EPAC-lung: pooled analysis of circulating tumour cells in advanced non-small cell lung cancer.

INTRODUCTION: We assessed the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with advanced non-small cell lung cancer (NSCLC) by undertaking a pooled analysis of individual patient data. METHODS: Nine European NSCLC CTC centres were asked to provide reported/unreported pseudo-anonymised data for patients with advanced NSCLC who participated in CellSearch CTC studies from January 2003 to March 2017. We used Cox regression models, stratified by centres, to establish the association between CTC count and survival. We assessed the added value of CTCs to prognostic clinicopathological models using likelihood ratio (LR) statistics and c-indices. RESULTS: Seven out of nine eligible centres provided data for 550 patients with prognostic information for overall survival. CTC counts of ≥2 and ≥ 5 per 7·5 mL were associated with reduced progression-free survival (≥2 CTCs: hazard ratio [HR] = 1.72, p < 0·001; ≥5 CTCs: HR = 2.21, p < 0·001) and overall survival (≥2 CTCs: HR = 2·18, p < 0·001; ≥5 CTCs: HR = 2·75, p < 0·001), respectively. Survival prediction was significantly improved by addition of baseline CTC count to LR clinicopathological models (log-transformed CTCs p < 0·001; ≥2 CTCs p < 0·001; ≥5 CTCs p ≤ 0·001 for both survival end-points), whereas moderate improvements were observed with the use of c-index models. There was some evidence of between-centre heterogeneity, especially when examining continuous counts of CTCs. CONCLUSIONS: These data confirm CTCs as an independent prognostic indicator of progression-free survival and overall survival in advanced NSCLC and also reveal some evidence of between-centre heterogeneity. CTC count improves prognostication when added to full clinicopathological predictive models.

A novel microfluidic platform for size and deformability based separation and the subsequent molecular characterization of viable circulating tumor cells.

Circulating tumor cells (CTCs) were introduced as biomarkers more than 10 years ago, but capture of viable CTCs at high purity from peripheral blood of cancer patients is still a major technical challenge. Here, we report a novel microfluidic platform designed for marker independent capture of CTCs. The Parsortix™ cell separation system provides size and deformability-based enrichment with automated staining for cell identification, and subsequent recovery (harvesting) of cells from the device. Using the Parsortix™ system, average cell capture inside the device ranged between 42% and 70%. Subsequent harvest of cells from the device ranged between 54% and 69% of cells captured. Most importantly, 99% of the isolated tumor cells were viable after processing in spiking experiments as well as after harvesting from patient samples and still functional for downstream molecular analysis as demonstrated by mRNA characterization and array-based comparative genomic hybridization. Analyzing clinical blood samples from metastatic (n = 20) and nonmetastatic (n = 6) cancer patients in parallel with CellSearch(®) system, we found that there was no statistically significant difference between the quantitative behavior of the two systems in this set of twenty six paired separations. In conclusion, the epitope independent Parsortix™ system enables the isolation of viable CTCs at a very high purity. Using this system, viable tumor cells are easily accessible and ready for molecular and functional analysis. The system's ability for enumeration and molecular characterization of EpCAM-negative CTCs will help to broaden research into the mechanisms of cancer as well as facilitating the use of CTCs as "liquid biopsies."

VEGFR-2, CXCR-2 and PAR-1 germline polymorphisms as predictors of survival in pancreatic carcinoma.

