Expression Of YAP1 Protein In ALK-fusion Lung Cancer And Role In Predicting The Efficacy Of Targeted Therapy

Echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase(EML4-ALK)fusion accounts for 3%-5% of non-small cell lung cancer(NSCLC)patients.After the advent of small molecule targeted drugs,the treatment efficacy and prognosis of lung cancer have been significantly improved.However,there are differences in the response of different patients to ALK-targeted therapy,and most patients with ALK-targeted therapy will inevitably develop resistance eventually,leading to tumor progression.The use of prognostic markers to monitor patient efficacy and timely change of treatment regimens,as well as individualized subsequent treatments based on drug resistance mechanisms,can effectively improve patient outcomes.Therefore,studying drug resistance mechanism to find new therapeutic targets has become a research direction and hotspot in recent years.Hippo-YAP pathway is a newly discovered signal transduction pathway in recent years,which plays an important role in the occurrence of various tumors.YAP(yes associated protein)is a transcriptional co-activator downstream of the Hippo pathway,which can interact with different transcription factors to activate the transcription and expression of genes.Some studies have shown that the Hippo-YAP pathway is also involved in the resistance of anti-tumor drugs.Therefore,this paper intends to explore the expression-related characteristics of YAP in ALK gene fusion lung cancer,efficacy on ALK-TKIs and predictive effect of YAP,providing a new theoretical basis for the targeted therapy of lung cancer.In this study,the baseline expression of YAP in ALK-fusion NSCLC tissues was systematically investigated through human histological specimen experiments using immunohistochemical staining.In vitro cytological experiments,CCK8 assay,scratch assay,lentivirus overexpression and gene silencing experiments were also involved.As well as clinical significance,combined with clinicopathological data and cell phenotype experiments were used to explore the possibility of YAP as a potential drug resistance mechanism.A total of 95 patients with advanced ALK fusion NSCLC were included in this study,mainly younger,non-smoking adenocarcinoma patients.The staining results of baseline pathological specimens showed that the positive staining of YAP1 mainly occurred in the cytoplasm and nucleus of lung cancer epithelial cells,the positive staining of phosphorylated YAP1 protein occurred mainly in the tumor cytoplasm.The high expression of nuclear YAP1 protein was correlated with age(P=0.02).The patients with high expression of phosphorylated YAP1 had a later T stage and more frequent contralateral lung metastasis(P<0.1),and the patients with high nuclear expression had a later N stage and lymph node The incidence of N2 and N3 metastasis was high(P=0.024).Correlation analysis showed that there was no significant correlation between YAP1 expression and PD-L1 expression;the nucleo-cytoplasmic ratio of phosphorylated YAP1 protein in the P53 protein positive expression group was lower(P<0.1).The expression of YAP1 and phosphorylated YAP1 protein in the cytoplasm of c-MET high expression group was higher(P<0.1).This study analyzed the best efficacy of 95 patients who received ALK-TKIs as first-line therapy,and the objective response rate of first-line ALK-TKIs in the total population was 62.1%,and the disease control rate was 94.7%.The ORR of first-line Crizotinib was 48.7%;the ORR of first-line Alectinib was 71.4%.In this patient cohort,a statistically significant difference was observed in ORR(P < 0.1)for firstline selection of different ALK-TKIs.There was a statistically significant difference in PFS between Crizotinib and Alectinib in first-line treatment(11.83 m vs.NR;HR:0.31,95%CI: 0.17-0.57,P<0.001).Patients with high baseline YAP1 nuclear expression had lower ORR with first-line ALK-TKIs(P < 0.1);first-line ALK-TKIs had shorter PFS(Median PFS: 23.4m vs.12.4m;HR: 0.50;95%CI: 0.234-1.057,P=0.02);subgroup analysis can also see that the PFS of the high-expression population treated with Crizotinib and Alectinib was shortened.YAP1 high nucleocytoplasmic ratio in the overall population(29.9m vs.16.3m;HR: 0.56;95%CI: 0.315-1.001,P=0.048)and in the crizotinib group(21.9m vs.11.8m;HR: 0.47;The PFS of 95%CI: 0.232-0.971,P=0.04)was also significantly reduced,indicating that the high nuclear expression of YAP1 is not conducive to survival.However,the cytoplasmic high expression of phosphorylated YAP1 protein decreased ORR(P<0.1)and PFS significantly shortened with first-line Alectinib treatment(NR vs.16.3m;HR: 0.39;95%CI: 0.138-1.101,P=0.02).Patients with negative expression of P53 could obtain better curative effect and longer survival(P<0.1).PD-L1 and c-MET had no predictive value for the overall population,crizotinib treatment group,and alectinib treatment group survival time.Univariate and multivariate analysis found that baseline YAP1 nuclear expression independently predicted PFS in this population with first-line treatment(P<0.1).This