Association of promoter methylation of RASSF1A and KRAS mutations in non-small cell lung carcinoma in Kashmiri population (India)

BACKGROUND: Non-small cell lung carcinoma (NSCLC) incidence and progression is increasing because of genetic and epigenetic changes. The mutations in the Kirsten rat sarcoma (KRAS) are the most frequently oncogene aberrations in lung carcinoma patients. A candidate tumor suppressor gene (TSG) Ras Association Domain Family 1 Isoform A (RASSF1A), is silenced by promoter hypermethylation in several human malignancies including non-small cell lung carcinoma (NSCLC). We hypothesized that RASSF1A methylation and KRAS mutations may play an important role in NSCLC. METHODS: Non-small cell lung carcinoma patients (n = 100) and equal number of healthy controls were assessed for activating KRAS (exon 2) mutations using allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) and promoter hypermethylation of RASSF1A using methylation specific PCR. RESULTS: The frequency of mutations in Kirsten rat sarcoma (KRAS) were found in 31% of NSCLC patients in the Kashmiri population and occur most commonly, but not exclusively, in adenocarcinoma histology and life-long smokers. The NSCLC patients in advanced stage reported the higher frequency of mutation in KRAS (exon 2). A significant higher frequency of this mutation was reported in patients with NSCLC (29.16%) who are positive for metastasis (P < 0.03). The frequencies of promoter hypermethylation at Ras Association Domain Family 1 Isoform A (RASSF1A) were 41% in cases and 3% in control samples. The frequency of KRAS mutation and RASSF1A promoter methylation were significantly different between adenocarcinomas (ADC) and squamous cell carcinomas (SCC) patients with NSCLC (P < 0.03). In addition, we reported that NSCLC patients having RASSF1A promoter methylation was significantly associated with smoking (P = 0.01). It was identified that NSCLC patients with RASSF1A promoter region hypermethylation had poorer survival and faster disease progression compared with those without hypermethylation of RASSF1A promoter region (P = 0.0001). The Median survivals among with cases containing promoter region hypermethylation of RASSF1A were 17.20 and 42.13 months for patients without promoter region hypermethylation of RASSF1A and the patients with KRAS mutation with or without hypermethylation of the promoter region of RASSF1A a tumor suppressor gene had poorer survival compared with those patients with wild type KRAS gene, with or without hypermethylation of RASSF1A promoter region. These differences were statistically significant based on Log-rank (Mantel-cox) test (P = 0.0001). The median survivals among patients with mutation in KRAS protooncogene were 16 months and 42 months for NSCLC patients with wild type KRAS gene. CONCLUSIONS: The aberrant RASSF1A gene promoter methylation with the subsequent mutation in KRAS gene (exon 2) plays a significant role in the pathogenesis and disease progression of non-small cell lung carcinoma (NSCLC).