BACKGROUND: Hypoxic environment of pancreatic cancer (PC) implicates high vascular in-growth, which may be influenced by angiogenesis-related germline polymorphisms. Our purpose was to evaluate polymorphisms of vascular endothelial growth factor receptor 2 (VEGFR-2), CXC chemokine receptor 2 (CXCR-2), proteinase-activated receptor 1 (PAR-1) and endostatin (ES) as prognostic markers for disease-free (DFS) and overall survival (OS) in PC. PATIENTS AND METHODS: Genotyping of 173 patients, surgically treated for PC between 2004 and 2011, was carried out by TaqMan(®) genotyping assays or polymerase chain reaction. Chi-square test, Kaplan-Meier estimator and Cox regression hazard model were used to assess the prognostic value of selected polymorphisms. RESULTS: VEGFR-2 -906 T/T and PAR-1 -506 Del/Del genotypes predicted longer DFS (P = 0.003, P = 0.014) and OS (VEGFR-2 -906, P = 0.011). CXCR-2 +1208 T/T genotype was a negative predictor for DFS (P < 0.0001). Combined analysis for DFS and OS indicated that patients with the fewest number of favorable genotypes simultaneously present (VEGFR-2 -906 T/T, CXCR-2 +1208 C/T or C/C and PAR-1 -506 Del/Del) were at the highest risk for recurrence or death (P < 0.0001). CONCLUSION: VEGFR-2 -906 C>T, CXCR-2 +1208 C>T and PAR-1 -506 Ins/Del polymorphisms are potential predictors for survival in PC.

DNA copy number loss and allelic imbalance at 2p16 in lung cancer associated with asbestos exposure.

Five to seven percent of lung tumours are estimated to occur because of occupational asbestos exposure. Using cDNA microarrays, we have earlier detected asbestos exposure-related genomic regions in lung cancer. The region at 2p was one of those that differed most between asbestos-exposed and non-exposed patients. Now, we evaluated genomic alterations at 2p22.1-p16.1 as a possible marker for asbestos exposure. Lung tumours from 205 patients with pulmonary asbestos fibre counts from 0 to 570 million fibres per gram of dry lung, were studied by fluorescence in situ hybridisation (FISH) for DNA copy number alterations (CNA). The prevalence of loss at 2p16, shown by three different FISH probes, was significantly increased in lung tumours of asbestos-exposed patients compared with non-exposed (P=0.05). In addition, a low copy number loss at 2p16 associated significantly with high-level asbestos exposure (P=0.02). Furthermore, 27 of the tumours were studied for allelic imbalances (AI) at 2p22.1-p16.1 using 14 microsatellite markers and also AI at 2p16 was related to asbestos exposure (P=0.003). Our results suggest that alterations at 2p16 combined with other markers could be useful in diagnosing asbestos-related lung cancer.

Gene expression and copy number profiling suggests the importance of allelic imbalance in 19p in asbestos-associated lung cancer.

Asbestos is a pulmonary carcinogen known to give rise to DNA and chromosomal damage, but the exact carcinogenic mechanisms are still largely unknown. In this study, gene expression arrays were performed on lung tumor samples from 14 heavily asbestos-exposed and 14 non-exposed patients matched for other characteristics. Using a two-step statistical analysis, 47 genes were revealed that could differentiate the tumors of asbestos-exposed from those of non-exposed patients. To identify asbestos-associated regions with DNA copy number and expressional changes, the gene expression data were combined with comparative genomic hybridization microarray data. As a result, a combinatory profile of DNA copy number aberrations and expressional changes significantly associated with asbestos exposure was obtained. Asbestos-related areas were detected in 2p21-p16.3, 3p21.31, 5q35.2-q35.3, 16p13.3, 19p13.3-p13.1 and 22q12.3-q13.1. The most prominent of these, 19p13, was further characterized by microsatellite analysis in 62 patients for the differences in allelic imbalance (AI) between the two groups of lung tumors. 79% of the exposed and 45% of the non-exposed patients (P=0.008) were found to be carriers of AI in their lung tumors. In the exposed group, AI in 19p was prevalent regardless of the histological tumor type. In adenocarcinomas, AI in 19p appeared to occur independently of the asbestos exposure.

Increased expression of high mobility group A proteins in lung cancer.

