Analysis Of Neoadjuvant Chemoimmunotherapy And Metabonomic Evolution In Non-Small-Cell Lung Cancer

Background and objectives: The postoperative prognosis of patients with stageⅡ-Ⅲ non-small cell lung cancer(NSCLC)remains poor.At present,multimodal treatments are considered as the standard treatment regime.However,neoadjuvant chemoradiotherapy and adjuvant chemoradiotherapy bring limited survival benefits.Targeted therapy is only sensitive to patients with specific gene mutations with a risk of treatment resistance.Recently,immunotherapy has been proved to significantly improve the prognosis of patients with a variety of malignant tumors.Neoadjuvant treatment of programmed cell death receptor 1(PD-1)inhibitor has also been proved to improve the pathological response of NSCLC patients.In the exploration of multimodal treatment,the pathological response of neoadjuvant PD-1 inhibitor combined with chemotherapy range highest.Chemotherapy can up-regulate the expression of programmed cell death ligand 1(PD-L1)in tumor microenvironment,and promote the release of tumor neoantigens and specific killing T cells.Neoadjuvant PD-1 inhibitor combined with chemotherapy is expected to be the most effective neoadjuvant treatment regime for NSCLC.However,the perioperative outcomes after neoadjuvant PD-1 inhibitor combined with chemotherapy had not been fully described.At present,clinical trials of neoadjuvant chemoimmunotherapy mainly analyzed NSCLC with resectable disease(RD),while lacking of analysis on NSCLC with potentially resectable disease(PRD),which could not be resected before treatment and obtain surgical opportunities through downstaging after neoadjuvant chemoimmunotherapy.The molecular characteristics and potential predictors of treatment efficacy were not clear.In addition,the immune evolution trajectory in lung adenocarcinoma described the progressive development of tumor immune escape,and the metabolic reprogramming in the microenvironment could affect the immunotherapy efficiency.Therefore,the trajectory of metabolomics in the evolution of adenocarcinoma needs to be further explored.The main contents included: 1)description of the perioperative outcomes in neoadjuvant chemoimmunotherapy,and the safety and feasibility of sleeve resection and video-assisted thoracoscopic surgery(VATS);2)feasibility of neoadjuvant PD-1inhibitor combined with chemotherapy in the treatment of PRD;3)the RNA-seq and predictors of neoadjuvant PD-1 inhibitor combined with chemotherapy in the treatment of NSCLC;4)the metabolomic trajectory in the evolution of lung adenocarcinoma.In the research,we aimed to confirm the safety and effectiveness of neoadjuvant PD-1inhibitor combined with chemotherapy,explained the transcriptome characteristics and explored the metabonomic mechanism to comprehensively understand the microenvironment and explore a more accurate and effective treatment mode for NSCLC.Methods: Firstly,we retrospectively analyzed the perioperative outcomes and therapeutic efficiency of NSCLC patients with neoadjuvant chemoimmunotherapy in Shanghai Pulmonary Hospital from April 2019 to April 2021,described the perioperative outcomes of sleeve resection and compared the perioperative outcomes of VATS with thoracotomy.We conducted the clinical trial,safety and effectiveness of neoadjuvant PD-1inhibitor(toripalimab)plus chemotherapy in stage Ⅱ-Ⅲ NSCLC: an open-label,singlearm,phase 2 trial.The inclusion criteria were mainly treated naive,clinical stage ⅡB-ⅢC and EGFR / ALK wild-type NSCLC patients.During the evaluation of multidisciplinary team(MDT),the patients were divided into RD and PRD.All patients received 2-4 cycles of toripalimab(240 mg q3w)plus carboplatin-based chemotherapy.After the second treatment cycle,all patients were reassessed,candidates eligible for surgery underwent surgery,otherwise they received the remaining treatment cycles.The primary endpoints were safety and major pathological response(MPR).The secondary endpoints were R0 resection rate,progression free survival(PFS),and overall survival(OS),to confirm the safety and effectiveness of toripalimab plus chemotherapy in the treatment of ⅡB-ⅢC NSCLC,and to analyze its feasibility in the neoadjuvant treatment of PRD.RNA sequencing was performed on the baseline and posttreatment samples of neoadjuvant PD-1 inhibitor plus chemotherapy to explore the transcriptomic characteristics and efficacy predictors.Through the large-scale targeted metabonomic analysis of resected atypical hyperplasia(AAH),adenocarcinoma in situ(AIS),minimally invasive adenocarcinoma(MIA)and invasive adenocarcinoma(IAC)tumor samples and matched normal tissues,to explore the metabonomic trajectory in the evolution of lung adenocarcinoma.We performed the cluster analysis of IAC,composed of lepidic(LEP)papillary(PAP),acinar(ACN),micropapillary(MIP)and solid(SOL),to explore the potential subtypes with different metabolic characteristics,and provide the theoretical support for a comprehensive understanding of the development mechanism of lung adenocarcinoma.Results: In the retrospective analysis of neoadjuvant chemoimmunotherapy in NSCLC,a total of 143 patients were included.Of them,131 patients received the radiological evaluation,23(17.6%)patients were complete response(CR),75(57.2%)partial response(PR)and 33(15.2%)stable disease(SD).In the 143 patients receiving pathological evaluation,74(51.7%)patients were MPR and 36(25.2%)pathological complete response(p CR).Histopathological diagnosis(P = 0.024)and radiological response(P = 0.002)were significantly