Study Of De Novo MET Overexpression In Non-small-cell Lung Cancer

Lung cancer is the leading cause of cancer-related deaths in the world,the overall 5-year survival rate is only 10-15%.More than 80%of lung cancers are of non-small cell lung cancer(NSCLC)histology includes the adenocarcinoma,squamous cell carcinoma and large cell carcinoma subtypes.Although platinum-based combination chemotherapy has improved median survival,the prognosis for these patients remains poor.With the intensive study of translational medicine,cancer treatment is being revolutionized by target-based therapeutic development since the gefitinib as the first EGFR-TKI(epidermal growth factor receptor tyrosine kinase inhibitor,EGFR-TKI)was used in 2002,targeted therapy has been the most active filed of lung cancer.The median over several has been increased to 24-30 months with the target therapy in EGFR mutation advanced non small cell lung cancer.Over the past decades,More than 50%driver genes were found in NSCLC,however,there are still such a number of patients all negative in gene types and how to overcome TKI resistance is the hot spot in the study of lung cancer.MET(Mesenchymal-epithelial transition)gene amplification has been shown to be another major mechanism of acquired resistance to EGFR-TKI by activating EGFR bypass,apart from T790M mutation in exon 20 in the EGFR gene.Previous clinical trials has demonstrated the safety and efficacy in combination EGFR-TKIs and MET inhibitors.Meanwhile,there are some reports about the de novo MET amplification patients showed the shrink response to receptor tyrosine kinases tyrosine kinase inhibitors(RTK-TKJs)for MET.OU et al.and Schwab et al.both reported the non-small cell Lung cancer patient with de novo MET amplification who achieved a rapid and durable response to crizotinib through a clinical trial(A8081001,ClinicalTrials.gov identifier:NCT00585195).The new data from 2014 ASCO displayed that 16 advanced c-Met-amplified NSCLC patients were enrolled,within 3 categories of amplification MET/CEP7 ratio>1.8-≤2.2(Low),>2.2<5(Intermediate)and>5(High).13 patients with c-MET-amplified NSCLC[Low(n=1),Intermediate(n=6)and High(n=6)],with 12 evaluable for response,To date 4 PRs have been observed[Low(n=0),Intermediate(n=1;20%)and High(3;50%)].Median duration of response was 35 weeks.Unlike the EGFR(epidermal growth factor receptor)、ALK(anaplastic lymphoma kinase)and ROS1(c-ros oncogene 1 receptor tyrosine kinase)play the initial driver gene in NSCLC,How does the MET work in NSCLC is still unknown.As the standards used to score IHC staining intensity vary,the MET overexpression rates differ among various studies.The criteria to evaluate c-Met gene amplification is the challenge puzzled many researchers.The MET gene is located on chromosome 7q21-31,and it is 120kb in length with 21 exons and 20 introns.It originally was isolated as the product of a human oncogene,tpr-met,which encodes an altered MET protein possessing constitutive kinase activity and transforming ability tyrosine-kinase,is a high-affinity receptor for hepatocyte growth factor/scatter factor(HGF/SF),and it is the only known ligand.Signaling via this receptor-ligand pair has been shown to affect a wide range of biological activities,including angiogenesis,cellular motility,growth,invasion,and morphogenic differentiation.The MET pathway is dysregulated via overexpression,gene amplification,ligand-dependent activation,and mutation.in vivo trials showed autocrine HGF/MET signaling plays a significant roles in the growth and differentiation of human lung adenocarcinoma cells MET and HGF have emerged as important biomarkers in NSCLC before therapy strategies.There are some MET tyrosine kinase inhibitors directly at the tyrosine kinase domain,or targeting the receptor effecter antagonist either alone or in combination showed the good reaction in clinical trials.It is still unknown how to define de novo MET activation and which molecular markers are optimal.Thus,this study aimed to evaluate the MET protein overexpression and to analyze their clinical features in Chinese lung cancer.And furthermore access its influence on chemotherapeutic efficacy in advanced NSCLC.And prospective evaluate the safety and efficacy of crizotinib to MET overexpression advanced NSCLC.To answer these questions will help to combine EGFR with Met targeted therapy.This study is