High mobility group A (HMGA) proteins play an important role in the regulation of transcription, differentiation, and neoplastic transformation. In this work, the expression of HMGA 1 and 2 in 152 lung carcinomas of mainly non-small-cell histological type has been studied by immunohistochemistry in order to evaluate their feasibility as lung cancer markers. In 17 lung cancer cases, the related bronchial epithelial changes were also studied for HMGA1 and 2 expression. RNA expression of HMGA1a and b isoforms and of HMGA2 was determined by real-time semi-quantitative RT-PCR in 23 lung carcinomas. High expression of HMGA1 and HMGA2 at both mRNA and protein levels was detected in lung carcinomas, compared with normal lung tissue. Nuclear immunostaining for HMGA1 and 2 proteins also occurred in hyperplastic, metaplastic, and dysplastic bronchial epithelium. Increased nuclear expression of HMGA1 and 2 correlated with poor survival (for adenocarcinomas, HMGA1, p=0.006; HMGA2, p=0.05). While the expression of HMGA2 was significantly associated with cell proliferation (p=0.008), HMGA1 expression did not show any association with proliferation or apoptotic index. Sequencing of HMGA2 transcripts from tumours with very high expression showed a normal full-length transcript. As HMGA proteins were expressed in about 90% of lung carcinomas and their expression was inversely associated with survival, they may provide useful markers for lung cancer diagnosis and prognosis.

L1CAM, INP10, P-cadherin, tPA and ITGB4 over-expression in malignant pleural mesotheliomas revealed by combined use of cDNA and tissue microarray.

Malignant pleural mesothelioma (MM) is a rare tumour with high mortality, which can exhibit various morphologies classified as epithelioid, biphasic and sarcomatoid subtypes. To investigate the molecular changes in these tumours, we studied gene expression patterns by combined use of cDNA arrays and tumour tissue microarrays (TMA). Deregulation of the expression of 588 cancer-related genes was screened in 16 MM comprising all three subtypes and compared with references, i.e. normal mesothelial cell lines and pleural mesothelium. Array data were analysed using three statistical methods; principal component analysis (PCA), permutation test and receiver operating characteristic (ROC) curves. Eleven genes were verified by real-time RT-PCR. Genes encoding two adhesion molecules [COL1A2 and integrin beta4 (ITGB4)] and a chemokine (INP10) were up-regulated in MM compared with both the cell lines and pleural mesothelium. There was a type-specific up-regulation of semaphorin E, ITGB4 and P-cadherin in epithelioid MM, matrix metalloproteinase 9 (MMP9) and tissue-type plasminogen activator (tPA) in sarcomatoid MM and neural cell adhesion molecule L1 (L1CAM) and INP10 in biphasic MM. Immunohistochemistry on TMA containing 47 MM (26 epithelioid, 15 sarcomatoid and six biphasic) was performed for five proteins, ITGB4, P-cadherin, tPA, INP10 and L1CAM. INP10 expression was increased in MM in general compared with normal mesothelium, while increased expression of P-cadherin, L1CAM and ITGB4 was more specific in MMs exhibiting an epithelioid growth pattern. The over-expression of tPA was more frequent in epithelioid MM despite higher mRNA levels in sarcomatoid and biphasic MM. We conclude that several proteins, associated with cell adhesion either directly (ITGB4, L1CAM, P-cadherin) or as a regulatory factor (INP10), are differentially expressed in MM. In particular, INP10, ITGB4 and COL1A2 were up-regulated in MM compared with both reference sample types, suggesting a relationship with development of these tumours.

Expression of myeloid-specific genes in childhood acute lymphoblastic leukemia - a cDNA array study.

Several specific cytogenetic changes are known to be associated with childhood acute lymphoblastic leukemia (ALL), and many of them are important prognostic factors for the disease. Little is known, however, about the changes in gene expression in ALL. Recently, the development of cDNA array technology has enabled the study of expression of hundreds to thousands of genes in a single experiment. We used the cDNA array method to study the gene expression profiles of 17 children with precursor-B ALL. Normal B cells from adenoids were used as reference material. We discuss the 25 genes that were most over-expressed compared to the reference. These included four genes that are normally expressed only in the myeloid lineages of the hematopoietic cells: RNASE2, GCSFR, PRTN3 and CLC. We also detected over-expression of S100A12, expressed in nerve cells but also in myeloid cells. In addition to the myeloid-specific genes, other over-expressed genes included AML1, LCP2 and FGF6. In conclusion, our study revealed novel information about gene expression in childhood ALL. The data obtained may contribute to further studies of the pathogenesis and prognosis of childhood ALL.

