The Correlation Between Intestinal Flora And The Efficacy Of First-line Immunotherapy Combined With Chemotherapy In Patients With Extensive Small Cell Lung Cancer

Objective:Lung cancer(LC)is a malignant tumor with the second highest morbidity and the first highest mortality in the world.Among them,small cell lung cancer(SCLC)accounts for about 15%,and about 70% of SCLC patients are in extensive stage(ES)at initial diagnosis,which with a 5-year survival rate of less than 10% and poor prognosis.The strategy of Immune Checkpoint inhibitors(ICIs)combined with chemotherapy improved the prognosis of ES-SCLC to a certain extent,but still faced with the situation of limited benefit population and lack of prognostic markers of efficacy.Recent studies have shown that intestinal microbiota can affect the efficacy of combination therapy by regulating the body’s immune system.The analysis of intestinal flora diversity and metabolite enrichment in patients with ES-SCLC has important scientific research and clinical significance for further optimizing the selection of advantageous population for ES-SCLC first-line immunization combined with chemotherapy.Therefore,this study mainly explored the possibility of intestinal flora as a predictor of first-line immunocombined chemotherapy efficacy by exploring the correlation between intestinal flora composition and the efficacy and survival benefit of ES-SCLC patients,and analyzed the expression of metabolites of intestinal flora in ES-SCLC patients to explore the correlation between intestinal flora related metabolites and the efficacy of immunocombined chemotherapy.Methods:1.Collection of clinical stool specimens: A total of 30 newly diagnosed ES-SCLC patients receiving PD-L1 inhibitor combined with chemotherapy were selected from the Department of Oncology Affiliated Hospital of Qingdao University.The enrolled patients were divided into PFS≥6 months group and PFS<6 months group.Fresh fecal samples were collected at baseline(before the first cycle of treatment)and after 2 cycles of treatment respectively,and clinical information of the enrolled patients was collected at the same time.2.Clinical efficacy and safety evaluation: Efficacy was evaluated according to the solid tumor response evaluation criteria(RECISIT)1.1.Adverse events were evaluated by using the National Cancer Institute Toxicity Evaluation Criteria(NCI-CTC)4.0.3.Microbial diversity analysis: The DNA of fecal samples from the enrolled patients was extracted.The V3-V4 highly variable region of intestinal flora 16 S r RNA gene was detected by Illumina Mi Seq high-throughput second-generation sequencing technology platform and bioinformation analysis platform.And the diversity of intestinal flora was analyzed.4.Microflora metabonomics analysis: Metabolites were extracted from stool samples of enrolled patients.The qualitative and quantitative results of metabolites were analyzed by LC/MS.Multivariate statistical analysis(PCA,OPLS-DA)and unidimensional statistical analysis(Wilcoxon rank sum test)were used to analyze the difference of bacterial metabolites.5.Statistical analysis: Nonparametric Mann-Whitney or Kruskal-Wallis tests were used to determine statistical differences in OTU count and α diversity index between the two groups.Welch’s t test and Wilcoxon rank-sum permutation test were used to evaluate the significant differences in intestinal flora composition of the patients.Pearson rank correlation analysis was used to calculate the correlation between bacterial flora and metabolites.Kaplan-Meier method was used to draw the survival curve.Cox regression model was used for multivariate analysis.P<0.05 was considered statistically significant.Results:1.Thirty patients were included in this study,who with a median age of 64.73 percent were male and 56 percent were smokers.There were 7 cases of craniocerebral metastasis and 8 cases of liver metastasis.Node efficacy evaluation: PR(40.0%)in 12 patients,SD(23.3%)in 7 patients,PD(13.3%)in 4 patients.The median PFS was 6.4 months(95% CI5.6-7.2 months),and the median OS was not reached.2.30 patients were divided into PFS ≥ 6 months group(19)and PFS < 6-month group(11).Sequencing of the baseline samples of enrolled patients based on Illumina showed that,compared with PFS< the 6-month group at the baseline level,the α diversity of intestinal flora in PFS ≥ 6 months group was higher,and the difference was statistically significant(P<0.05).Bacterial β diversity of the PFS ≥ 6 months group was higher than PFS< 6 months group,but there was no statistical difference(P<0.05).3.Wilcoxon rank sum test was used to evaluate the significant difference in intestinal flora composition between the two groups.At baseline,there were 15 gut flora higher mean relative abundance in the PFS ≥ 6 months group,such as Ruminococcu,UCG-002 and norank＿f＿Eubacterium＿coprostanoligenes＿group,which was statistically significant.The difference of intestinal flora composition between the two groups at baseline and after 2 cycles of treatment was analyzed.In the PFS ≥ 6 months group,Coprococcus increased,Akkermansia and Desulfovibrio decreased after treatment(P<0.05).While in the PFS <6-month group,after treatment,the number of bacteria under norank＿f＿Muribaculaceae and norank＿f＿Lachnospiraceae increased,and Veillonella decreased(P<0.05).4.Non-targeted metabolomics studies of LC-MS have shown that,compared with PFS <6-month group,7,8-dihydroxycoumarin,Parasorbic acid and DG(PGJ2/0:0/8:0)of intestinal flora metabolites in the PFS≥ 6-month group were significantly decreased,while Cortivazol,Prostaglandin I2,Blumealactone B,Citroside A,Physagulin B,Lyso PC(18:4(6Z,9Z,12 Z,15Z)/0:0)and Cichorioside K were significantly increased(P<0.05).5.Multivariate analysis of Cox regression model showed that Christensenllaceae＿R-7＿group was an independent factor affecting the prognosis of immunocombined chemotherapy(P<0.05).Conclusions:1.The intestinal flora diversity and composition of ES-SCLC patients were different bewteen the the PFS≥6 months group and PFS<6 months group.α diversity and βdiversity of intestinal flora in PFS ≥ 6 months group were higher than those in PFS<6-month group.There were differences in intestinal flora composition between the two groups before treatment,and within each group,there were also differences in intestinal flora composition before and after treatment.2.The metabolites of intestinal flora in ES-SCLC patients were different in the expression levels between the PFS≥6 months group and PFS<6 months group.The expression levels of 7,8-Dihydroxycoumarin,Parasorbic acid and DG(PGJ2/0:0/8:0)were lower in patients with PFS≥6 months,and the expressions of Cortivazol and Prostaglandin I2 were higher than those of PFS<6 months group.The expression level of metabolites in intestinal flora of patients in each group changed before and after treatment.3.In this study,COX regression analysis showed that Christensenllaceae＿R-7＿group was the only independent factor that had a statistically significant impact on the prognosis of patients with extensive small-cell lung cancer receiving first-line immunotherapy combined with chemotherapy.