divided into three parts as following:Charpterl The prognosis significance of MET protein overexpression and the relationships with the clinical parameters in patients with NSCLC by different scored standards1.Objective:To investigate the clinicopathological features of MET overexpression and its prognosis significance in de novo lung cancer patients in China.2.Methods:Three hundred and ninety seven frozen and paraffin samples were obtained from patients who had been diagnosed with lung cancer between December 2013 and October 2014 including surgery resections lung puncture biopsy and lymph node.All the patients were without company with EGFR、ALK、K-ras、Rosl driver gene mutation before biopsy and had written informed consent.MET was scored according to H-score[range:<60(no expression),0-300,60-<100(Low expression),≥100-<300(Intermediate expression),300(High expression)]and by scoring system used in the MetMab trial(≥50%of cells with moderate or strong staining).All statistical analyses were performed using the SPSS 13.0.The Chi-square test was used to assess the relationships between MET and clinicopathologic parameters.All statistical tests were two-sided,and statistical significance was defined as P<0.05.3.Results:According to two approached to evaluate MET expression.1)MetMab scoring criteria,IHC positively rate is 27%(107/397),and it is associated with pathologic type,adenocarcinoma is more frequency than squamous cell carcinoma(34.8%vs.5.9%.X2=30.155,P=0.001)and smoking status(no smoking 31.9%vs.smoking22.6%,x2=4.298,p=0.038),but not association with gender(female 26.9%vs.male 27%;x2<0.001,P=0.986)and age(x2=0.319,P=0.572),stage(x2=4.244,P=0.236).2)Median MET H-score was 60(0-300),CDMET is highly expressed in 216 cases(54.4%),and lowly expressed in 181 cases(45.6%).MET expression is correlated with pathologic type,adenocarcinoma is more frequency than squamous cell carcinoma(61.6%vs.38.4%),and the squamous cell carcinoma is more than adenocarcinoma in MET negative(x2=20.740,P<0.001),it is not associated with smoking status(x2=0.770,P=0.380)、gender(X2=0.020,P=0.887)、age(x2=0.634,P=0.426)and stage(x2=4.332,P=0.228).②Further analysis the details of the status of H-score as below 60(MET negative),≥60-<100(Low expression),≥100-<300(Intermediate),300(High expression);the rate of low is 60(15.10%),intermediate 138(34.80%)and 18(4.50%)in high expression.The adenocarcinoma is more frequency than squamous cell carcinoma in intermediate and high expression,but the squamous cell carcinoma is more than adenocarcinoma in MET negative and low expression(X2=30.493,P＜0.001).but not association with smoke(x2=6.880,P=0.076)、gender(x2=5.369,P=0.147)、age(x2=0.841,P=0.840)and stage(x2=6.033,P=0.110).4.Conclusions:Incidence is different in two scoring criteria for de novo MET overexpression.H-score has higher IHC-positive rate than MetMab.de novo MET overexpression is not associated with clinical parameters such as age、gender and clinical stage.There were more patients with adenocarcinoma in the MET-positive group compared with the MET-negative group.H-score can be the better cut-off values by further classification of MET IHC score.Charpter2 The relationships between de novo M MET protein overexpression and the first-line chemotherapy in patients with advanced non-small-cell lung cancer1.Objective:To investigate de novo MET protein overexpression and the correlations with first-line chemotherapy of advanced NSCLC.2.Methods:A total of 112 advanced NSCLC who accepted first-line chemotherapy were screened from 397 patients in chaerpterl.RECIST 1.0 criteria was used to access the effective as complete response(CR)、partial response(PR)、stable disease(SD)、progressive disease(PD).The Chi-square test was used to assess the relationships between MET expression and effective of first-line chemotherapy.Univariate analysis was analyzed by using Kaplan-Meier method and significance differences among subgroups were compared by using the Log-rank test.Multivariate analysis were performed using Cox proportional hazards model to identify independent prognostic factors.All statistical tests were two-sided,and statistical significance was defined as P<0.05.Results:Evaluation curative effect of 112 cases of first-line chemotherapy in patients.1)As MetMab trail system in 29 MET overexpression patients(25.9%)5 7 of 29 are PR(24.10%),20 were SD(69.00%),2 of