Correlation Research Of ALK,ROS1 And EGFR Gene Mutations And Clinicopathological Features In NSCLC

Background:The incidence of lung cancer is the highest in the world,and it is the leading cause of malignant tumor death in many countries.It is one of the most harmful malignant tumors to human health.Lung cancer is one of the most common cancers in China,it has become the leading cause of death for malignant tumors,accounting for 22.7%of all malignant tumor deaths,and the morbidity and mortality are still rising.The diagnosis and treatment of this disease has been a research hotspot,and traditional treatment methods can not meet the requirements.Previous study found that in recent years new oncogenes,EML4-ALK(echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase)、ROS1(C-ros oncogene 1,receptor tyrosine kinase)and EGFR(Epidermal growth factor receptor)mutations in the process of the occurrence of NSCLC,EML4-ALK、ROS1 and EGFR were the target molecular for drug,which has become the focus in the treatment of NSCLC.Pre-clinical studies,as well as the treatment for cancer patients with ALK and ROS1 translocation and EGFR mutation confirmed that a variety of tyrosine kinase inhibitors(Tyrosine kinase,inhibitors,TKI)including crizotinib、gefitinib and so on can made NSCLC patients with the ALK、ROS1 fusion and EGFR mutation got significantly relieved and survival benefit.At present,molecular targeted therapy is mainly aimed at tyrosine kinase inhibitors of ALK,ROS1 and EGFR mutations.Targeted therapy for these three genes has achieved significant therapeutic effects in NSCLC patients.However,Clinically,quite a few patients have lymph node metastasis when they come to doctor for treatment,and the consistency of ALK expression between primary foci and metastatic foci directly affects the therapeutic effect of targeted therapy in metastatic patients.But the mechanism of metastasis in NSCLC patients is not clear.Whether EMT and Wnt pathways play a role in metastasis,and the relationship between them and ALK has not been reported in the literature.Herceptin for HER2 gene amplification breast cancer has a good survival benefit for treatment,but there were little research about the relationship between Her2 and metastasis in NSCLC.Clinically,there are many patients without these gene mutations,but relatively few studies are related to them,especially for pathological features.Objective:To detect the expression of ALK,Her2,β-catenin,E-cadherin and Vimentin and compare the difference of them in primary and metastatic lesions of NSCLC;To further discuss the role of genes and pathological subtypes in ALK positive and negative NSCLC patients with metastasis;To investigate the clinicopathological features of ALK translocation,ROS1 fusion and EGFR mutation and three non-mutation cases.Methods:In this study,Patients were selected from a pathological database of lung cancer cases between September 2013 and January 2016 undergoing surgical resection of the primary tumors and the corresponding lymph node metastatic sites at the Pathology Department of The First Affiliated Hospital of Zhejiang University,and the primary and paired metastatic lymphatic section had to contain greater than 100 tumor cells.The primary foci of 45 patients with ALK arrangement and 33 patients without ALK arrangement were screened by VENTANA in this study.The primary and metastatic lesions were detected by VENTANA and FISH respectively,and the differences between primary foci and metastatic foci were compared.VENTANA and FISH were used to detect ALK and Her2 in primary and metastatic foci respectively,expression of beta catenin and EMT markers E-cadherin and Vimentin was detected by immunohistochemistry,comparison of the expression and histopathological differences between primary and metastatic foci,to discuss the relationship between genes and ALK。In this study,356 NSCLC patients from a pathological database of lung cancer cases between December 2011 and September 2015 at this single center were included in retrospective analysis,all of them were tested for ALK、ROS1 and EGFR.The mutation status and clinical pathological data of the patients were analyzed statistically.The clinicopathological features of three mutation cases cases were discussed.Results:Pathological morphology of ALK positive patients were more stable than those with negative ones when metastases(P<0.05);There was no significant difference in ALK and Her2 gene status between primary and metastatic foci.(P>0.05);The expression ofβ-catenin and Vimentin in metastatic foci was significantly stronger than primary foci(P<0.05);The expression of E-cadherin in metastatic foci was weaker than that in primary ones,but there was no significant difference(P>0.05);The amplification ratio of Her2 gene in ALK negative lung cancer was significantly higher than that of ALK positive lung cancer.(P<0.001);The expression ofβ-catenin protein in ALK-positive lung cancer was stronger than that in ALK-negative lung cancer,but there was no significant difference.(P>0.05);The expression level of E-cadherin protein in ALK positive lung cancer was significantly weaker than that in ALK negative lung cancer(P<.0.05);The expression level of Vimentin protein in ALK positive lung cancer was significantly stronger than that in ALK negative lung cancer(P<0.001);The difference between the two detection methods of IHC and FISH is concentrated in cases of Cerbb2 1+ and Cerbb2 2+。Among the 356 cases,22 cases with ALK fusion gene,positive rate was 6.2%,it happened more frequently in the pathological type of solid and mucinous adenocarcinoma(P<0.05);15 cases with ROS1 fusion,positive rate was 4.2%,it happened more frequently in the pathological type of mucinous adenocarcinoma(P<0.05);206 cases with EGFR mutation,the mutation rate was 57.9%,it happened more frequently in female,no-smoke,pathological types of glandular(P<0.05);No double mutation was found in 356 cases.Among the 356 patients,73 died,283 survived or lost follow-up,and the median survival time was 41 months in the non-mutation group.The correlation of between gene and OS was calculated with the Kaplan-Meier survival curve.The results showed that the OS of EGFR mutant patients was longer than that of the EGFR wide type patients(P<0.05);The correlation between ALK fusion,ROS1 fusion,EGFR gene mutation and DFS was calculated with the Kaplan-Meier survival curve.The results showed that the DFS time of EGFR mutant patients was longer than that of the EGFR wild type patients(P<0.05);The correlation of between ALK fusion,ROS1 fusion,EGFR gene mutation and PFS was calculated with the Kaplan-Meier survival curve.The results showed that the PFS ofEGFR mutant patients was longer than that of the EGFR wild type patients(P<0.05);All patients were divided into two groups,Ⅰa-Ⅲa and Ⅲb-Ⅳ according to the clinical stages,and the relationship between EGFR mutation and OS,DFS,PFS was calculated by Kaplan-Meier survival curve.The results showed that the OS,DFS and PFS of patients with EGFR mutation were longer than patients without EGFR mutation except for DFS in Ⅲb-Ⅳ phase(the number of nonsurgical cases in the Ⅰa-Ⅲa and surgical cases in the Ⅲb-Ⅳ was too few)。Conclusion:1、There was no significant difference in the expression of ALK between primary lesions and metastatic foci,ALK detection of primary lesions can be used as a basis for the treatment of advanced metastatic non-small cell lung cancer.;The expression of β-catenin.Vimentin and E-cadherin were higher in ALK positive patients than those in negative patients,while the expression ofβ-catenin、Vimentin in metastatic lesions was higher than that in primary lesions.,the expression of E-cadherin in metastatic foci was lower than that in primary tumors,all these indicate that ALK positive patients are more likely to invade and metastases;Wnt/β-catenin signalingpathway may be a new way to block tumor metastasis;The role of Her2 gene in ALK positive NSCLC with metastasis is still unclear needed to be further studied。2、The positive rate of ALK fusion gene was 6.2%,the positive rate of ROS1 fusion was 4.2%,the mutation rate of EGFR was 57.9%;ALK is more common in solid and mucinous adenocarcinoma.ROS1 is more common in mucoid adenocarcinoma,EGFR-mutation NSCLC patients are more likely to be women,never-smoked and glandular type and adherent type,EGFRmutation group had a longer OS than wide type group.