correlated with MPR.All patients underwent R0 resection,112(78.3%)patients underwent lobectomy,23(16.1%)patients underwent sleeve resection and 8(5.6%)patients underwent pneumonectomy.The operation time of sleeve resection was significantly longer than that of lobectomy(P =0.003),but there was no significant difference in intraoperative bleeding,drainage time and postoperative hospital stay(P > 0.05).In the 63(44.1%)patients receiving VATS,2(3.2%)were converted to thoracotomy due to extensive pleural adhesion.The postoperative drainage time was shorter in VATS group(P = 0.001).There was no significant difference in intraoperative bleeding,operation time and postoperative hospital stay between VATS group and thoracotomy group(P > 0.05).Postoperative complications occurred in 11(7.7%)patients,of which 1 died of bronchopleural fistula(BPF).A total of 50 eligible patients were enrolled in the clinical trial,of which 12(24.0%)were evaluated as RD and 38(76.0%)PRD.Treatment related adverse events(TRAEs)occurred in 48(96.0%)patients.Severe TRAEs occurred in 3(6.0%)patients,including myelosuppression,drug-induced liver injury and death of hemoptysis.The objective response rate(ORR)was 76.0%,including 8(16.0%)patients with CR,30(60.0%)patients with PR,10(20.0%)patients with stable disease(SD)and 2(4.0%)patients with progressive disease(PD).Eventually,12(100%)patients with RD and 25(65.8%)patients with PRD were re-evaluated to be eligible for surgical treatment,while 1(2.0%)patient with PRD refused surgery.Therefore,36(100%)patients who received surgery underwent R0 resection.20(55.6%)patients achieved MPR and 10(27.8%)patients achieved p CR.There was a significant correlation between radiological response and pathological response(P < 0.001),but there was no significant difference between MPR and non-MPR in gender,age,smoking history,baseline PD-L1,TRAEs and pathological type(P > 0.05).The median follow-up time was 14.0 months(95% CI:11.0-16.0 months),and the 12-month OS of all patients was 92.7%(95% CI: 85.0%-100.0%),and the PFS was 75.6%(95% CI: 63.1%-90.6%).OS(P = 0.016)and PFS(P = 0.006)in the surgery group were significantly improved compared with those in the non-surgery group,OS(P = 0.019)and PFS(P = 0.004)in the response group(CR+ PR)were significantly improved compared with those in the non-response group(SD+ PD).PFS(P = 0.048)in 16(44.4%)patients receiving immune maintenance treatment was significantly improved compared with those not,but there was no significant difference in OS.Through the RNA sequencing analysis of the baseline tumor samples in the neoadjuvant PD-1 inhibitor plus chemotherapy,we found the expression level of chitinase-3-like protein 1(CHI3L1)could predict the treatment response(AUC =0.795),and the PFS of patients with higher expression was significantly better than that with lower expression(P = 0.012).Through analyzing the baseline and posttreatment tumor samples,we found that the expression of VEGFB in the non-response group was up-regulated after treatment,and no immune-related pathway was found by gene set enrichment analysis(GSEA).In the response group,the expression of immune related pathways such as "antigen processing and presentation" in tumor samples was upregulated.T cell immunoglobulin mucin 3(TIM-3)in lymph node samples was upregulated,while neutrophil count was down-regulated.Through analyzing the posttreatment tumor samples,we found that the expression level of TIM-3 in the response group was higher than that in the non-response group,while the estrogen related pathway was higher in the non-response group.We performed the immunohistochemical verification and found that the cell counts of CD4,CD8 and CD20 in tumor samples of MPR were higher than those of non-MPR.Through analyzing the AAH(n = 12),AIS(n = 22),MIA(n = 19)and IAC(n =148)tumor samples and matched normal samples,we found that the expression levels of arginine,proline and purine metabolism showed a progressive change in the evolution process.Through cluster analysis of IAC composed of LEP(n = 28),ACN(n= 57),Pap(n = 17),sol(n = 22)and MIP(n = 24),we found 3 subtypes with significantly different clinical characteristics and prognosis.Through the Kyoto Encyclopedia of genes and genomes(KEGG)analysis,we found the expression level of bile acid metabolism showed a progressive increasing trend in the 3 subtypes.Through immunohistochemical semi-quantitative analysis,we found the expression of G protein coupled bile acid receptor 1(TGR5)in type III was significantly higher than that in type I and type II,and the prognosis of patients with higher expression was significantly worse than that with lower expression.Conclusions: Surgery after neoadjuvant chemoimmunotherapy,even sleeve resection,was safe and feasible.The perioperative outcomes of VATS and thoracotomy were similar.Neoadjuvant PD-1 inhibitor plus chemotherapy in the treatment of stageⅡB-ⅢC NSCLC had high MPR and manageable TRAEs,which could even downstage and convert the initially PRD into RD.Disparate transcriptomic characteristics of therapeutic efficiency were observed,and CHI3L1 expression predicted therapeutic response in RNA-seq analysis.Perturbed metabolic pathways emerged early in premalignant lesions,and progressive changes of metabolic trajectories were involved in lung adenocarcinoma evolution.IAC could be divided into 3 subtypes with distinct clinical characteristics by cluster analysis,and the expression level of bile acids was significantly different in the 3 subtypes and closely related to the prognosis.