Glutathione-S-transferase M1, M3, T1 and P1 polymorphisms and susceptibility to non-small-cell lung cancer subtypes and hamartomas.

Polymorphic glutathione-S-transferase (GST) genes causing variations in enzyme activity may influence individual susceptibility to lung cancer. In this case-control study (consisting of 389 Caucasian lung cancer patients, including 151 adenocarcinomas (ACs) and 172 squamous cell carcinomas (SCCs), and 353 hospital control subjects without malignant disease, genotype frequencies for GSTM1, GSTM3, GSTP1 and GSTT1 were determined by polymerase chain reaction (PCR)/ restriction fragment length polymorphism (RFLP)-based methods. While adjusted odds ratios (ORs) indicated no significantly increased risk for lung cancer overall due to any single GST genotype, the risk alleles for GSTM1, GSTM3 and GSTP1 conferring reduced enzyme activity were present at higher frequency in SCC than in AC patients. This is consistent with a reduced detoxification of carcinogenic polycyclic aromatic hydrocarbons (PAHs) from cigarette smoke that are more important for the development of SCC than for AC. An explorative data analysis also identified statistically significantly increased ORs for the combinations GSTT1 non-null and GSTP1 GG or AG for lung cancer overall (OR 2.23, CI 1.11-4.45), and for SCC (OR 2.69, CI 1.03-6.99). For lung cancer overall, and especially among SCC patients, the GSTT1 null genotype was underrepresented (SCC 11.2% v. control subjects 19%, P = 0.026, OR 0.57, CI 0.30-1.06). Additionally, in 28 patients with hamartomas, the GSTT1 null genotype was also protective (P = 0.013), while GSTP1 variant allele carriers were overrepresented (OR 2.48, CI 1.06-6.51). In conclusion, GST genotypes may act differently, either by detoxifying harmful tobacco carcinogens and/or by eliminating lung cancer chemopreventive agents. The latter role for GSTT1 would explain the observed lower risk of SCC and hamartoma associated with GSTT1 null. Further confirmatory studies are required.

Modulation of DNA and protein adducts in smokers by genetic polymorphisms in GSTM1,GSTT1, NAT1 and NAT2.

The formation of DNA and protein adducts by environmental pollutants is modulated by host polymorphisms in genes that encode metabolizing enzymes. In our study on 67 smokers, aromatic-DNA adduct levels were examined by nuclease P1 enriched 32P-postlabelling in mononuclear blood cells (MNC) and 4-aminobiphenyl-haemoglobin adducts (4-ABP-Hb) by gas chromatography-mass spectroscopy. Genetic polymorphisms in glutathione S-transferase M1 (GSTM1), T1 (GSTT1) and N-acetyl-transferase 1 (NAT1) and 2 (NAT2) were assessed by polymerase chain reaction-based methods. DNA adduct levels, adjusted for the amount of cigarettes smoked per day, were higher in GSTM1(-/-) individuals (1.30 +/- 0.57 adducts per 108 nucleotides) than in GSTM1(+) subjects (1.03 +/- 0.56, P = 0.05), higher in NAT1 slow acetylators (1.58 +/- 0.54) than in NAT1 fast acetylators (1.11 +/- 0.58, P = 0.05) and were also found to be associated with the NAT2 acetylator status (1.29 +/- 0.64 and 1.03 +/- 0.46, respectively, for slow and fast acetylators, P = 0.06). An effect of GSTT1 was only found in combination with the NAT2 genotype; individuals with the GSTT1(-/-) and NAT2-slow genotype contained higher adduct levels (1.80 +/- 0.68) compared to GSTT1(+)/NAT2 fast individuals (0.96 +/- 0.36). Highest DNA adduct levels were observed in slow acetylators for both NAT1 and NAT2 also lacking the GSTM1 gene (2.03 +/- 0.17), and lowest in GSTM1(+) subjects with the fast acetylator genotype for both NAT1 and NAT2 (0.91 +/- 0.45, P = 0.01). No overall effects of genotypes were observed on 4-ABP-Hb levels. However, in subjects smoking less than 25 cigarettes per day, 4-ABP-Hb levels were higher in NAT2 slow acetylators (0.23 +/- 0.10 ng/g Hb) compared to fast acetylators (0.15 +/- 0.07, P = 0.03). These results provide further evidence for the combined effects of genetic polymorphisms in GSTM1, GSTT1, NAT1 and NAT2 on DNA and protein adduct formation in smoking individuals and indicate that, due to the complex carcinogen exposure, simultaneous assessment of multiple genotypes may identify individuals at higher cancer risk.

