P53 AND RAS GENE MUTATIONS IN PATIENTS WITH NON-SMALL CELL LUNG CARCINOMAS IN SERBIA

Vesna Minic*, Gordana Andjelic,Vojkan  Stanic and Zvonko Magic
Institute of Medical Research, Military Medical Academy, Crnotravska 17,
11000 Belgrade, Serbia and Montenegro
*uranium@drenik.net

INTRODUCTION. Lung cancer is one of the leading causes of cancer-related deaths in the world and despite advances in clinical management, mortality and incidence have shown similar increasing trends over the last two decades. The poor survival may be ascribed to its biological aggressiveness and a relative lack of symptoms attributable to early disease, combined with the rather poor sensitivity of the classical approaches for early detection (1). Therefore, efforts aimed at early identification and interventions in lung cancer are of the highest importance. Mutations in p53 tumor suppressor gene and ras oncogenes appear to play a significant role in the development of Non-Small Cell Lung Carcinoma (NSCLC), which accounts for ~75-80% of all lung cancer cases. We analyzed 50 patients with histologically confirmed NSCLC, different histological subtypes, TNM stages and histological grades to determine the incidence of p53, H-ras and K-ras mutations.

METHOD. We analyzed primary tumors obtained from 50 patients with NSCLC who underwent curative operations at the Military Medical Academy, Belgrade. Tumors were frozen at liquid nitrogen and stored at -80⁰C until DNA extraction. Genomic DNA was extracted by a digestion with proteinase K and phenol extraction - ethanol precipitation method (2). DNA fragments covering the coding regions of exons 5, 6, 7 and 8 and exon 1 H-ras and K-ras genes were amplified by PCR, using specific oligonucleotide primers for all genes. After amplification, PCR products were analyzed by SSCP method, on 10% non-denaturing polyacrylamide gels, which were stained with silver nitrate. The statistical significance of the association between histological subtype, grade and stage and the founded frequency of gene mutations was determined by the c²-test.

RESULTS. Mutations in p53 gene were detected in 24 out of 50 samples (48.0%) with higher incidence in squamous cell carcinomas (SCC) and large cell carcinomas (LCC). Difference between mutations detected in SCC and AC (adenocarcinomas) was statistically significant (c²=7.3, p<0.01). Mutations in H-ras gene were found only in 2 of 50 analyzed carcinomas (4.0%).  Both mutations were found in SCC making the overall incidence in this histological subtype 7.4% (2 of 27). K-ras mutations were detected in 36.0% (18/50) of carcinomas, with higher incidence in stage I carcinomas (45%). While 47.4% carcinomas with histological grade G2 carried mutations in K-ras gene, only 18.2% G3 carcinomas carried such a mutation. The difference in a number of mutations founded in these G2 and G3 carcinomas was statistically significant (c²=4.01; p<0.05).

DISCUSSION. Our results agree with the reported incidence of p53 mutations in NSCLC (3) indicating that these mutations are common in NSCLC. Since the mutations are more frequent in early-stage of this carcinoma, it appears that mutations in p53 gene could be an early event in lung carcinogenesis. Furthermore, it seems that mutations in K-ras gene also present an early event in genesis of NSCLC, and not only in AC, that majority of previously reports indicated (3), but in SCC as well. Based on the facts that we find the high incidence of K-ras mutations in stage I carcinomas and higher frequency in G2 than G3 carcinomas, we could conclude that the oncogene activation of K-ras is an early but not an initial event in the genesis of NSCLC. At last, H-ras mutations seem to play an inferior role in lung carcinogenesis and appear only in SCC.

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