| ObjectiveThe most common lung malignancy worldwide is the non-small cell lung cancer (NSCLC) occupied 85% of lung cancer, which is also one of the leading causes of cancer related deaths today. Although early-stage NSCLC patients are treated with complete resection, 30% to 60% will develop recurrence and die as a result of their disease. Clinical trials have tested the ability of adjuvant cisplatin-based chemotherapy to improve survival after complete resection of NSCLC. To date, adjuvant cisplatin-based chemotherapy for completely resected NSCLC patients is becoming a criterion. While an absolute benefit of 4.1% in 5-year overall survival is quite low, the majority couldn't benefit from this chemotherapy. To assign the patients to get benefits from chemotherapy is becoming hot spots in lung cancer study.With the development of molecular bio-technique, the research on cancer has already studied on molecular level. The basic and clinical researchs on cancer include molecular mechanism, molecular diagnosis and molecular prognosis so on. Up to date, the only prospectively validated prognosis and predictive factors that can be used as a guide in the clinics is pathological stage (such as TNM stage). Patients with higher stage tumors with poor prognosis have the greatest potential to benefit from adjuvant cisplatin-based therapy. However, there is no accepted biomarker of prognosis or prediction of outcome. Evaluation of further molecular events associated with carcinogenesis is required to determine who among patients stands the best chances to benefit from this therapeutic approach. As a series of new techniques developing and putting into practices (such as gene array, tissue array and protein array, etc), many molecular biomarker alone or combination are found in lung cancer prognosis analysis. To combine TNM stage and molecular markers to provide more powerful data for NSCLC appropriate treatment protocols. Now more and more attentions are focus on the prognosis and predictive value of bio-marker on lung cancer chemotherapy.It is generally accepted that cytotoxicity of cisplatin is mediated through induction of apoptosis and arrest of cell cycle resulting from its interaction with DNA, such as the formations of cisplatin-DNA adducts, which activates multiple signaling pathways, including those involving p53, etc. Increased expression of anti-apoptotic genes and mutations in the intrinsic apoptotic pathway may contribute to the inability of cells to detect DNA damage or to induce apoptosis. Of the genes that are frequently mutated in lung cancers, TP53, KRAS and EGFR are among the most common. TP53 mutations occur in at least 50% of stage II-III lung cancers. Most mutations are missense, turning the p53 protein into a transcriptionally inactive form that often accumulates within cells. In contrast, KRAS mutations at codon 12 and 13 occur in about 20-25% of adenocarcinomas and in a low proportion on other NSCLC. And EGFR mutations at Exon18 and Exon21 were found in around 40-80% of NSCLC, especially in adenocarcinoma with no smoking in women. In this study, we have investigated whether mutations in TP53, KRAS or EGFR could provide prognostic indications or predict a survival benefit from cisplatin-based adjuvant chemotherapy in completely resected non–small-cell lung cancer in international adjuvant lung cancer trial (IALT).MethodsSpecimens 783 paraffin-embedded tumor samples had participated in the IALT, which compared adjuvant cisplatin-based chemotherapy with observation among patients with non–small cell lung cancer. Twenty-eight centers in 14 countries contributed specimens. Approval was obtained from the local institutional review boards, according to the legal regulations in each participating country. All tumors were reviewed according to the histopathological classification system adopted by the World Health Organization (WHO) in 2004.Detection and analysis of TP53 Mutations Mutation analyses were conducted using genomic DNA isolated from 783 paraffin-embedded archived tissue sections. DNA was extracted by standard QIAamp DNA extraction Kit (QIAGEN S.A.) and exons5 to 8 of TP53 were amplified by polymerase chain reaction (PCR) using their respective primer sequences. PCR products were directly DHPLC or sequenced. PCR products with mutation were reanalyzed by independent PCR and sequencing to confirm the presence and nature of mutations for TP53. The codon distribution and pattern of TP53 mutations were compared with those of the IARC TP53 database, which compiles all mutations described in TP53 in the literature (IARC TP53 database).Detection and analysis of KRAS Mutations Exon1 of KRAS were amplified by polymerase chain reaction (PCR) using its primer sequences. KRAS mutations at codon 12 and 13 were analyzed by directly sequencing and confirmed by sequencing or Restriction Fragment length Polymorphism (RFLP). The prevalence and patterns of KRAS mutations were compared with those of the COSMIC database, which compiles all mutations described in KRAS in the literature (COSMIC database).Detection and analysis of EGFR Mutations Exon18 to 21 of EGFR were amplified by polymerase chain reaction (PCR) using their respective primer sequences. EGFR mutations at exons18 to 21 were analyzed by sequencing and confirmed by a