Genetic polymorphisms of MPO, GSTT1, GSTM1, GSTP1, EPHX1 and NQO1 as risk factors of early-onset lung cancer.

Early-onset lung cancer diagnosed up to the age of 50 is a very rare disease, with an increasing incidence rate. Differences in aetiology, characteristics and epidemiology of early and older onset lung cancer have been described previously, suggesting the importance of genetic factors in early-onset lung cancer aetiology. A case-control study was conducted to investigate the effects of genetic polymorphisms in the MPO, EPHX1, GSTT1, GSTM1, GSTP1 and NQO1 genes on the risk of early-onset lung cancer development. Six hundred thirty-eight Caucasian patients under the age of 51 with confirmed primary lung cancer and 1,300 cancer free control individuals, matched by age and sex, were included in this analysis. Seventeen single nucleotide polymorphisms and two deletion polymorphisms were genotyped. No significant association was found for any of the analyzed polymorphisms and overall lung cancer risk. Nonsignificantly decreased risk of lung cancer was observed for carriers of 1 or 2 copies of GSTM1. Subgroup analysis revealed gender- and/or smoking-specific effects of EPHX1 rs2854455 (IV-1464C > T) and rs2234922 (His139Arg), GSTT1 deletion, GSTP1 rs1695 (Ile105Val), rs947895 (+991C > A) and rs4891 (Ser185Ser) and NQO1 rs1800566 (Pro187Ser) polymorphisms. However, none of the observed effects were confirmed by interaction tests nor were they significant after Bonferroni correction for multiple testing. In summary, our study suggested a modifying effect of polymorphisms in EPHX1, GSTP1, GSTT1, GSTM1 and NQO1 genes on the risk of early-onset lung cancer. To confirm these observations and to eliminate possible bias in our analyses, larger studies are warranted.

CYP450 polymorphisms as risk factors for early-onset lung cancer: gender-specific differences.

Cytochrome P450 (CYP) enzymes, involved in metabolism of tobacco carcinogens, are also involved in estrogen metabolism and many are regulated by estrogens. These genes may thus be of relevance to gender-specific differences in lung cancer risk, particularly in early-onset lung cancer, where a high proportion of women is observed. We conducted a case-control study to investigate genetic polymorphisms in cytochromes that might modify the risk of developing early-onset lung cancer. In total, 638 Caucasian patients under the age of 51 with primary lung cancer and 1300 cancer-free control individuals, matched by age and sex, were included in this analysis. Thirteen polymorphisms in the CYP1A1, CYP1B1, CYP2A13, CYP3A4 and CYP3A5 genes were analyzed. No significant association was found for any of the analyzed polymorphisms and lung cancer risk overall. However, among women, a significantly increased risk of early-onset lung cancer was observed for carriers of the minor allele of CYP1B1 SNP rs1056836 [odds ratio (OR) 1.97; 95% confidence interval (CI) 1.32-2.94; P < 0.001]. Also, a non-significant increase in lung cancer risk was observed in the group of women carriers of the minor allele of CYP2A13 SNP rs1709084 (OR 1.64; 95% CI 1.00-2.70; P = 0.05). The effect of these two polymorphisms was shown to be modified by smoking. Haplotype analysis was performed for CYP1B1 and CYP2A13. No differences between cases and controls were observed for both genes (P = 0.63 and P = 0.42 for CYP1B1 and CYP2A13, respectively). Our results suggest that the CYP1B1 and the CYP2A13 genotypes may contribute to individual susceptibility to early-onset lung cancer in women.

A multiplex real-time PCR method for detection of GSTM1 and GSTT1 copy numbers.

OBJECTIVES: Deletion polymorphisms of Glutathione-S-transferase (GST) M1 and T1 are considered risk factors for various diseases. However, most previous studies only distinguished "null" and "non-null" genotypes. Our aim was to develop a reliable, high-throughput GSTM1/T1 genotyping method able to determine allele copy numbers. DESIGN AND METHODS: We developed a multiplex real time PCR method to distinguish between heterozygous (1/0) and homozygous (1/1) GSTM1 and GSTT1 genotypes. The principle of relative quantification was applied and an expectation-maximisation (EM) algorithm was developed to assign one of 3 possible genotypes: 1/1, 1/0 or 0/0 for each of the two genes. RESULTS: 1320 Caucasians were genotyped using the newly developed method. The observed genotype distributions did not deviate from the expected and were in Hardy-Weinberg equilibrium. GSTM1 duplication was detected in one sample. CONCLUSION: This new semiquantitative genotyping method is a sensitive and promising tool for large-scale molecular epidemiological and clinical studies.