Glutathione S-transferase genotypes and allergic responses to diisocyanate exposure.

Diisocyanates are the most common low molecular weight chemicals to cause occupational asthma. However, only some 5-10% of exposed workers develop asthma, which suggests an underlying genetic susceptibility. Diisocyanates and their metabolites may be conjugated with glutathione by glutathione S-transferases (GSTs). We examined whether polymorphisms in the GSTM1, GSTM3, GSTP1 and GSTT1 genes modify allergic responses to diisocyanate exposure. The study population consisted of 182 diisocyanate exposed workers, 109 diagnosed with diisocyanate-induced asthma and 73 without asthma. Lack of the GSTM1 gene (null genotype) was associated with a 1.89-fold risk of diisocyanate-induced asthma [95% confidence interval (CI) 1.01-3.52]. Moreover, among the asthma patients, the GSTM1 null genotype was associated with lack of diisocyanate-specific immunoglobulin (Ig)E antibodies [odds ratio (OR) 0.18, 95% CI 0.05-0.61] and with late reaction in the specific bronchial provocation test (OR 2.82, 95% CI 1.15-6.88). Similarly, GSTM3 AA genotype was related to late reaction in the specific bronchial provocation test (OR 3.75, 95% CI 1.26-11.2). The GSTP1 Val/Val genotype, on the other hand, was related to high total IgE levels (OR 5.46, 95% CI 1.15-26.0). The most remarkable effect was seen for the combination of GSTM1 null and the GSTM3 AA genotype which was strongly associated with lack of diisocyanate-specific IgE antibodies (OR 0.09, 95% CI 0.01-0.73) and with late reaction in the bronchial provocation test (OR 11.0, 95% CI 2.19-55.3). The results suggest, for the first time, that the polymorphic GSTs, especially the mu class GSTs, play an important role in inception of ill effects related to occupational exposure to diisocyanates.

Relevance of N-acetyltransferase 1 and 2 (NAT1, NAT2) genetic polymorphisms in non-small cell lung cancer susceptibility.

The highly polymorphic N-acetyltransferases (NAT1 and NAT2) are involved in both activation and inactivation reactions of numerous carcinogens, such as tobacco derived aromatic amines. The potential effect of the NAT genotypes in individual susceptibility to lung cancer was examined in a hospital based case-control study consisting of 392 Caucasian lung cancer patients [152 adenocarcinomas, 173 squamous cell carcinomas (SCC) and 67 other primary lung tumours] and 351 controls. In addition to the wild-type allele NAT1*4, seven variant NAT1 alleles (NAT1*3, *10, *11, *14, *15, *17 and *22) were analysed. A new method based on the LightCycler (Roche Diagnostics Inc.) technology was applied for the detection of the polymorphic NAT1 sites at nt 1088 and nt 1095. The NAT2 polymorphic sites at nt 481, 590, 803 and 857 were detected by polymerase chain reaction-restriction fragment length polymorphism or LightCycler. Multivariate logistic regression analyses were performed taking into account levels of smoking, age, gender and occupational exposure. An increased risk for adenocarcinoma among the NAT1 putative fast acetylators [odds ratio (OR) 1.92 (1.16-3.16)] was found but could not be detected for SCC or the total case group. NAT2 genotypes alone appeared not to modify individual lung cancer risk, however, individuals with combined NAT1 fast and NAT2 slow genotype had significantly elevated adenocarcinoma risk [OR 2.22 (1.03-4.81)] compared to persons with other genotype combinations. These data clearly show the importance of separating different histological lung tumour subtypes in studies on genetic susceptibility factors and implicate the NAT1*10 allele as a risk factor for adenocarcinoma.

hOGG1 polymorphism and loss of heterozygosity (LOH): significance for lung cancer susceptibility in a caucasian population.

