Mutational analysis of EGFR-related genes in non-small cell lung cancer

Abstract

The genetic abnormalities of non-small cell lung cancer (NSCLC) are heterogeneous and dependent on factors including tumor type, tobacco exposure, ethnicity, gender, etc. The epidermal growth factor receptor (EGFR) pathway is important for normal lung tissue growth and repair. In lung cancers, EGFR mutations are frequently observed in adenocarcinomas (AD) from non-smoking women especially of Asian origin. Other genes interacting with EGFR and/or converge on common downstream effectors may be involved in lung cancers. In this study, we investigated for mutations of EGFR and its dimer partner HER2, KRAS and BRAF which converge on the downstream MAPK pathway, and MET which converges on STAT3 pathway in 175 NSCLC including 135 AD, 27 squamous cell carcinoma (SCC), 8 lymphoepithelioma-like carcinomas (LELC) which are etiologically related to Epstein-Barr virus, and 5 adenosquamous carcinomas. There were 74 non-smokers, 62 smokers, 12 passive and 27 ex-smokers comprising 82 women and 93 men. For EGFR, mutations involving the kinase domain (exons 18-21) were found in 59 of 135 AD (43.7%) and 4 of other tumors. The mutations were more frequent in female (45/82, 54.9%) than male (18/93; 19.4%; P<.001), and for AD, more frequent in non- and passive smokers (47/77, 61.0%) compared to ex- and current smokers (12/58; 20.7%; P<.001). KRAS (codon 12) mutations were found in AD (18/135; 13.3%) only, and were more frequent in smokers and ex-smokers (14/89; 15.7%) than non-smokers and passive smokers (4/86; 4.65%; P=.023). HER2 (exon 18-21) and BRAF (exon 11 and 15) mutations were found in 2.22% (3/135) and 1.48% (2/135), respectively when all AD were considered; and 5.17% (3/58) and 1.72% (1/58), respectively when only AD with EGFR/KRAS wild-types were considered. For MET, RT-PCR was performed to detect juxtamembrane domain changes spanning exon 14. Four tumors (2.96%) of all 135 AD, or (6.90%) of 58 AD with wild-type EGFR and KRAS showed MET exon 14 deletion. Further sequencing analysis using intronic primers and genomic DNA showed that in all 4 mutants, the deletions were due to somatic intronic mutations at different positions leading to splicing abnormalities. Fluorescence in situ hybridization (FISH) analysis using a MET locus-specific probe showed increased MET signal number compared to normal lymphocytes in two tumors for which tissues were available for analysis. Overall, apart from EGFR mutations, all other mutations were found in AD only. Mutations of EGFR, KRAS, HER2 and MET were found in mutual exclusion to one-another. One tumor harbored both BRAF and KRAS mutations. There was no correlation of HER2, BRAF or MET mutant tumors with gender, smoking history or other clinicopathological data but the numbers of mutant cases were too few for meaningful analysis. The results suggested that of the genes studied in our NSCLC, EGFR and KRAS mutations were the major oncogenic drivers while HER2, BRAF and MET contribute only a small proportion in our population.