Polymorphisms in ABCG2, ABCC3 and CNT1 genes and their possible impact on chemotherapy outcome of lung cancer patients.

The prognosis of lung cancer patients treated with chemotherapy is poor, motivating the search for predictive factors. Single nucleotide polymorphisms (SNPs) in membrane transporter genes could influence the pharmacokinetics of cytostatic drugs and therefore affect treatment outcome. We examined 6 SNPs with known or suspected phenotypic effect: ABCG2 G34A, C421A; ABCC3 C-211T, G3890A, C3942T and CNT1 G565A. For 349 Caucasian patients with primary lung cancer [161 small cell lung cancer (SCLC), 187 nonsmall cell lung cancer (NSCLC) and 1 mixed] receiving first-line chemotherapy 3 different endpoints were analyzed: response after the 2nd cycle (R), progression-free survival (PFS) and overall survival (OS). The prognostic value of the SNPs was analyzed using multivariable logistic regression, calculating odds ratios (ORs) when comparing genotype frequencies in responders and nonresponders after the 2nd cycle. Hazard ratios (HRs) for PFS and for OS were calculated using Cox regression methods. In all lung cancer patients, none of the investigated polymorphisms modified response statistically significant. The only significant result in the histological subpopulations was in SCLC patients carrying the ABCC3 -211T allele who showed significantly worsened PFS (HR: 1.79; 95% confidence interval (CI) 1.13-2.82). In an exploratory subgroup analysis significantly worse OS was seen for carriers of the ABCG2 421A-allele treated with platinum-based drugs (HR: 1.60; 95% CI 1.04-2.47; n = 256). In conclusion, this study prioritizes ABCC3 C-211T and ABCG2 C421A as candidate transporter SNPs to be further investigated as possible predictors of the clinical outcome of chemotherapy in lung cancer patients.

Effect of NAT1 and NAT2 genetic polymorphisms on colorectal cancer risk associated with exposure to tobacco smoke and meat consumption.

N-Acetyltransferases 1 and 2 (NAT1 and NAT2), both being highly polymorphic, are involved in the metabolism of aromatic and heterocyclic aromatic amines present in cigarette smoke and red meat cooked by high-temperature cooking techniques. We investigated the effect of differences in acetylation capacity, determined by NAT1 and NAT2 genotypes, on colorectal cancer risk associated with exposure to tobacco smoke or red meat consumption. In this population-based case-control study in Germany, 505 patients with incident colorectal cancer and 604 age- and sex-matched control individuals with genotyping data and detailed risk factor information were included. Genotyping of NAT1 and NAT2 genetic polymorphisms was done using a fluorescence-based melting curve analysis method. The association between genotypes, environmental exposures, and colorectal cancer risk was estimated using multivariate logistic regression. Colorectal cancer risk associated with active smoking was elevated after accumulation of 30(+) pack-years of smoking [odds ratio (OR), 1.4; 95% confidence interval (95% CI), 0.9-2.2] but not significantly modified by either NAT1 or NAT2 genotype. Exposure to environmental tobacco smoke was associated with an increased risk for colorectal cancer only among NAT2 fast acetylators (OR, 2.6; 95% CI, 1.1-5.9 for exposure in childhood and adulthood). Frequent consumption of red meat significantly increased colorectal cancer risk for the group comprising all NAT2 fast acetylators or carriers of the NAT1*10 allele (OR, 2.6; 95% CI, 1.1-6.1) but not among those with "slow" NAT1 and NAT2 genotypes. Our findings indicate that NAT1 and NAT2 genotypes may contribute jointly to individual susceptibility and that heterocyclic aromatic amines may play an important role in colorectal cancer associated with red meat and possibly also exposure to environmental tobacco smoke.

Identification and functional characterization of the natural variant MRP3-Arg1297His of human multidrug resistance protein 3 (MRP3/ABCC3).

