (Ritalin) use for treatment of attention deficit/hyperactivity disorder (ADHD) in children. However, there is a paucity of information on whether this drug poses any potential health risks, such as mutagenicity or carcinogenicity, for humans. To address this issue, we investigated whether this central nervous system stimulant produces cytogenetic abnormalities in pediatric patients at therapeutic levels. In a population composed of twelve children treated with therapeutic doses of methylphenidate, we analyzed three cytogenetic endpoints in peripheral blood lymphocytes obtained before and three months after initiation of treatment with this drug. In all participants, treatment induced a significant 3, 4.3 and 2.4-fold increase in chromosome aberrations, sister chromatid exchanges and micronuclei frequencies, respectively (P=0.000 in all cases). These findings warrant further investigations of the possible health effects of methylphenidate in humans, especially in view of the well-documented relationship between elevated frequencies of chromosome aberrations and increased cancer risk.

Gender differences in genetic damage induced by the tobacco-specific nitrosamine NNK and the influence of the Thr241Met polymorphism in the XRCC3 gene.

The rapid increase in adenocarcinoma of the lung and mortality amongst women strongly suggests that gender differences exist in sensitivity to certain tobacco carcinogens. In the current study, we performed the mutagen-sensitivity assay, with the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), to test the hypothesis that women are more sensitive to the genotoxic effects of NNK than men. Chromosome aberration (CA) frequencies in peripheral blood lymphocytes (PBLs) from 99 patients were evaluated before and after in vitro exposure to NNK. Because the Thr241Met polymorphism in the DNA-repair gene XRCC3 is associated with increased risk of tobacco-related cancers, especially among women, we also tested the hypothesis that individuals who inherit the homozygous variant 241Met allele are more sensitive to the genotoxic effects of NNK. CA frequency was significantly higher 1 hr after NNK treatment in women, compared with men (P = 0.02). When smoking and gender were considered together, a significant interaction was observed. PBLs from female smokers had significantly higher frequencies of NNK-induced CA, compared with female nonsmokers 1 hr after treatment (P = 0.02). We observed no overall effect of the Thr241Met polymorphism on NNK-induced CA in men, women, smokers, or nonsmokers. Overall, our data indicate that women are more sensitive to the genotoxic effects of NNK than men. Because in past years smoking among women has increased, and in view of the close correlation between NNK exposure and adenocarcinoma of the lung, our data provide a plausible explanation for the recent increase in the incidence of this cancer among women.

Establishment and characterization of a new cell line of canine inflammatory mammary cancer: IPC-366.

Canine inflammatory mammary cancer (IMC) shares epidemiologic, histopathological and clinical characteristics with the disease in humans and has been proposed as a natural model for human inflammatory breast cancer (IBC). The aim of this study was to characterize a new cell line from IMC (IPC-366) for the comparative study of both IMC and IBC. Tumors cells from a female dog with clinical IMC were collected. The cells were grown under adherent conditions. The growth, cytological, ultrastructural and immunohistochemical (IHC) characteristics of IPC-366 were evaluated. Ten female Balb/SCID mice were inoculated with IPC-366 cells to assess their tumorigenicity and metastatic potential. Chromosome aberration test and Karyotype revealed the presence of structural aberration, numerical and neutral rearrangements, demonstrating a chromosomal instability. Microscopic examination of tumor revealed an epithelial morphology with marked anysocytosis. Cytological and histological examination of smears and ultrathin sections by electron microscopy revealed that IPC-366 is formed by highly malignant large round or polygonal cells characterized by marked atypia and prominent nucleoli and frequent multinucleated cells. Some cells had cytoplasmic empty spaces covered by cytoplasmic membrane resembling capillary endothelial cells, a phenomenon that has been related to s vasculogenic mimicry. IHC characterization of IPC-366 was basal-like: epithelial cells (AE1/AE3+, CK14+, vimentin+, actin-, p63-, ER-, PR-, HER-2, E-cadherin, overexpressed COX-2 and high Ki-67 proliferation index (87.15 %). At 2 weeks after inoculating the IPC-366 cells, a tumor mass was found in 100 % of mice. At 4 weeks metastases in lung and lymph nodes were found. Xenograph tumors maintained the original IHC characteristics of the female dog tumor. In summary, the cell line IPC-366 is a fast growing malignant triple negative cell line model of inflammatory mammary carcinoma that can be used for the comparative study of both IMC and IBC.