separately sequencing.The prevalence and patterns of EGFR mutations were compared with those of the COSMIC database, which compiles all mutations described in EGFR in the literature (COSMIC database).Statistical Analyses The prognostic values of TP53,KRAS and EGFR status and chemotherapy for survival were studied using a Cox model in order to incorporate factors related to survival.Survival rate was studied by Kaplan—Meier method. All other factors that were statistically related to the TP53,KRAS and EGFR status in the multivariate Log-Rank (P <0 .05) were added to the survival Cox model. All analyses were performed using SAS software, version 8.2.Results1. Results of TP53The prevalence of TP53 mutation was 46% (240/524). The overall prevalence of TP53 mutations in this series is compatible with data in NSCLC from the IARC TP53 database. The codon distribution and mutation patterns of TP53 mutations (in terms of base changes) were consistent with those reported for lung cancers in patients with tobacco smoking history. Specifically, G to T transversion represented 29.97% of all detected mutations, an observation that has been shown to represent a typical mutagen signature of components of tobacco smoke. When all significant variables are put together in a logistic model stratified by centre, only age, sex, pathological TNM stage, T of TNM and quality after final H&E are retained to explain TP53 status.For 5-year survival analysis, TP53 mutation status had no prognostic or predictive value for survival in all NSCLC grouped together. However, cisplatin-based therapy showed a trend to benefit in TP53 wild type and to be harmful in TP53 mutated patients (P =0.18 for Overall Survival (OS) and P =0.13 for Disease Free Survival (DFS). A borderline significant interaction between TP53 and treatment was observed in non-adenocarcinoma patients (test for interaction, P =0.05 for DFS and P =0.25 for OS).Eight-year survival results support those of the 5-year analysis while TP53 mutation has a predictive value at P =0.04 for disease free survival on the whole group. Notably, a significant interaction between TP53 and treatment was observed in non-adenocarcinoma patients (test for interaction, P =0.02 for DFS and P =0.09 for OS).2. Results of KRASThe prevalence of KRAS mutation was 14% (98/718, including 90 mutations at codon 12 and 8 mutations at codon 13). The overall mutation pattern was similar to the one already reported in Cosmic database in lung cancer, with an excess of G to T transversion. More TP53 mutations were found in KRAS wild-type samples than in KRAS mutated samples (48% vs 27%, P =0.002). When all significant variables are put together in a logistic model stratified by centre, only T of TNM, histological type and final H&E are retained to explain KRAS status.For 5-year survival analysis, KRAS mutation has a borderline prognostic effect on overall survival (P =0.08) and disease-free survival (P =0.02) on the whole group of patients ;mutation of KRAS was not predictive of the effect of chemotherapy. The percentage of KRAS mutation in lung adenocarcinoma was clearly higher than that in non-adenocarcinoma NSCLC(P<0.0001). KRAS mutation has prognostic role for survival in non-adenocarcinoma patients(P=0.006,for 5-year OS;P=0.0009,for 5-year DFS).3. Results of EGFRIn 208 adenocarcinoma lung cancers, the prevalence of EGFR mutation was 11%(22/208). The overall EGFR mutation pattern was similar to the one already reported in Cosmic database in adenocarcinoma lung cancer. When all variables are put together in a logistic model stratified by centre stratum, only Pleural invasion (P=0.01) is retained to explain EGFR status.EGFR mutation has neither prognostic nor predictive role for survival in IALT adenocarcinoma patients(P >0.05). Since the rate of mutation is low (11%), the power for showing prognostic or predictive effects is low in this series of about 200 patients. It will be very important to perform a pooled analysis in order to study EGFR mutation with enough power.Conclusions1. TP53 mutation status had no prognostic for both 5 year survival and 8 year survival in NSCLC.2. TP53 mutation has a predictive value at P =0.04 for disease free survival on the whole group for 8 year analysis with a trend to benefit in TP53 wild type and to be harmful in TP53 mutated patients.3. The analyses of 5 year survival and 8 year survival accordantly suggested that a significant interaction between TP53 and treatment was observed in non-adenocarcinoma patients on disease free survival.4. KRAS is not predictive of the effect of chemotherapy and has a borderline prognostic effect on disease-free survival on the whole group of patients.5. Prognostic effect of KRAS mutation was different among different histology groups, with the worst prognostic effect in non-adenocarcinoma NSCLC.6. EGFR mutation has neither prognostic nor predictive role for survival in IALT adenocarcinoma patients.Above all, mutation detection of hot genes in NSCLC may help in assigning patients to get benefits from adjuvant cisplatin-based chemotherapy. The results of this study, TP53, KRAS and EGFR mutation analysis, will provide theory basis for finding molecular markers for adjuvant chemotherapy for resected NSCLC.
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