The human multidrug resistance protein 3 (MRP3, symbol ABCC3) is an ATP-binding cassette transporter that mediates the efflux of organic anions, including lipophilic substances conjugated with glucuronate, sulphate or glutathione, across the basolateral membrane of polarized cells (e.g. hepatocytes) into blood. Genetic variants of MRP3 may affect the transport of these substances out of cells. The aims of this study were: (i) to identify MRP3 polymorphisms; (ii) to functionally characterize one relatively frequent MRP3 polymorphism; and (iii) to establish whether MRP3 transports bilirubin glucuronosides. Exonic nucleotide variants in the ABCC3 gene were identified by single-strand conformation polymorphism analysis. The 3890G>A mutation, resulting in MRP3-ArgHis, was introduced into the ABCC3 cDNA which was stably transfected into MDCKII cells. For the functional characterization of MRP3-ArgHis in comparison with MRP3, ATP-dependent transport was analysed in isolated membrane vesicles. Two non-synonymous MRP3 variants were identified with an allele frequency of 0.003 for 1643T>A (MRP3-LeuGln) and 0.08 for 3890G>A (MRP3-ArgHis). Because of the high frequency of the 3890G>A mutation, and because of the close proximity of Arg to the second nucleotide-binding domain, we pursued the functional characterization of the MRP3-ArgHis polymorphic variant. MRP3-ArgHis was correctly localized to the basolateral membrane of polarized MDCKII cells. We identified monoglucuronosyl bilirubin, bisglucuronosyl bilirubin and leukotriene C4 as substrates for both MRP3 and MRP3-ArgHis. Dehydroepiandrosterone-3-sulphate and 17beta-glucuronosyl oestradiol were transported with similar kinetics by MRP3 and MRP3-ArgHis. This experimental setup provides a useful tool to analyse the functional consequences of polymorphic variants of MRP3.

Genotype relationships in the CYP3A locus in Caucasians.

Genetic polymorphisms of the human CYP3A family affect clinical drug efficacy and may modify cancer risk. CYP3A genes show high sequence similarity that had previously lead to misallocation of CYP3A polymorphisms. Recent studies indicated a high degree of or even complete linkage for certain CYP3A alleles. Reliable LightCycler-based genotyping methods were developed and their degree of linkage in a large Caucasian population (n = 1210) investigated. Strong linkage disequilibrium was confirmed between CYP3A4, CYP3A5, and CYP3AP1 (each at P < 10(-5)). Contrary to some previous results claiming complete linkage between the phenotypically relevant CYP3A5*1 and a variant in a pseudogene promoter region CYP3AP1*1 we found among 428 controls (15 of 66) and 782 lung cancer cases (25 of 115) approximately 22% of CYP3AP1*1 carriers to be homozygous for CYP3A5*3 We conclude that contrary to previous assumptions, the CYP3AP1 genotype is not a reliable predictor for CYP3A5 activity.

The CYP3A4*1B allele increases risk for small cell lung cancer: effect of gender and smoking dose.

CYP3A isozymes are involved in tobacco carcinogen- and steroid-metabolism, and are expressed in human lung tissue showing interindividual variation in expression and activity. The CYP3A4*1B allele has been associated with a two-fold higher promoter activity and with high-grade prostate cancers. The very frequent intron 3 polymorphism in the CYP3A5 gene (CYP3A5*3) results in decreased CYP3A5 protein levels. A case-control study was conducted in 801 Caucasian lung cancer patients that included 330 adenocarcinomas, 260 squamous cell carcinomas, 171 small cell lung cancers (SCLC) and 432 Caucasian hospital-based controls. CYP3A-genotyping was performed by capillary polymerase chain reaction followed by fluorescence-based melting curve analysis. A significantly increased SCLC risk for CYP3A4*1B allele carriers [odds ratio (OR) 2.25, 95% confidence interval (CI) 1.11-4.55, P=0.02] was found. After dividing cases and controls by gender, an increased lung cancer risk for CYP3A4*1B carriers (OR 3.04, 95% CI 0.94-9.90, P=0.06) for women but not for men (OR 1.00, 95% CI 0.56-1.81) was revealed. Heavier smoking men (> or =20 pack-years) with the CYP3A4*1B allele had a significant OR for lung cancer of 3.42 (95% CI 1.65-7.14, P=0.001) compared to *1A/*1A carriers with lower tobacco exposure (<20 pack-years). For women, the respective OR was 8.00 (95% CI 2.12-30.30, P=0.005). Genotype frequencies were generally in Hardy-Weinberg equilibrium, except for CYP3A5 where a greater than expected number of CYP3A5*1 homozygotes was observed among cases (P=0.006). In addition, we observed linkage disequilibrium of CYP3A4 and CYP3A5 (P<0.00001), but a non-significantly increased lung cancer risk was only found for homozygous CYP3A5*1 allele carriers (OR 5.24, 95% CI 0.85-102.28, P=0.14) but not for heterozygotes. To confirm our observation that the CYP3A4*1B allele increases SCLC risk and modifies the smoking-related lung cancer risk in a gender-specific manner, further studies, including CYP3A haplotype analysis, will be necessary.

