Peter Drings

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 Ser326Cys polymorphism was found to be common (allele frequency 0.22) in Caucasians. However, no major difference in Ser326Cys genotype distribution could be detected between cases and controls. Two 5`‐end polymorphisms previously found in Japanese as well as Arg131Gln could not be detected in this population. An Ala85Ser polymorphism was found in 2 controls, whereas Arg46Gln 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 Ser326 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. Int. J. Cancer 88:932–937, 2000.

Altered DNA repair capacity and bleomycin sensitivity as risk markers for non-small cell lung cancer.

DNA repair capacity in human peripheral blood lymphocytes was monitored by the repair rate of bleomycin‐induced DNA damage using an alkaline single‐cell gel electrophoresis assay (comet assay). DNA repair capacity, after 15 min repair time, in lymphocytes of non‐small cell lung cancer patients (n = 160) and controls (n = 180) was 67% and 79.3%, respectively (p < 0.0004). Bleomycin sensitivity defined as the tail moment of bleomycin‐treated peripheral blood lymphocytes, without allowing time for DNA repair, was significantly higher in lung cancer patients than in tumor‐free hospital controls (p < 0.0001). There was no correlation, in either patient or control group, between the bleomycin sensitivity and DNA repair capacity with age or gender. The median values of DNA repair capacity and sensitivity in controls were used as the cut‐off points for calculating odds ratios (OR). After adjustment for age, gender and smoking status, the cases vs. controls had reduced DNA repair capacity (OR = 2.1; 95% confidence limit [CL] 1.1–4.0) and increased bleomycin sensitivity (OR = 4; 95% CL 2.2–7.4). For current smokers, the adjusted risk associated with bleomycin sensitivity was 2.3 (95% CL 1.1–4.9). We conclude that our standard comet assay as a phenotypical repair test has sufficient sensitivity and rapidity allowing application to both native and cryopreserved lymphocytes. Bleomycin sensitivity and DNA repair capacity were found to be 2 independent susceptibility markers for non‐small cell lung cancer, confirming similar investigations with different marker end points. The latter were much more time consuming than the method used in our study. Thus, the comet assay is more suitable for screening large numbers of individuals in epidemiological studies. Validation of this assay in large prospective studies for the identification of subjects at high risk for non‐small cell lung cancer is now warranted.

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.

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.

Sensitive detection of rare cancer cells in sputum and peripheral blood samples of patients with lung cancer by preproGRP-specific RT-PCR

RT‐PCR‐based amplification of transcripts expressed in cancer but not in normal non‐neoplastic cells is increasingly used for the sensitive detection of rare disseminated or exfoliated cancer cells to improve cancer staging and early detection protocols. However, these assays are frequently hampered by false‐positive test results due to low‐level transcription of the marker genes in normal cells. To overcome these limitations, target transcripts have to be identified that are tightly suppressed in normal non‐neoplastic tissues, whereas they should be actively transcribed in the respective cancer cells. Here, we tested RT‐PCR assays for 7 neuroendocrine marker transcripts including NCAM, PGP 9.5, gastrin, gastrin receptor, synaptophysin, preprogastrin‐releasing peptide (preproGRP) and GRP‐receptor to detect rare exfoliated tumor cells in peripheral venous blood and sputum samples from patients with lung cancer. Among these preproGRP RT‐PCR was the only assay with which illegitimate transcription in blood or sputum samples from healthy donors or patients with unrelated diseases did not interfere. However, it reproducibly detected up to 10 small‐cell lung cancer cells diluted in either 10 ml blood or 5 ml sputum samples. Single blood and sputum samples were collected directly before diagnostic bronchoscopy from 175 patients suspected to have lung cancer. Twenty‐six of these had small‐cell lung cancer (SCLC). Thereof, 13 patients (50%) tested positive in the blood sample and 5 of 23 patients (22%) tested positive in the sputum sample. Moreover, among 92 patients with non‐small‐cell lung cancer (NSCLC) 25 patients (27%) had disseminated cancer cells in peripheral blood. Amplification of preproGRP transcripts from clinical samples is a sensitive and specific assay to detect disseminated or exfoliated lung cancer cells either in peripheral blood or sputum samples.

Do genetic factors protect for early onset lung cancer? A case control study before the age of 50 years.

BackgroundEarly onset lung cancer shows some familial aggregation, pointing to a genetic predisposition. This study was set up to investigate the role of candidate genes in the susceptibility to lung cancer patients younger than 51 years at diagnosis.Methods246 patients with a primary, histologically or cytologically confirmed neoplasm, recruited from 2000 to 2003 in major lung clinics across Germany, were matched to 223 unrelated healthy controls. 11 single nucleotide polymorphisms of genes with reported associations to lung cancer have been genotyped.ResultsGenetic associations or gene-smoking interactions was found for GPX1(Pro200Leu) and EPHX1(His113Tyr). Carriers of the Leu-allele of GPX1(Pro200Leu) showed a significant risk reduction of OR = 0.6 (95% CI: 0.4–0.8, p = 0.002) in general and of OR = 0.3 (95% CI:0.1–0.8, p = 0.012) within heavy smokers. We could also find a risk decreasing genetic effect for His-carriers of EPHX1(His113Tyr) for moderate smokers (OR = 0.2, 95% CI:0.1–0.7, p = 0.012). Considered both variants together, a monotone decrease of the OR was found for smokers (OR of 0.20; 95% CI: 0.07–0.60) for each protective allele.ConclusionSmoking is the most important risk factor for young lung cancer patients. However, this study provides some support for the T-Allel of GPX1(Pro200Leu) and the C-Allele of EPHX1(His113Tyr) to play a protective role in early onset lung cancer susceptibility.