Oxidative damage is implicated in several chronic diseases including cancer. 8-Hydroxyguanine (8-oxoG) is one of the major promutagenic DNA lesions, which is produced by reactive oxygen species, causes G:C to T:A transversions and is excised by OGG1, an 8-oxoG specific DNA glycosylase/AP-Lyase. In a nested case-control study, gDNA from 105 Caucasian primary non-small cell lung cancer cases and 105 matched controls was screened for 6 possible new polymorphic sites in the human OGG1 gene, detected previously mainly in tumour tissue. The previously described Ser(326)Cys polymorphism was found to be common (allele frequency 0.22) in Caucasians. However, no major difference in Ser(326)Cys genotype distribution could be detected between cases and controls. Two 5;-end polymorphisms previously found in Japanese as well as Arg(131)Gln could not be detected in this population. An Ala(85)Ser polymorphism was found in 2 controls, whereas Arg(46)Gln was detected in only 1 case. As the hOGG1 gene is mapped (3p26.2) to a region frequently lost in primary lung tumours, the frequency of loss of heterozygosity (LOH) was investigated. Forty-three percent of the studied lung tumours exhibited loss of one of the hOGG1 alleles. The wt Ser(326) allele was not predominantly lost in our sample set, which suggests a minor role of this polymorphism in tumourgenesis. Our results show that LOH at the hOGG1 gene locus is a very common occurrence in lung tumourgenesis, possibly leading to increased mutational damage due to ROS in smokers. However, the hOGG1 polymorphisms studied are probably not major contributors to individual lung cancer susceptibility in Caucasians.

CYP2D6 gene polymorphism in caucasian smokers: lung cancer susceptibility and phenotype-genotype relationships.

Individual susceptibility to smoking-related cancers is proposed to partly depend on a genetically determined ability to metabolise tobacco carcinogens. We previously reported on the association between the activity of the xenobiotic-metabolising enzyme CYP2D6 and lung cancer risk in a hospital-based case-control study among French Caucasian smokers. Here we extended the study to address the effect of four gene-inactivating mutations (CYP2D6(*)3, (*)4, (*)5 and (*)16) and the gene duplication of the CYP2D6 gene (CYP2D6(*)2x2 or CYP2D6(*)1x2) on lung cancer risk in the same population (150 patients with primary lung carcinoma of squamous cell or small cell histology and 172 controls). The risk of lung cancer associated with the CYP2D6 poor metaboliser genotype (odds ratio 1.5, 95% confidence interval 0.5-4.3) did not differ from that in the reference category of extensive metaboliser and ultra-rapid metaboliser genotypes combined. Lung cancer risks for the CYP2D6 PM genotype amongst light smokers (tobacco consumption 20 g/day) were not significantly different. The present findings agree with the discrepancy between the phenotype-based and genotype-based studies indicated by the recent meta-analyses.

Genetic polymorphism of CYP genes, alone or in combination, as a risk modifier of tobacco-related cancers.