study also preliminarily explored the effect of YAP1 expression on ALK fusion non-small cell cancer cell lines H2228 and H3122 through cell phenotype experiments.The results suggested that overexpression of wild-type YAP1 would significantly enhance the proliferation,migration and invasion abilities of H3122 and H2228(P <0.1),and reduce the drug susceptibility of crizotinib and alectinib;while down-regulation of YAP1 can significantly reduce the proliferation,migration and invasion ability of stable transfected strains,which is expected to become a potential target for overcoming ALK inhibitor resistance.Part I:Correlation between YAP1 expression and other clinicopathological features in patients with ALK fusion lung cancerPurpose:To explore the expression and clinicopathological characteristics of YAP1 protein in ALK fusion NSCLC,and to explore the correlation between YAP and PD-L1,TP53 and c-MET expression.Method:Tissue specimens from patients with ALK fusion non-small cell lung cancer diagnosed in Shanghai Pulmonary Hospital from January 2018 to March 2020 were collected before treatment,and specimens with unclear diagnosis and incomplete data were removed.Data,IHC staining was performed on the histopathological specimens before baseline treatment,and the expression intensity was quantitatively analyzed by interpreting the staining results and calculating Hscore.Result:1.A total of 95 patients with advanced ALK fusion NSCLC were included in the study,mainly younger,non-smoking patients with adenocarcinoma.Bone metastasis was the most common occurrence in advanced patients,accounting for 41.1%,and metastases occurred in multiple bones throughout the body.Foci predominated;the staining results of baseline pathological specimens showed that the positive immunostaining results of YAP1 mainly occurred in the cytoplasm and nucleus of lung cancer epithelial cells,and only negative or weak YAP1 expression was detected in the mesenchymal cells surrounding the cancer cells;phosphorylation Positive staining for YAP1 protein occurred mainly in the tumor cytoplasm,with partial staining observed in the nucleus.2.The high expression of YAP1 protein in the nucleus was correlated with age(P=0.02).Patients over 65 years old more often showed high expression of YAP1 in the nucleus,while male patients more often showed high expression of YAP1 in the cytoplasm(P=0.08);YAP1 The T staging with high nuclear-cytoplasmic ratio was later(P=0.07);the patients with high cytoplasmic expression of phosphorylated YAP1 had later T staging and higher incidence of lung metastasis(P<0.1),and the patients with high nuclear expression had later N staging,the incidence of lymph node N2 and N3 metastasis was higher(P=0.024);the incidence of pulmonary metastasis was higher in the phosphorylated YAP1 low NCR group(P=0.09),and the incidence of extrapulmonary metastasis in the high NCR group was higher(P=0.08).3.PD-L1 negative patients accounted for 54.7%,P53 protein expression negative accounted for 68.4%,and c-MET high expression accounted for 51.6%;PD-L1expression was related to the pathological stage of patients(stage III vs.IV,P=0.02),T stage(P=0.04)was correlated with intrapulmonary metastases(P=0.08),pleural metastases(P=0.02)and metastases in other sites(P=0.06);correlation analysis indicated that there was no significant correlation between YAP1 expression and PDL1 expression.Correlation;in the ALK fusion non-small cell lung cancer population,the nucleocytoplasmic ratio of phosphorylated YAP1 protein in the P53 protein positive expression group was lower(P<0.1),and the expression of phosphorylated YAP1 in the nucleus of the positive group was lower(P=0.08).;The expression of YAP1 and phosphorylated YAP1 protein in the cytoplasm of the c-MET high expression group was higher(P<0.1).Conclusion:The study included 95 cases of ALK fusion NSCLC.Analysis of YAP1 protein expression in baseline tissue samples found that high nuclear YAP1 protein expression was correlated with age;correlation analysis showed that the nucleocytoplasmic ratio of phosphorylated YAP1 protein in the P53 protein positive expression group was lower,the expression of YAP1 and phosphorylated YAP1 protein in the cytoplasm of the c-MET high expression group was higher.Part II:The relationship between YAP1 protein expression and targeted therapy efficacy in ALK-fusion NSCLCPurpose:To explore the effect of YAP1 protein expression and related clinicopathological features on the efficacy and survival prediction of first-line ALK-TKIs in patients with ALK-fusion NSCLC.Method:Tissue specimens of patients with ALK fusion non-small cell lung cancer diagnosed in Shanghai Pulmonary Hospital from January 2018 to March 2020 were collected before treatment,and the specimens with unclear diagnosis and incomplete data were removed,and the baseline clinical data were collected retrospectively for the patients after screening.Data,IHC staining was performed on the histopathological specimens before baseline treatment,and Hscore was used to interpret the staining results to quantitatively analyze the