Effect of XPD/ERCC2 polymorphisms on chromosome aberration frequencies in smokers and on sensitivity to the mutagenic tobacco-specific nitrosamine NNK.

Polymorphisms in DNA-repair genes could contribute to the interindividual differences in cancer susceptibility in smokers. By reducing DNA-repair capacity, these polymorphisms may influence the net level of smoking-induced genetic damage significantly, a critical step in the cascade of events leading to cancer. In this biomonitoring study, we examined the relationship between polymorphisms in the DNA-repair gene XPD/ERCC2 and genetic damage. We tested the hypothesis that coding polymorphisms in XPD/ERCC2 limit DNA-repair efficiency in humans leading to increased frequencies of chromosome aberration (CA) in their lymphocytes. We also used the mutagen-sensitivity assay, with the tobacco-specific nitrosamine NNK as a model mutagen, to determine whether lymphocytes from individuals with the variant XPD alleles are more sensitive to this tobacco-specific carcinogen. We calculated odds ratios (ORs) as estimates of relative risk of increased frequencies of CA associated with two XPD polymorphisms (Asp312Asn in exon 10 and Lys751Gln in exon 23). We observed a 2.57-fold (95% confidence limit [CL] = 0.88-7.50; P = 0.10) increase in risk of elevated in vivo frequencies of CA associated with the variant 312Asn allele in the total population. The relative risk was more pronounced in smokers (OR = 4.67; 95% CL = 1.04-20.90; P = 0.04) and in all subjects >48 years old (OR = 7.33; 95% CL = 1.53-35.10; P = 0.01). Similarly, elevations in NNK-induced aberrations were significantly associated with the 312Asn allele (OR = 3.69; 95% CL = 1.29-10.56; P = 0.02). The risk was higher in smokers (OR = 4.62; 95% CL = 1.14-18.70; P = 0.04) and in subjects >48 years old (OR = 5.76; 95% CL = 1.30-25.41; P = 0.03). No significant effect was observed with the 715Gln variant allele in relation to either in vivo or NNK-induced CA. These data suggest that the Asp312Asn polymorphism may alter the phenotype of the XPD protein, resulting in reduced DNA-repair capacity.

The cytokinesis-blocked micronucleus assay as a strong predictor of lung cancer: extension of a lung cancer risk prediction model.

BACKGROUND: There is an urgent need to improve lung cancer outcome by identifying and validating markers of risk. We previously reported that the cytokinesis-blocked micronucleus assay (CBMN) is a strong predictor of lung cancer risk. Here, we validate our findings in an independent external lung cancer population and test discriminatory power improvement of the Spitz risk prediction model upon extension with this biomarker. METHODS: A total of 1,506 participants were stratified into a test set of 995 (527 cases/468 controls) from MD Anderson Cancer Center (Houston, TX) and a validation set of 511 (239 cases/272 controls) from Massachusetts General Hospital (Boston, MA). An epidemiologic questionnaire was administered and genetic instability was assessed using the CBMN assay. RESULTS: Excellent concordance was observed between the two populations in levels and distribution of CBMN endpoints [binucleated-micronuclei (BN-MN), binucleated-nucleoplasmic bridges (BN-NPB)] with significantly higher mean BN-MN and BN-NPB values among cases (P < 0.0001). Extension of the Spitz model led to an overall improvement in the AUC (95% confidence intervals) from 0.61 (55.5-65.7) with epidemiologic variables to 0.92 (89.4-94.2) with addition of the BN-MN endpoint. The most dramatic improvement was observed with the never-smokers extended model followed by the former and current smokers. CONCLUSIONS: The CBMN assay is a sensitive and specific predictor of lung cancer risk, and extension of the Spitz risk prediction model led to an AUC that may prove useful in population screening programs to identify the "true" high-risk individuals. IMPACT: Identifying high-risk subgroups that would benefit from screening surveillance has immense public health significance.

Use of the cytokinesis-blocked micronucleus assay to detect gender differences and genetic instability in a lung cancer case-control study.

