Elin H. Kure

Comparative synchronous fluorescence spectrophotometry and 32P-postlabelling analysis of PAH-DNA adducts in human lung and the relationship to TP53 mutations

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were studied in human lung from 39 lung cancer patients by synchronous fluorescence spectrophotometric (SFS) and 32P-postlabeling assays. Regression analysis of the samples failed to detect any correlation between benzo[a]pyrene-diolepoxide (BPDE)-DNA adducts detected by SFS and the BPDE co-migrating spot detected by 32P-postlabeling. We have also analyzed the relationship between adduct levels and TP53 mutations. By postlabeling diagonal radioactive zone (DRZ) adducts were detected in 37 of 39 (95%) lung tissues from lung cancer patients and the adduct level ranged from 6.81 to 108.50 adducts/10(8) nucleotide. Thirty-three of 39 (85%) had detectable levels of BPDE-DNA adducts (> 1 adduct/10(9) nucleotide). Current heavy smokers (> 20 cigarettes/day) have significantly higher DRZ adduct levels compared to individuals smoking less than 20 cigarettes/day. By SFS combined with immunoaffinity column (IAC), 11 of 39 (28%) samples had detectable adduct levels, and 6 of 11 (55%) were detectable by SFS following purification of benzo[a]pyrene (BP)-tetrols by high pressure liquid chromatography (HPLC). Six of 33 (18%) samples were positive for BPDE-DNA adducts by both postlabeling and HPLC/SFS. No correlation was observed between the SFS and 32P-postlabeling assays for the detection of BPDE-DNA adducts. However, there was a good correlation between adduct levels detected by IAC/SFS and HPLC/SFS. We found a weak association between total PAH-DNA adduct levels in lung tissue and TP53 mutations.

Influence of GSTM1, GSTT1, GSTP1, NAT1, NAT2, EPHX1, MTR and MTHFR polymorphism on chromosomal aberration frequencies in human lymphocytes.

We have studied the influence of genetic polymorphisms in the xenobiotic-metabolizing genes GSTM1, GSTP1, GSTT1, EPHX1, NAT1 and NAT2 and the folate-metabolizing genes MTR and MTHFR on the frequencies of cells with chromosomal aberrations (CAs) in peripheral lymphocytes of Norwegian men. Log-linear Poisson regression models were applied on 357 subjects of whom data on all the polymorphisms examined were available. Total CAs and chromosome-type aberrations (CSAs) were significantly increased by higher age alone, whereas chromatid-type aberrations (CTAs) were elevated by the GSTT1-null genotype and MTHFR codon 222 variant allele and chromatid gaps (CTGs) by EPHX1 high activity genotype and occupational exposure. Stratification by smoking and age (<40 and ≥40 years) showed that the effect of the GSTT1 null and EPHX1 high activity genotypes only concerned (older) smokers, in agreement with the roles of the respective enzymes in detoxification and metabolic activation. The MTHFR codon 222 variant allele was associated with high CTGs in smokers, the MTR codon 919 variant allele with high CTAs in older smokers and the NAT2 fast acetylator genotype with high CTGs in older subjects. Among younger nonsmokers, however, carriers of the MTHFR codon 222 and MTR codon 919 variant alleles showed a decrease in the level of CTGs and total CAs, respectively. In conclusion, polymorphisms of GSTT1, EPHX1, MTHFR, MTR and NAT2 differentially affect the frequency of CTAs, CSAs and CTGs, showing interaction with smoking and age. It appears that CA subtypes rather than total CAs should be considered in this type of studies.

Abstract 2398: KRAS mutation status is a strong prognostic factor in some but not all subtypes of non-small cell lung cancer.

Non-small cell lung cancer is cancer killer #1, and consists of a variety of histological subtypes of which adenocarcinoma, squamous cell carcinoma and large cell carcinoma are most frequent. Genetic aberrations are regarded both prognostic and predicitive in NSCLC, and are found with various frequency and different significance in the subtypes. Mutation in the KRAS gene is a well-known factor in NSCLC, but the prognostic impact is still debated, and the role as target of therapy is under investigation. We intended to study the frequency and prognostic importance of KRAS mutations in a large number of early stage lung cancers of all major histological subtypes. Material and methods Tumor tissue was obtained from 321 consecutively operated lung cancer patients admitted to Oslo University Hospital-Rikshospitalet in the period 2006-2012. Tissue was taken from the excised tumours, snap frozen in liquid nitrogen in the operation room, and stored at -80oC until DNA isolation. The tumour cell content in the specimens was found to be more than 70% in most samples. DNA was isolated according to standard procedures, and mutation analysis was done with a TaqMan-based wobble-enhanced amplification refractory mutation system, for detecting the seven most commonly reported mutations of clinical importance in the KRAS gene; g.34G>C (p.G12R), g.34G>A (p.G12S), g.34G>T (p.G12C), g.35G>A (p.G12D), g.35G>C (p.G12A), g.35G>T (p.G12V), and g.38G>A (p.G13D). After thorough follow-up (median 34.6 months, range 6.3-80.6), relapse-free survival was calculated with the Kaplan-Meier method using Chi-Square for significance assessment. Results Tissues from 154 females and 167 males were analyzed; 212 (66%) adenocarcinomas, 89 (28%) squamous cell carcinomas and 20 (6%) large cell carcinomas. 182 (57%) were in stage I, 88 (27%) II and 51 (16%) stage III. 24 (7,5%) were never-smokers. Overall, 89 patients (27.7%) harbored a KRAS mutation, 78 (36.8%) of adenocarcinomas, 5 (5.6%) of squamous cell carcinomas and 6 (30%) of large cell carcinomas. The most frequent mutation subtype was G12C (51.7%). In the total group there was a significant relapse-free survival difference (p=0.021) in favor of the wild-type. In the adenocarcinoma subgroup, however, there was no survival difference (p=0.316), whereas there was a major difference in the non-adenocarcinoma group (p Conclusions KRAS mutations confer a survival disadvantage in non-small cell lung cancers. This effect is only seen in non-adenocarcinoma subtypes, which might have implications in therapy strategies. Citation Format: Odd Terje Brustugun, Marius Lund-Iversen, Maria M. Bjaanaes, Ann Rita Halvorsen, Martina L. Skrede, Lars H. Jorgensen, Elin H. Kure, Aslaug Helland. KRAS mutation status is a strong prognostic factor in some but not all subtypes of non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2398. doi:10.1158/1538-7445.AM2013-2398