Analysis Of Correlation Among Some Genes Of 60 Cases Of Large Cell Lung Carcinoma (LCLC) And Connection Between Prognosis And Them

Objectives:Activating mutations in the EGFR tyrosine kinase domain, increased EGFR gene copy numbers and EGFR protein overexpression have been found associated with favorable response to EGFR-TKIs, while K-Ras gene mutation has been reported to predict primary resistance to EGFR-TKIs and associate with poor response. High levels of VEGF protein expression are correlated with increased probability of response to anti-VEGF therapy. ERCC1 protein positive expression was a useful marker for clinical resistance to platinum chemotherapy in NSCLC. To observe EGFR, K-Ras proto-oncogene, ERCC1 and VEGF genes mutational or expressional status, the correlation among them and prognosis in LCLC, preliminarily explore the potential prevalent or recessive patients of EGFR-targeted therapy, chemotherapy and anti-VEGF therapy. Meanwhile, evaluate the clinical reliability of real-time quantitative PCR method.Methods:Assess potential biomakers included EGFR exonl8-21 and K-Ras exon2 mutations by DNA direct sequencing; EGFR, RCC1 and VEGF protein expression by IHC; EGFR gene copy number by FISH in a cohort of 60 cases of LCLC, then compared with follow-up data to analyze the correlation among them and influence of prognosis and clarify the molecular basis. The results of EGFR and K-Ras mutations which detected by RT-PCR method were compared with DNA direct sequencing.Results:1 (1.7%) patient had EGFR L858M point mutation in exon 21, K-Ras mutations were found in 3 (5.0%) patients (2 cases were G12C point mutation and another was G12D point mutation),6 EGFR mutations (1 case with in-frame deletion in exon19,1 case was G719C mutation and 4 cases were L858R point mutations) 4 K-Ras mutations (including G12C in 3 patients and G12V in 1 patient) were revealed by RT-PCR, only 2 cases K-Ras mutation were both detected by two methods.19.6% (10/51) patients had high EGFR gene copy number (FISH positive), positive expression rates of EGFR, ERCC1 and VEGF proteins were 38.3%,56.7% and 70.0% respectively. EGFR FISH positive was more seen in patients who had EGFR protein positive expression (Spearman's correlation coefficient=0.390, P=0.005). A trend was found that EGFR FISH positive was more frequently found in patients who had ERCC1 protein positive expression (P=0.433) and K-Ras wild type (P=0.486), K-Ras mutation was more frequently found in patients who had ERCC1 protein negative expression (P=0.411) and VEGF protein positive expression (P=0.252), but there was no statistically significant. No statistically significant correlations were observed among EGFR, K-Ras, ERCC1 and VEGF genes status. Compared with lymph node metastasis negative patients, VEGF protein positive expression was more frequently found in patients who had lymph node metastasis (84.6% vs 58.8%, P=0.046). There were no statistically significant differences in EGFR protein expression, EGFR FISH status, K-Ras mutation and ERCC1 expression related to any of the analyzed clinicopathologic variables. In the univariate analysis of LCLC patients, advanced staging (P=0.000) and the presence of lymph node metastasis (P=0.049) were significantly meaningful indicators of a poor prognosis. The Cox regression model revealed that only TNM staging was an independent prognostic factor for overall survival (hazard ratio [HR]=3.347,95%CI=1.791-6.254; P= 0.000). EGFR, K-Ras, ERCC1 and VEGF genes status did not influence on overall survival.Conclusions:Compared with lung adenocarcinoma, the rare incidence of increased EGFR gene copy number and the low rate of EGFR mutation observed in our series suggest that patients with LCLC are likely to obtain little benefit from EGFR-TKIs therapy. EGFR gene amplification is one of the important mechanisms for EGFR protein overexpression. No conclusive correlations were observed among EGFR, ERCC1 and VEGF genes. VEGF protein overexpression maybe an negative prognostic factor in LCLC. EGFR, K-Ras, ERCC1 and VEGF genes status did not influence on overall survival. Only TNM stages and lymph node metastasis status had a significant impact on survival of LCLC, the Cox regression model revealed that TNM staging was an independent prognostic factor for survival. RT-PCR technique as another method to detect major EGFR and K-Ras mutations from formalin-fixed paraffin-embedded samples was not accord with the results of DNA direct sequencing method, the further research for clinical application of RT-PCR technique is needed.