BACKGROUND: Although tobacco exposure is the predominant risk factor for lung cancer, other environmental agents are established lung carcinogens. Measuring the genotoxic effect of environmental exposures remains equivocal, as increases in morbidity and mortality may be attributed to coexposures such as smoking. METHODS: We evaluated genetic instability and risk of lung cancer associated with exposure to environmental agents (e.g., exhaust) and smoking among 500 lung cancer cases and 500 controls using the cytokinesis-blocked micronucleus (CBMN) assay. Linear regression was applied to estimate the adjusted means of the CBMN endpoints (micronuclei and nucleoplasmic bridges). Logistic regression analyses were used to estimate lung cancer risk and to control for potential confounding by age, gender, and smoking. RESULTS: Cases showed significantly higher levels of micronuclei and nucleoplasmic bridges as compared with controls (mean ± SEM = 3.54 ± 0.04 vs. 1.81 ± 0.04 and mean ± SEM = 4.26 ± 0.03 vs. 0.99 ± 0.03, respectively; P < 0.001) with no differences among participants with or without reported environmental exposure. No differences were observed when stratified by smoking or environmental exposure among cases or controls. A difference in lung cancer risk was observed between nonexposed male and female heavy smokers, although it was not statistically significant (I(2) = 64.9%; P value for Q statistic = 0.09). CONCLUSIONS: Our study confirms that the CBMN assay is an accurate predictor of lung cancer and supports the premise that heavy smoking may have an effect on DNA repair capacity and in turn modulate the risk of lung cancer. IMPACT: Identifying factors that increase lung cancer risk may lead to more effective prevention measures.

Modulation of Radiation-Induced Genetic Damage by HCMV in Peripheral Blood Lymphocytes from a Brain Tumor Case-Control Study.

Human cytomegalovirus (HCMV) infection occurs early in life and viral persistence remains through life. An association between HCMV infection and malignant gliomas has been reported, suggesting that HCMV may play a role in glioma pathogenesis and could facilitate an accrual of genotoxic damage in the presence of g-radiation; an established risk factor for gliomas. We tested the hypothesis that HCMV infection modifies the sensitivity of cells to γ-radiation-induced genetic damage. We used peripheral blood lymphocytes (PBLs) from 110 glioma patients and 100 controls to measure the level of chromosome damage and cell death. We evaluated baseline, HCMV-, γ-radiation and HCMV + γ-radiation induced genetic instability with the comprehensive Cytokinesis-Blocked Micronucleus Cytome (CBMN-CYT). HCMV, similar to radiation, induced a significant increase in aberration frequency among cases and controls. PBLs infected with HCMV prior to challenge with γ-radiation led to a significant increase in aberrations as compared to baseline, γ-radiation and HCMV alone. With regards to apoptosis, glioma cases showed a lower percentage of induction following in vitro exposure to γ-radiation and HCMV infection as compared to controls. This strongly suggests that, HCMV infection enhances the sensitivity of PBLs to γ-radiation-induced genetic damage possibly through an increase in chromosome damage and decrease in apoptosis.

The L84F and the I143V polymorphisms in the O6-methylguanine-DNA-methyltransferase (MGMT) gene increase human sensitivity to the genotoxic effects of the tobacco-specific nitrosamine carcinogen NNK.

O-Methylguanine-DNA-methyltransferase (MGMT) is a direct-reversal DNA repair protein that removes DNA adducts formed by alkylating mutagens found in tobacco smoke. Several coding single nucleotide polymorphisms (cSNPs) in the MGMT gene have been reported. However, their effect on the levels and types of genetic damage induced by specific environmental carcinogens remains to be fully elucidated. We developed two novel genotyping techniques and used them, in conjunction with the mutagen-sensitivity assay, to test the hypothesis that the L84F and I143V cSNPs in the MGMT gene confer increased sensitivity to genetic damage induced by the alkylating tobacco-specific nitrosamine carcinogen NNK. Lymphocytes from 114 healthy volunteers were exposed in vitro to NNK, and the genotoxic response was assessed by measuring chromosome aberration (CA) frequencies. A significant (P<0.02) increase in NNK-induced CA was observed in cells from individuals with the 84F polymorphism compared to cells from individuals homozygous for the referent L84 allele. A significant positive interaction between this cSNP and smoking, gender and age was observed (P<0.03). In subjects with the variant 143V allele, significantly higher levels of NNK-induced CA were observed in males and in young subjects (<43 years old) compared to subjects homozygous for the referent I143 allele (P<0.02). Individuals who inherited two cSNPs had significantly higher levels of NNK-induced CA compared to individuals with none or with one cSNP (P<0.002). These new data suggest that the 84F and 143V cSNPs may alter the function characteristics of the MGMT protein, resulting in suboptimal repair of genetic damage induced by NNK.