Study On The Clinical Efficacy Of Traditional Chinese Medicine Combined With Cryoablation In The Treatment Of Advanced NSCLC And Its Effect On Tumor Immune Microenvironmen

ObjectiveClinical part:We count the short-term and long-term efficacy of traditional Chinese medicine combined with cold ablation and single cold ablation,compare the efficacy differences between the two treatment methods,further explore the relevant predictive factors affecting the survival time after cold ablation,and summarize the characteristics of the dominant population in the traditional Chinese medicine combined with cold ablation group,so as to provide reference for clinical work.Experimental part:Based on PET-CT of small animals,to explore the effect of Yangfei Decoction combined with cold ablation on the distribution and density of tumor infiltrating immune cells in Lewis tumor bearing mice,and whether it affects the immune environment in mice by affecting JAK2-STAT3 pathway.MethodClinical part:Using the method of retrospective cohort study,the patients with stage III B-ⅣNSCLC who were diagnosed as deficiency of Qi and Yin and mutual obstruction of phlegm and blood stasis in Dongfang Hospital of Beijing University of Traditional Chinese Medicine oncology department from January 2012 to January 2019 and who only received Yangfei Decoction combined with cold ablation,and the patients with stage Ⅲ B-ⅣNSCLC who only received cold ablation in the respiratory department or oncology department of the Naval General Hospital of the Chinese People’s Liberation Army and Luoyang Central Hospital of Henan Province hospital were collected.After being included in the cases according to strict acceptance and exclusion criteria,they were matched to ensure the comparability of the two teams,The efficacy was evaluated from three aspects:short-term efficacy(tumor local control rate,peripheral immune inflammatory factor level,KPS score,main tumor marker level),long-term efficacy(postoperative survival,survival rate)and safety(mainly postoperative complications).The effects of different factors on cold ablation were further compared,and the dominant population characteristics of traditional Chinese medicine combined with cold ablation group were screened to better guide clinical work.Experimental part:(1)In Experiment 1,the Lewis tumor bearing mouse model was established and 24 Lewis lung cancer model mice were randomly divided into ① traditional Chinese medicine combined with cold ablation group,②cold ablation group,③traditional Chinese medicine group and④ model group.24 mice were examined by micro 18F-FDG PET-CT to explore the changes of SUVmax,SUVmean,tumor volume and the proportion of 40%SUVmax sketched volume before,7 days and 14 days after operation.The changes of the density of infiltrating immune cells CD8+T cells,CD86+Ml macrophages,CD206+M2 macrophages and Ly6G+neutrophils in the tumor of mice 7 days and 14 days after operation were explored by immunohistochemical method,Through the grating and pin to accurately locate the image and pathological section,explore the difference of the distribution of tumor infiltrating immune cells in the high metabolic area and low metabolic area,and further analyze the correlation between tumor infiltrating immune cells and SUVmax value.(2)Experiment 2:20 Lewis lung cancer model mice were randomly divided into①traditional Chinese medicine combined with cold ablation group,② cold ablation group,③traditional Chinese medicine group and④model group.They were killed after 14 days of intervention,and the IFN-γ and IL-10 in the tumor was detected by ELISA,RT-qPCR and Western blot were used to detect the expression levels of JAK2,STAT3 mRNA and JAK2,STAT3 and p-STAT3 protein in mouse tumors after different interventions.Research resultsClinical part:According to the inclusion and exclusion criteria,78 cases were included in the traditional Chinese medicine combined with cold ablation group and 83 cases in the simple cold ablation group.After the propensity score is matched,70 cases were included in each of the two groups.(1)Short-term effects:①esponse evaluation criteria in solid tumors:ORR(28.6%VS 17.1%)and DCR(87.1%VS 82.9%)in the traditional Chinese medicine combined with cold ablation group were higher than those in the single cold ablation group,but there was no significant difference.② The levels of peripheral immune inflammatory factors:NLR1,PLR1 and SⅡ1 in the traditional Chinese medicine combined with cold ablation group after one month of treatment were significantly lower than those in the simple cold ablation group(P<0.05);At the same time,the differences ΔNLR,ΔPLR and ΔSⅡ were also significantly lower than those in the single cold ablation group(P<0.05).③ Changes of tumor markers:after one month of treatment,the values of CEA,NSE and CYFRA21-1 in traditional Chinese medicine combined with cold ablation group and single ablation group decreased in varying degrees,but there was no significant difference.④ KPS score changes:after one month of treatment,the KPS scores of the two groups were improved,but there was no significant difference between the two groups.(2)Long term efficacy:①The median postoperative survival time of traditional C hinese medicine combined with cold ablation group was significantly higher than that of single cold ablation group(8.25 months VS 7.03 months,P=0.042).②The survi val rates of 6 months,12 months and 18 months in the traditional Chinese medicine combined with cold ablation group were higher than those in the cold ablation group,but only the survival rate of 18 months was statistically significant.