them PD(6.90%),83 MET negative patients 27 are PR(32.50%),46 were SD(55.4%),10 of them were PD(12.00%).But it is not found associations with PFS(x2=1.7,P=0.427,P=0.143).Further cox regression analysis show that the personal status is correlated with the PFS of the first-line chemotherapy in NSCLC patients(HR＝2.578,95%CI:1.205-5.518,P=0.015).PS≥2 is an independent poor prognosis factor in advanced NSCLC first-line chemotherapy(HR=4.025,95%CI:2.164-7.486,P=0.000).2)As H-score system,54 of 112 patients are MET overexpression(48.21%),it is not found associations with PFS(x2＝4.570,P＝0.206);In the cell model,the MET-positive cell showed no difference in chemotherapy but with a increase in percentage of growth inhibition upon treatment with MET inhibitor INC280 compared to MET-negative cell(DOC GI50 48.2nM;107.5nM;SN38 GI50 11.3;14.5 nM;INC280 GI50 146.2nM,>1uM;PF04217903 GI50 147.2nM,>3uM)。In Western blot,the MET TKI inhibt the MET overexpression cell line by MET-Akt-Erk1/2 downstream pathway.3.Conclusions:MET overexpression is not associated with first-line chemotherapy in advanced non-small-cell lung cancer.But MET overexpression can be a predict biomarker to MET-TKI in in vitro.PS≥2 is an independent poor prognosis factor in advanced NSCLC first-line chemotherapy.Charpter3 Crizotinib in de novo c-Met overexpression advanced non small cell lung cancer1.Objective:MET gene amplification has been identified as de novo activation in advanced non small cell lung cancer(NSCLC).However,it is not clear that MET overexpression could be as the biomarker for de novo.2.Methods:Advanced NSCLC patients with de novo MET expression in charpterl were detected by immunohistochemistry.≥50%tumor cells with moderate to high intensity staining were defined as MET positive.The statuses of EGFR5 ALK,KRAS and ROS1 were also tested.Gene copy numbers have been detected by FISH(By Cappuzzo scoring system&MET/CEP7 ratio).≥5 copies were positive or MET/CEP7 ratio≥1.8 was defined as MET amplification with low≥1.8-≤2.2,Intermediate>2.2-<5 and High>5;All statistical analyses were performed using the SPSS 13.0.The Nonparametric test were used to assess the correlations between effective and clinicopathologic parameters.All statistical tests were two-sided,and statistical significance was defined as P<0.05.3.Results:From December 2013 to October 2014,29 patients got the therapy of crizotinib,and 16 eligible patients were evaluable to access.All patients are adenocarcinoma,3 are female(18.8%),13 are male(81.2%).8 of them achieved partial response(PR,50.0%),2 of them were stable disease(SD,12.5%)and 6 were PD(37.5%).15 of 16 patients got the FISH test.5 of them achieved partial response in 6 MET amplification,9 of them were PR in MET non-amplification.Adverse events of grade 3 QT prolongation have been found in 1 patient.For one death with interstitial lung disease,causality to crizotinib was not ruled out.The other most frequent drug-related AEs were gradel-25 including nausea(9/16),loss of appetite(9/16),vomiting(7/16),diarrhea(4/16),trouble swallowing(4/16).EGFR,ALK,KRAS and ROSI were all negative.The response is not association with ageN gender、PS and smoking status.As H-score system,the median score is 280(110-300)in PR,SD is 175(110-300)and PD is 242(190-300);The score is higher in PR,but the difference hasn’t statistical significance(P＝0.623);neither with gender(P=0.781)、age(P=0.406)、smoke status(P=0.931)、PS(P=0.800)and MET amplification(P=0.538).4.Conclusions:MET overexpression could be as a biomarker for de novo.MET inhibitor against de novo c-Met overexpressed patients is a good strategy.And it is not association with clinicopathology.INNOVATION POINTS:1.This research including retrospective study and Prospective observational,Clinical data and in vitro outcome.2.Compare two MET scoring criteria to value MET overexpression and its prognosis significance in de novo non-small-cell lung cancer patients in China for the first time.H-score can be the better cut-off values by further classification of MET IHC score.3.To confirm that MET overexpression is not associated with first-line chemotherapy in advanced non-small-cell lung cancer,but can be a predict biomarker to MET-TKI in in vitro..Provide the reference value to future clinical trials.4.Prospective observe crizotinib targeted the MET overexpression NSCLC,and could be as the biomarker for de novo.