expression intensity.The efficacy and survival prediction of protein expression on ALK-TKIs.Result:1.The study first analyzed the best efficacy of 95 patients who received ALK-TKIs in the first line.The objective response rate of first-line ALK-TKIs in the general population was 62.1%,and the disease control rate was 94.7%.First-line crizotinib was used in 39 patients,with an ORR of 48.7%;first-line alectinib had an ORR of 71.4%.In this patient cohort,statistically significant differences were observed in ORR(P < 0.1)and DCR(P = 0.089)for first-line treatment with different ALK-TKIs.There was a statistically significant difference in PFS between crizotinib and alectinib first-line treatment(11.83 m vs.NR;HR: 0.31,95%CI: 0.17-0.57,P<0.001).2.Patients with high baseline YAP1 nuclear expression had lower ORR(P<0.1)and shorter PFS with first-line ALK-TKIs(P=0.02).YAP1 high nucleocytoplasmic ratio in the overall population(29.9m vs.16.3m;HR: 0.56;95% CI: 0.315-1.001,P=0.048)and in the crizotinib group(21.9m vs.11.8m;HR: 0.47;95%CI: 0.232-0.971,P=0.04),the PFS was significantly reduced.While the cytoplasmic high expression of phosphorylated YAP1 protein decreased ORR(P<0.1)and PFS significantly shortened with first-line alectinib treatment(NR vs.16.3m;HR: 0.39;95%CI: 0.138-1.101,P=0.02).3.The ORR of first-line treatment in patients with positive expression of P53 protein and high expression of c-MET was significantly decreased(P<0.1).The median progression-free survival and overall survival of the first-line ALK-TKIs between the negative and positive expression of P53 protein were statistically significantly different,and the crizotinib and alectinib groups also obtained similar results,TP53 negative Expression patients could obtain better curative effect and longer survival(P<0.1).PD-L1 and c-MET had no predictive value for the overall population,crizotinib treatment group,and alectinib treatment group survival time.4.Univariate analysis and multivariate analysis found that the baseline YAP1 nuclear expression could independently predict the PFS of first-line treatment in this population(P<0.1).Conclusion:Nuclear YAP1 expression can significantly predict the efficacy and survival of firstline ALK-TKIs,and is an independent predictor of PFS in this population.The efficacy prediction effect of other indicators still needs longer follow-up and further research and exploration.Part III: The expression of YAP1 protein affects the proliferation,migration,invasion ability and drug sensitivity of ALK-fusion cell linesPurpose:To explore the effect of YAP1 protein expression on the proliferation,migration,invasion and drug sensitivity of ALK fusion lung cancer cell lines.Method:In this study,lentiviral transfection was used to screen stably transfected cell lines that overexpressed wild-type YAP1 and down-regulated YAP1 protein.The stably transfected H3122 and H2228 cells were subjected to clone formation assay,scratch assay,Transwell invasion assay and CCK8 assay.The proliferation,migration and invasion ability and drug sensitivity of the strain were explored.Result:1.The study first tested the proliferation ability of ALK fusion cell lines after overexpression and downregulation of YAP1 by detecting the ability of single tumor cells to form colonies by clonogenic assay.The results suggest that overexpression of wild-type YAP1 can enhance the single-cell clone proliferation ability of H2228 and H3122 cell lines,and the colony formation is significantly more than that of the negative control group;while down-regulation of YAP can significantly reduce the proliferation ability of the two cell lines,the colony formation is less,P<0.1.2.By analyzing the degree of wound healing of H2228 and H3122 cell lines cultured for 24 h and 48 h,it was found that the migration ability of H2228 and H3122 cell lines was enhanced after overexpression of wild-type YAP1 protein,while down-regulation of YAP1 significantly reduced the migration ability of both cell lines,the migration speed decreased,and the wound healing slowed down,P<0.1.3.Transwell invasion assay found that overexpression of wild-type YAP1 significantly enhanced the invasion ability of H3122 and H2228 stably transfected cell lines,and the number of cells penetrating Matrigel was significantly higher than that of the negative control group;while down-regulation of YAP1 significantly reduced the invasion of ALK fusion cell lines capacity,the number of cells passing through the Transwell chamber was significantly reduced,P<0.1.4.The sensitivity of H3122 and H2228 stably transfected cell lines overexpressing wild-type YAP1 to different concentrations of crizotinib and alectinib was detected by CCK8 assay.The IC50 curve for crizotinib and alectinib treatment shifted to the right,and the IC50 value increased,suggesting a decrease in drug sensitivity.Conclusion:YAP1 expression affects the proliferation,migration,and invasion abilities of ALKpositive non-small cell lung cancer cell lines,as well as drug sensitivity to ALKTKIs,and is expected to be a potential target for overcoming ALK inhibitor resistance.