Tobacco use is causally associated with cancers of the lung, larynx, mouth, esophagus, kidneys, urinary tract, and possibly, breast. Major classes of carcinogens present in tobacco and tobacco smoke are converted into DNA-reactive metabolites by cytochrome P450 (CYP)-related enzymes, several of which display genetic polymorphism. Individual susceptibility to cancer is likely to be modified by the genotype for enzymes involved in the activation or detoxification of carcinogens in tobacco and repair of DNA damage. We summarize here the results of case-control studies published since 1990 on the effects of genetic variants of CYP1A1, 1A2, 1B1, 2A6, 2D6, 2E1, 2C9, 2C19, 17, and 19 alone or in combination with detoxifying enzymes as modifiers of the risk for tobacco-related cancers. The results of studies on gene-gene interactions and the dependence of smoking-related DNA adducts on genotype were also analyzed. Some CYP variants were associated with increased risks for cancers of the lung, esophagus, and head and neck. The risk was often increased in individuals who also had GSTM1 deficiency. For breast cancer in women, a few studies suggested an association with CYPs related to metabolism of tobacco carcinogens and steroidal hormones. The overall effects of common CYP polymorphisms were found to be moderate in terms of penetrance and relative risk, with odds ratios ranging from 2 to 10. Some CYP1A1/GSTM1 0/0 genotype combinations seem to predispose the lung, esophagus, and oral cavity of smokers to an even higher risk for cancer or DNA damage, requiring, however, confirmation. Future strategies in molecular cancer epidemiology for identifying such susceptible individuals are discussed with emphasis on well-designed larger studies.

Glutathione S-transferase GSTM3 and GSTP1 genotypes and larynx cancer risk.

Glutathione S-transferases (GSTs) are involved in detoxification of reactive metabolites of carcinogens and, therefore, could be potentially important in susceptibility to cancer. The associations between larynx cancer risk and GSTM3 and GSTP1 gene polymorphisms, either separately or in combination with GSTM1 and GSTT1 gene polymorphisms, were evaluated using peripheral blood DNA from 129 cancer patients and 172 controls, all regular smokers. The frequencies of GSTM3 AA, AB, and BB genotypes were 60.5%, 36.4%, and 3.1% in cases and 72.7%, 24.4%, and 2.9% in controls, respectively. The frequencies of GSTP1 AA, AG, and GG genotypes were 48.1%, 40.3%, and 11.6% in cases and 50.0%, 37.2%, and 12.8% in controls, respectively. Multivariate logistic regression analyses did not reveal any association between the GSTP1 (AG or GG) genotype and larynx cancer [odds ratio, 1.1; 95% confidence interval (CI), 0.7-2.0]. In contrast, a significant increase in risk was related to the GSTM3 (AB or BB) genotype (odds ratio, 2.0; 95% CI, 1.1-3.4). The combined GSTM3 (AB or BB) and GSTM1-null genotype conferred a 4-fold risk (95% CI, 1.6-10.1) of larynx cancer as compared with the combined GSTM3 AA and GSTM1-positive genotype. However, the effect of GSTM3 (AB or BB) genotype was similar among individuals with GSTM1-positive or GSTM1-null genotypes.

Glutathione S-transferase GSTM1, GSTM3, GSTP1 and GSTT1 genotypes and the risk of smoking-related oral and pharyngeal cancers.

Several polymorphic glutathione S-transferase enzymes are involved in the detoxification of active metabolites of many potential carcinogens from tobacco smoke and may therefore be important in modulating susceptibility to smoking-related cancers. As part of a hospital-based case-control study performed in France among Caucasian smokers, we studied GSTM1, GSTM3, GSTP1 and GSTT1 gene polymorphisms in 121 patients with oral cavity and pharyngeal cancers and 172 hospital controls using peripheral blood DNA. An increase in risk was found among carriers of the GSTP1 (AG or GG) genotype (OR 1.6, 95% CI 1.0-2.8, p = 0.07) or the GSTT1 null genotype (OR 2.0, 95% CI 1.0-4.0, p = 0.05). The effect of these at-risk genotypes was most marked in subjects with a history of more than 30 years of smoking, among whom the respective ORs were 2.0 (95% CI 1.0-3.9) and 3.3 (95% CI 1.3-8.1), though the interaction tests between these genotypes and duration of smoking were not significant. In contrast, neither the GSTM1 null genotype nor the GSTM3 AA genotype was associated with oropharyngeal cancer risk (OR 0.9, 95% CI 0.5-1.5 and OR = 1.3, 95% CI 0.7-2.3, respectively). Our results thus suggest that GSTP1 and GSTT1 gene polymorphisms modulate susceptibility to smoking-related cancers of the oral cavity and pharynx.

