| PART 1 A prospective cohort study of 406 patients with advanced colorectal cancer after failure of conventional treatmentObjective1.To evaluate the clinical effect of traditional Chinese medicine in the treatment of advanced colorectal cancer after conventional treatment failure,and to validate and discuss the role of traditional Chinese medicine in the treatment of advanced colorectal cancer.2.To sum up the clinical characteristics of the preponderant population who benefit from TCM treatment,and to analyze the prognostic factors that influence survival.MethodsA prospective,multicenter,cohort study was conducted in 9 large Chinese medicine and Western medicine centers to include patients with advanced colorectal cancer who had failed conventional treatment,the patients were divided into three groups:TCM Group,integrated TCM and wm group and wm group.The follow-up period was 1 cycle every 3 months,beginning with the patient's admission and ending with the patient's death or termination of the study.The main outcome measure was the total survival time(Os),and the secondary outcome measure was PFS.Results1.General InformationThe data analysis deadline was November 30,2019,a total of 406 patients were included in the analysis,and the final formation of the cohort by statistics,139 people in the Western medicine group,155 people in the integrated Chinese and Western Medicine Group,112 people in the Chinese Medicine Group.The median follow-up time was 19.67 months in the group of Western medicine,20.47 months in the Group of integrated traditional and Western medicine,and 20.4 months in the group of traditional Chinese medicine.The median exposure time was 6.43 months and the longest exposure time was 29.73 months.The median exposure time was 3.73 months and the longest exposure time was 33 months.In the population information,and the tumor basic information three groups are basically balanced.2.Survival Analysis2.1 Overall median overall survival analysisThe overall median overall survival time was 11.37 months in the western medicine group,14.03 months in the integrated Chinese and western medicine group,and 6.33 months in the integrated traditional Chinese medicine group(P<0.001).Compared with the western medicine group,the risk of death in the integrated traditional Chinese and western medicine group was reduced by about 26%(HR=0.74,95%CI 0.55-0.98,p=0.038).Compared with the traditional Chinese medicine group,the risk of death in the integrated traditional Chinese and western medicine group was reduced by about 49%(HR=0.51,95%CI 0.38,0.69,P<0.001).Compared with the traditional Chinese medicine group,the western medicine group reduced the risk of death by about 47%(HR=1.47,95%CI 1.09,1.99,p=0.012).2.2 Overall progression-free survival analysis.Through the analysis of the overall progression-free survival time(PFS,progression-free survival),it was found that the PFS of the western medicine group was 5.37months,the PFS of the integrated traditional Chinese and western medicine group was 5.8months,and the PFS of the traditional Chinese medicine group was 5.03months.There was no significant difference in the risk of death among the three groups(P>0.05).2.3 Survival analysis of subgroups of integrated traditional Chinese and western medicine group and western medicine group.Compared with the western medicine group,the integrated traditional Chinese and western medicine group significantly prolonged OS(P<0.05)and reduced the risk of death in female,KPS score? 80,metastatic organs? 2,KRAS mutation and previous antineoplastic therapy?3 lines(P<0.05).2.4 Survival analysis of subgroups of western medicine group and traditional Chinese medicine group.The survival analysis of western medicine group and traditional Chinese medicine group showed that in male,age? 65 years old,right colon,diagnosis period ?<18 months,previous anti-tumor therapy ?2 lines,number of metastatic organs?2,liver metastasis,KRAS mutation,unknown BRAF status,past targeted therapy,TCM syndrome type of spleen and kidney deficiency and internal stasis syndrome,OS was significantly longer in the western medicine group than in the traditional Chinese medicine group.And significantly reduce the risk of death(P<0.05).2.5 Survival analysis of integrated traditional Chinese and western medicine group and subgroup of traditional Chinese medicine group.In the survival analysis of the integrated traditional Chinese and western medicine group and the traditional Chinese medicine group,regardless of sex,age,left and right colon,diagnosis time of stage IV,liver metastasis and previous targeted therapy,the survival time of the integrated traditional Chinese and western medicine group was significantly longer than that of the traditional Chinese medicine group(P<0.05).And reduce the risk of death(P<;0.05).For KPS score<80,KRAS mutation,wild and unknown state of BRAF,previous antineoplastic therapy?2 lines,low exposure group of traditional Chinese medicine,subgroup of deficiency of spleen and kidney syndrome,OS could be significantly prolonged and the risk of death could be reduced(P<0 05).2.6 Survival subgroup analysis of the last evaluation state.In the progressive subgroup survival analysis,the survival of the patients in the integrated traditional Chinese and western medicine group was significantly longer than that in the traditional Chinese medicine group(13.10 monthsvs6.87 months,P=0.006),but there was no significant difference between the integrated traditional Chinese and western medicine group and the western medicine group,the traditional Chinese medicine group and the western medicine group.The analysis of intolerant survival subgroup showed that the survival in the integrated traditional Chinese