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.

Early onset lung cancer, cigarette smoking and the SNP309 of the murine double minute-2 (MDM2) gene

The polymorphism SNP309 (rs2279744) in the promoter region of the MDM2 gene has been shown to alter protein expression and may play a role in the susceptibility to lung cancer. The MDM2 protein is a key inhibitor of p53 and several mechanisms of MDM2/p53 interactions are presently known: modulating DNA-repair, cell-cycle control, cell growth and apoptosis.We used 635 Caucasian patients diagnosed with lung cancer before 51 years of age and 1300 healthy gender and age frequency matched population Caucasian controls to investigate the association between the MDM2 SNP309 and the risk of developing early onset lung cancer. Conditional logistic models were applied to assess the genotype-phenotype association, adjusted for smoking.Compared to the GG genotype, the adjusted ORs for the TG and TT genotype were 0.9 (95% CI: 0.7–1.5) and 1.0 (95% CI: 0.7–1.5), respectively. Also no association was found for histological subtypes of lung cancer. The strength of this study is that within young cases the genetic component to develop lung cancer may be greater. Our results indicate that the MDM2 SNP309 is not significantly associated with lung carcinogenesis but point towards gender-specific differences.

Biology and clinical use of GM-CSF in lung cancer

Administration of granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to induce an increase in production of neutrophils, monocytes, and eosinophils. This is reflected in an accelerated recovery of myelopoiesis following cytotoxic chemotherapy and radiation. Human trials completed so far have used patients with many tumor types. It was clearly demonstrated that the leukocyte responses are dose and schedule dependent and that the route of factor administration is important. The results of clinical trials suggest that the acute toxicity of cytotoxic chemotherapy may be decreased. This may result in a higher dose of drugs or in a reduction of treatment interval, and it may increase the rate of complete remissions and the number of patients with long-term disease free survival, particularly in small cell lung cancer. However, up to now a definitive statement as to the value of this supportive treatment in solid tumor chemotherapy is not possible.

Sensitive detection of few vital small cell lung cancer (SCLC) cells in blood and sputum by RT-PCR

REGIONAL DETERMINATION OF ATP, GLUCOSE AND LACTATE 1N CERVICAL CANCER BIOPSIES G. Schwickert, S. Walenta, W. Mueller-Klieser and E.K. Rofstad Objectives and Methods l0 biopsies of patients with cervical cancer in different clinical stages were taken prior to radiation therapy and investigated using metabolic imaging with a bioluminescent technique. Frozen tissue sections adhered to cover glasses were brought into contact with an enzymatic solution that cuntait~s a specific luciferase. The metabolite of interest, i.e. ATP, glucose or lactate was converted in biochemical reactions resulting in a local, concentration-depandent emission of light. The distribution of light intensity was registered with a highly sensitive video system (Argus 100, Hamamatsu Photonies). Absolute tissue concentration values were obtained by calibration with appropriate standards. Digital image analysis allowed for a correlation of metabolites in parallel sections with regard to histological structures. Results Mean metabolite concentrations of all three metabolites varied between the biopsies over a wide range. This was even true for the comparison of two biopsies from the same tumor. On the other hand, deviations between sections of the same biopsy were much less. The concentration of blcod glucose correlated well with the mean glucose concentrations of bioluminescenco analysis. The distribution pattern of metabolites could be clearly assigned to the histological structures, that were divided into neoplasm, stroma and necrosis. These areas were reflected in histograms of pixel concentration values. In most cases, pixel correlations of metabolite concentrations in parallel sections yielded positive values for Spearman s rank correlation coefficient for all substrates. In some tumors strongly negative values were obtained, ff these correlations were restricted to histologically defined areas. Tumors were classified according to staging, grading and occurrence of metastnses. Correlations between metabolites were better in tumors with documented metastases, than in those without tumor spread. The concentration of lactate was higher in those tumors that were associated with metastases. A similar difference was obtained for glucose, if only areas of neoplasm were considered. So far no obvious relation could be found between the concentration or distribution of metabolites and the staging or grading classes. Conclusions Quantitative biohiminescence and single photon imaging with computerized image analysis allows for the measurement of metabolites in histologically well-defined tumor ater~. Moreover, metabolites in different tumors or in different regions of the same tumor can be compared on a quantitative base. The metabolic milieu in the cervix tumors investigated, is mainly dctermiued by the nutritive supply which can be concluded from the positive correlation between glucose concentration in the blood and in tumor tissue. High glucose and lactate concentrations are associated with a higher incidence of documented metastases. This finding will be substantiated by measurements in a higher number of cancers and different tumor entities. Underlying mechanism have to be investigated in the near future.