N-acetyltransferase NAT1 and NAT2 genotypes and lung cancer risk.

Acetyltransferases, encoded by the NAT1 and NAT2 genes, are involved in the activation/inactivation reactions of numerous xenobiotics, including tobacco-derived aromatic amine carcinogens. Several allelic variants of NAT1 and NAT2, which cause variations in acetylation capacity, have been detected. The NAT2 slow acetylator phenotype/genotype has been inconsistently associated with lung cancer and, to date, the role of NAT1 polymorphism in lung cancer has not been reported. The effect of NAT1 and NAT2 genetic polymorphisms on individual lung cancer risk was evaluated among 150 lung cancer patients and 172 control individuals, all French Caucasian smokers. The NAT1 alleles (*3, *4, *10, *11, *14, and *15) and the NAT2 alleles (*4, *5, *6, *7) were differentiated by polymerase chain reaction-based restriction fragment length polymorphism methods using DNA extracted from peripheral white blood cells. Genotypes were classified according to current knowledge of the functional activity of the variant alleles. The NAT1*10 and NAT1*11 alleles were considered as rapid alleles, the NAT1*4 and the NAT1*3 as normal alleles and NAT1*14 and NAT1*15 as slow-acetylation alleles. Logistic regression analyses were performed taking into account the age, sex, smoking and occupational exposures. A significant association was observed between lung cancer and NAT1 genotypes (P(homogeneity) < 0.02) with a gene dose effect (P(trend) < 0.01); compared with homozygous rapid acetylators, the lung cancer risk was 4.0 (95% confidence interval 0.8-19.6) for heterozygous rapid acetylators, 6.4 (95% confidence interval 1.4-30.5) for homozygous normal acetylators and 11.7 (95% confidence interval 1.3-106.5) for heterozygous slow acetylators. None of the individuals were homozygous slow acetylators. Similar results were obtained whatever the adjustment considered. No significant association was found between NAT2 genotype and lung cancer. The NAT1 polymorphism may thus be an important modifier of individual susceptibility to smoking-induced lung cancer.

Role of glutathione S-transferase GSTM1, GSTM3, GSTP1 and GSTT1 genotypes in modulating susceptibility to smoking-related lung cancer.

Glutathione S-transferases GSTM1, GSTM3, GSTP1 and GSTT1 are involved in the detoxification of active metabolites of several carcinogens in tobacco smoke. We studied the potential role of GSTM3 and GSTP1 gene polymorphisms either separately, or in combination with GSTM1 and GSTT1 gene polymorphisms, in susceptibility to lung cancer using peripheral blood DNA from 150 lung cancer patients and 172 control individuals, all regular smokers. The frequencies of GSTM3, AA, AB and BB genotypes were 70.7%, 24.0% and 5.3% in cases and 72.7%, 24.4% and 2.9% in control individuals respectively. The frequencies of GSTP1, AA, AG and GG genotypes were 44.7%, 44.0% and 11.3% in cases and 50.0%, 37.2% and 12.8% in control individuals respectively. When studied separately, neither GSTM3 nor GSTP1 genotypes contributed significantly to the risk of lung cancer. Although failing to reach statistical significance, the combined GSTM3 AA and GSTP1 (AG or GG) genotype conferred a nearly threefold risk when the GSTM1 gene was concurrently lacking (odds ratio 2.9, 95% confidence interval 0.7-12.1). Significant interactions were observed between pack-years of smoking and the combined GSTM3 AA and GSTP1 (AG or GG) genotype, or the combined GSTM3 AA, GSTP1 (AG or GG) and GSTM1 null genotype. The combination of these three a priori at risk genotypes conferred an increased risk of lung cancer among smokers with a history of at least 35 pack-years (odds ratio 2.7, 95% confidence interval 1.2-6.0), but not in lighter smokers, probable because of the lower average number of pack-years of smoking found among control individuals with this genotype combination.