(3)Postoperative complications:within one month,the main postoperative complications of the two groups were cough,blood in sputum,fever and pain.The incidence of cough and blood in sputum was the highest in the two groups,which could be relieved after symptomatic and routine treatment.There was no significant difference in the incidence of postoperative complications between the two groups(P<0.05).(4)Analysis of risk factors after cold ablation:univariate analysis found that pathological type,N stage,M stage,treatment method,peripheral blood immune inflammatory factors before treatment and the difference before and after treatment had statistically significant effects on the prognosis.Multivariate analysis showed that only M stage(M0 VS M1c),SⅡ0 and ΔNLR were independent prognostic factors affecting the prognosis of advanced NSCLC;SⅡ0 and ΔNLR are divided into high and low groups according to the cut-off point values of ROC curve 593.86 and 0.34 respectively.In the traditional Chinese medicine combined with cold ablation group,the median postoperative survival time of SⅡ0low and ΔNLRlow is significantly higher than that of SⅡ0hi and ΔNLRhi patients(P<0.05);In the simple cold ablation group,there was no significant difference in the median postoperative survival between SⅡ0low and SΔ0hi patients,while the median postoperative survival of ΔNLRlow patients was significantly higher than that of ΔNLRhi patients(P=0.003).(5)Analysis of dominant population in traditional Chinese medicine combined with cold ablation group:SⅡ0<593.86 are characteristics of dominant population with long survival time.Experimental part:Experiment 1(1)Traditional Chinese medicine,cold ablation and traditional Chinese medicine combined with cold ablation can effectively inhibit the tumor growth of mice,improve the thymus index of mice,and reduce the SUVmax,SUVmean value and the proportion of high metabolic area.The therapeutic effect of traditional Chinese medicine combined with cold ablation is significantly better than that of cold ablation alone or traditional C hinese medicine.(2)There are great differences in the distribution of immune cells in high and low metabolic areas.The distribution of CD8+T cells in low metabolic areas is significantly more than that in high metabolic areas,while the distribution of CD206+M2 macrophages and Ly6G+neutrophils is significantly less than that in high metabolic areas.(3)Traditional Chinese medicine and cold ablation therapy can regulate the immune microenvironment and enhance the immune response in tumor by acting on different immune cells in different zones.In particular,traditional Chinese medicine combined with cold ablation therapy can increase the density of CD8+T cells and M1 macrophages in low metabolism area and reduce the density of M2 macrophages in high metabolism area at the same time,and the effect is better than cold ablation alone or traditional Chinese medicine therapy.(4)SUVmax was negatively correlated with CD8+T cells,positively correlated with M2 macrophages and neutrophils,but not with M1 macrophages,indicating that SUV value can well predict the distribution of infiltrating immune cells in tumors.Experiment 2(1)Traditional Chinese medicine combined with cold ablation can significantly improve IFN-y in tumor and reduce the level of IL-10,and the effect is better than cold ablation alone or traditional Chinese medicine treatment alone.(2)Traditional Chinese medicine combined with cold ablation,cold ablation and traditional Chinese medicine treatment can inhibit the expression of JAK2,STAT3 and p-STAT3 protein and the mRNA level of JAK2 and STAT3 in JAK2-STAT3 pathway.Traditional Chinese medicine combined with cold ablation has a stronger effect than cold ablation alone or traditional Chinese medicine treatment.ConclusionClinical part:(1)compared with simple cold ablation,traditional Chinese medicine Yangfei Decoction combined with cold ablation can improve the local control rate of tumor,more effectively reduce the level of peripheral immune inflammatory factors,regulate the peripheral immune environment,improve KPS score,reduce the level of tumor markers,prolong the postoperative survival time and improve the postoperative survival rate.(2)M stage(M0 VS M1c),SII0 and ΔNLR are independent prognostic factors affecting the prog nosis of advanced NSCLC after cold ablation.(3)In the traditional Chinese medicine combined with cold ablation group,ΔNLR<0.34 and SII0<593.86 are the dominant population characteristics with long postoperative survival.Experimental studies:(1)Compared with simple cold ablation and simple traditional Chinese medicine,traditional Chinese medicine combined with cold ablation can better inhibit the tumor growth of Lewis tumor bearing mice,improve the thymus index of mice,and reduce the SUVmax,SUVmean value and the proportion of high metabolic area.(2)Traditional Chinese medicine combined with cold ablation may increase the density of CD8+T cells and M1 macrophages in low metabolism area,reduce the density of M2 macrophages in high metabolism area and increase IFN-y While reducing the level of IL-10,so as to enhance the immune response in tumor.(3)Traditional Chinese medicine combined with cold ablation may regulate the immune microenvironment in tumor by inhibiting the expression of molecular mRNA and protein in JAK2-STAT3 pathway,so as to improve the antitumor effect on Lewis tumor bearing mice.