Myeloperoxidase (MPO) genotype and lung cancer histologic types: the MPO -463 A allele is associated with reduced risk for small cell lung cancer in smokers.

MPO participates in the metabolic activation of tobacco carcinogens such as PAHs. A frequent MPO -463 G-->A polymorphism in the promoter region reduces MPO transcription and has been correlated with >4-fold lower benzo[a]pyrene-DNA adduct levels in the skin of coal tar-treated patients. Four of 7 case-control studies found significantly reduced lung cancer risk associated with the A allele. Due to their different etiologies, we examined whether the MPO genotype affects histologic lung cancer types differentially. A case-control study was conducted in 625 ever-smoking lung cancer patients, including 228 adenocarcinomas, 224 SCCs, 135 SCLCs and 340 ever-smoking hospital controls. MPO genotyping was performed by capillary PCR followed by fluorescence-based melting curve analysis. Combining the MPO -463 (G/A+A/A) genotypes, a protective effect approaching significance (OR = 0.75, 95% CI 0.55-1.01) was observed when comparing all lung cancer cases to controls. Among histologic types of lung cancer, a weak protective effect was found for both adenocarcinoma (OR = 0.81, CI 0.55-1.19) and SCC (OR = 0.82, CI 0.56-1.21); a stronger and significant effect was found for SCLC (OR = 0.58, CI 0.36-0.95; p = 0.029). Our results also suggest that the MPO genotype varies among inflammatory nonmalignant lung diseases. In conclusion, our results emphasize the need for a separate analysis of lung cancer histologic types and an adjustment for inflammatory nonmalignant lung diseases in future MPO-related studies. We confirm that the MPO -463 A variant affords a protective effect against lung cancer risk in smokers, which was strongest for SCLC patients.

Differential effect of NAT2 on the association between active and passive smoke exposure and breast cancer risk.

BACKGROUND: Polymorphisms in the N-acetyltransferase 2 (NAT2) gene influence the rate of metabolism of aromatic and heterocyclic amines present in tobacco smoke. Because the physicochemical composition of mainstream and sidestream smoke differ, we conducted a case-control study to assess a possible differential effect of NAT2 genotype on the relationship between active/passive smoke exposure and breast cancer risk. METHODS: Breast cancer patients diagnosed by 50 years of age and population-sampled controls were interviewed to obtain detailed lifetime active and passive smoking history. NAT2 genotype was determined in 422 breast cancer patients and 887 controls. Multivariate logistic regression analysis was performed to estimate breast cancer risk in relation to smoking history by acetylator status and interaction effects. RESULTS: Compared with women never regularly exposed to tobacco smoke, odds ratios (ORs) for current smoking and ex-smoking were 1.7 [95% confidence interval (CI): 1.0-2.9] and 1.2 (95% CI, 0.7-2.0) in slow acetylators, and not increased in rapid acetylators. Active smoking variables, such as pack-years, duration of smoking, and time since cessation, showed significant dose-response relationships with breast cancer risk among slow acetylators but not rapid acetylators. In contrast, passive smoking was associated with higher risk in rapid than in slow acetylators, with ORs of 2.0 (95% CI, 1.0-4.1) and 1.2 (95% CI, 0.7-2.0), respectively. CONCLUSIONS: Our results suggest that the NAT2 status has a differential effect on the association of active and passive smoking with breast cancer and demonstrate the need to consider possible different mechanisms associated with exposure to main- and sidestream tobacco smoke.