Clinicopathologic And Molecular Characterization Of MET Aberration In NSCLC Patients

Background:Lung cancer is a leading cause of cancer death with high morbidity and mortality in China.Non small cell lung cancer(NSCLC)is the most common histological subtype.With the intensive study and development of molecular genetics and targeted therapies,the treatment of NSCLC has entered a new era of individualized medicine.Since the mesenchymal-epithelial transition factor(MET(gene was discovered in 1984,the molecular mechanism of MET aberration has been gradually concerned.The main mechanisms of MET aberration include MET skipping mutation and amplification.Despite important advances in treatment of NSCLC cases with MET aberration,the extended use of MET Tyrosine kinase inhibitors(TKI)partially limited duing to the inevitable emergence of drug resistance.Based on the present research,targeted therapy for MET aberration is an effective strategy.Unfortunately,patients with MET aberration are ofen identified at advanced stage.Therefore,it is imperative for early detection of MET aberration.Objective:This study sought to illustrate the clinicopathologic and molecular characterization of NSCLC with MET aberration and to clarify the prognosis of MET aberration.Furthermore,to find out effectively screening methods for early detection of MET aberration,we explored the correlation between MET aberration and pathomorphological features,and investigated the concordance between fluorescence in situ hybridization(FISH),Immunohistochemistry(IHC)and polymerase chain reaction(PCR)or next generation sequencing(NGS).Moreover,the expression of enhancer of zeste homolog 2(EZH2)protein in NSCLC with MET aberration was also explored as a potential biomarker that might indicate MET aberration.Methods:(1)From 2019 to 2022,a total of 1247 NSCLC patients who were initially diagnosed and treated in Qilu Hospital of Shandong University were enrolled,identified by polymerase chain reaction(PCR)or next generation sequencing(NGS).Then,the frequency of common mutations and the clinicopathological characterizations of the patients were obtained and analyzed statistically.(2)We screened the samples of NSCLC with MET skipping mutation(23 cases),MET amplification(3 cases)and wild subtype(34 cases)out of them,and analyzed the clinicopathological characterizations of the samples.Subsequently,by following up their survival status,we analyzed the correlation between MET aberration and survival prognosis at different clinical stages,and the prognostic factors in NSCLC patients by univariate and multivariate Cox-regressive analyses.(3)Two pathologists reviewed the tissue sections of samples,evaluated the histopathological subtype and cell morphology,and further analyzed the correlation between pathomorphological characterization and MET aberration.(4)The samples of NSCLC with MET aberration were detected by PCR and NGS for uniformity.FISH and IHC were used to detect the frequency of occurrence of MET aberration in NSCLC patients,and to systematically explore the consistency with PCR or NGS.Among them,the expression of MET protein was interpreted by MetMab Score and H-Score.The receiver operating characteristic(ROC)curve was plotted based on H-Score results,and the Cut off value was calculated.(5)GSEA analyzed the expression of the difference in the PRC2 EZH2 UP.V1 DN pathway of MET skipping mutation and amplification samples from TCGA database.IHC was adopted to explore the expression of EZH2 protein in MET skipping mutation,amplification and wild group of NSCLC.Results:1.The clinicopathologic information and frequency of driving gene mutation in NSCLC patients.Of the 1247 NSCLC patients,747(59.90%)were aged 60 years or older and 633(50.76%)were female.The most common histological subtype was adenocarcinoma(1164,93.35%).The most patients presented with TNM stage O+Ⅰ+Ⅱ(700,56.13%)and metastases(534,42.82%).It’s confirmed that EGFR mutation occurred most frequently(54.21%),followed by the KRAS mutation(10.10%).The incidence of MET aberration was 3.69%(n=46),and the mutation rate of MET 14 exon skipping mutation and amplification were 1.84%、0.24%,respectively.2.Clinicopathologic characterizations of NSCLC with MET aberration.The proportions of NSCLC patients in the MET aberration group and the MET wild group in the elderly patients(≥60 years old)were respectively 80.77%and 50.00%,with a statistical difference(P=0.038).Compared with the MET wild group,MET aberration group was more common at III-IV stage patients with NSCLC(63.64%vs.29.41%),and more prone to metastasis(52.17%vs.5.88%),with statistically significant differences(P<0.01).3.The influence of MET aberration on the prognosis and independent analysis of prognostic factors in patients with NSCLC.In the MET aberrant group,the overall survival(OS)of patients with NSCLC means 28.22±4.08 months,and the median survival was 41.50±16.28 months.At the clinical Ⅲ-Ⅳ stage,the OS of NSCLC patients with MET aberration was significantly worse(P=0.010),compared with MET wild.Howerver,there was no significant difference in OS between the groups(P=0.712),at the clinical Ⅰ-Ⅱ stage.In addtion,univariate Cox-regressive analysis showed that TNM stage and MET gene status played important roles on the patients’ OS,with statistical difference(P<0.05 and HR>1).Multivariate Cox-regression analysis showed that MET gene status was an independent factor affecting the prognosis of NSCLC patients(P=0.036).4.Histomorphologic characterization of MET aberration in NSCLC.In the MET skipping and amplification groups,solid adenocarcinoma was predominant(42.86%,100.00%),while acinoid subtype adenocarcinoma was the most common in the MET wild group(29.63%).At the same time,there was a statistically significant difference between the MET amplification group and the MET wild group,in terms of poor differentiation and solid growth of tumor cells(P<0.001).The difference also exists between the MET skipping group and the MET wild group in the characterization of poorly-differentiated tumor cells(P=0.011).5.Consistency between PCR and NGS.The consistency was 100%,in which 26 NSCLC samples with MET aberration were detected by NGS and PCR.6.Consistency between FISH and PCR or NGS.The FISH results of 13 NSCLC samples were consistent with those of NGS,displayed that 10 samples with MET amplification were positive,2 samples with MET skipping and 1 sample with MET wild were negative.7.Consistency between IHC and PCR or NGS.According to MetMab Score and H-Score,the frequency of MET over-expression in the MET aberration group was 83.33%and 52.10%,respectively.Compared with the wild group(38.46%and 7.6%),the incidence of MET over-expression in the MET aberration group was higher,with a statistically significant difference(P<0.01).The over-expression of MET protein could indicate the aberration of MET.According to H-Score score,ROC Curve was plotted in which the Area Under Curve(AUC)was calculated as 0.774 and the Cut off value was 214.8.GSEA enrichment analysis of TCGA database samples performed that the expression of MET amplification group was up-regulated(FDR<0.05).The expression of EZH2 protein in MET amplified NSCLC samples was higher than that in MET skipping group and wild group.The difference between MET amplified and wild groups was statistically significant(P=0.018<0.05),while it wasn’t observed between MET amplified and skipping groups(P=0.09>0.05).Conclusions:1.MET aberration is characterized by older age in NSCLC patients and is frequently observed in adenocarcinoma,with poorly differentiated and solid growth.2.MET aberration is an independent factor predictive of poor prognosis in patients with NSCLC.3.There is a high consistency between NGS and PCR in detecting MET aberration.FISH has advantage in detecting MET amplification,with consistency of 100%with NGS or PCR.4.Nearly all MET IHC-positive cases were positive for MET amplification or METex14 mutations,suggesting IHC appears to be an inefficient screen for these genomic changes.5.EZH2 is overexpressed in MET amplified NSCLC,suggesting that EZH2 might be used as a potential biomarker indicating the amplified status of MET gene.