and western medicine group was significantly longer than that in the traditional Chinese medicine group and the western medicine group(P<0.05),but there was no significant difference in survival between the western medicine group and the integrated traditional Chinese and western medicine group(P>0.05).3.COX multivariate regression analysis.3.1 results of multivariate analysis of the population:The primary location of tumor,KPS score and previous targeted therapy were independent prognostic risk factors.The risk of death in patients with right colon was 1.4 times higher than that in patients with left colon,and the risk of death in patients with KPS score less than 80 was 1.96 times higher than that in patients who had not received targeted therapy.Traditional Chinese medicine exposure(HR=0.718,95%CI0.535-0.962,P=0.0267)and western medicine anti-tumor treatment(HR=0.55,95%CI0.402-0.751,P<0.001)were independent protective factors,which could reduce the risk of death.3.2 Multivariate analysis of subgroup population.a.Integrated traditional Chinese and western medicine group and western medicine group.The independent risk factors affecting survival were KPS<80,past anti-tumor treatment lines 3 lines,and the diagnosis time of stage ?>18 months and integrated traditional Chinese and western medicine treatment were independent protective factors affecting survival.b.Integrated traditional Chinese and western medicine group and traditional Chinese medicine group.Multivariate analysis of the population in the integrated traditional Chinese and western medicine group and the traditional Chinese medicine subgroup:KPS<80 and previous targeted therapy were the independent risk factors affecting the survival of the patients,while receiving integrated traditional Chinese and western medicine treatment and exposure of traditional Chinese medicine were the independent protective factors affecting the survival of the patients.c.Traditional Chinese medicine group and western medicine group.KPS score<80,the primary location of the tumor in the right colon and the number of lines of previous anti-tumor therapy were independent risk factors.4.Quality of life evaluation.a.FACT-C(v4.0).From the point of view of the score after entering the group,the score of the traditional Chinese medicine group was the lowest,the integrated traditional Chinese and western medicine group was the best,and the western medicine group was the second,and the difference among the three groups was statistically significant(P=0.0003).After 1 cycle,the test outcome index was still dominant in the integrated traditional Chinese and western medicine group.b.TCM symptom rating scale.In terms of the effective rate of symptom improvement in traditional Chinese medicine,the advantages of integrated traditional Chinese and western medicine group,western medicine group and traditional Chinese medicine group were 24%,70%,13%and 9.5%,respectively.Conclusions1.There was no significant difference in the improvement of PFS in advanced colorectal cancer after routine treatment.2.Under the premise that the demographic information,primary location of tumor,pathological type and degree of differentiation of the three groups were basically the same,the combination of traditional Chinese and western medicine treatment significantly prolonged the overall median OS,and improved the quality of life of patients compared with western medicine treatment and traditional Chinese medicine treatment.3.In the subgroup analysis,for the advanced colorectal cancer patients with long disease progression,poor effect after multi-line treatment,heavy tumor load and poor prognosis of gene status,the treatment of integrated traditional Chinese and western medicine still achieved better clinical benefits than that of western medicine alone.4.In the face of heavy tumor load and non-standard anti-tumor treatment,simple traditional Chinese medicine treatment is difficult to compare with western medicine in many factors with strong poor prognosis.Patients who cannot tolerate chemotherapy should choose the treatment of integrated traditional Chinese and western medicine to reduce toxicity and increase efficiency in order to maximize the survival and prognosis.5.Among the overall prognostic factors,the right colon,KPS score<80 and previous targeted therapy were independent prognostic factors,while traditional Chinese medicine exposure and western medicine anti-tumor therapy were independent prognostic factors,and subgroup independent prognostic factors.PART 2 Study on the relationship between ctDNA status and prognosis of 83 cases of failed conventional treatment of Advanced Colorectal Cancer treated with traditional Chinese MedicineObjectiveTo explore the ctDNA status mutation gene and its correlation with prognosis in patients with advanced colorectal cancer treated with traditional Chinese medicine(TCM)by NGS technique.MethodsPatients with advanced colorectal cancer who had failed conventional treatment for more than 3 months were collected and 10ml was extracted from peripheral blood.The program provided by the clinical testing center was used to detect ctDNA tumor-related mutation genes in peripheral blood,and the customized NGS detection panel covered 416 tumor-related genes.Results1.General information.A total of 83 patients from four centers of Xiyuan Hospital,Guanganmen Hospital,Beijing traditional Chinese Medicine Hospital and Peking University Cancer Hospital of the Chinese Academy of traditional Chinese Medicine were included,of which 29(34.94%)were older than 65 years old and 54(65.05%)were less than 65 years old.There were 48 males(57.83%)and 35 females(42.17%).Traditional Chinese medicine group(n=37)and integrated traditional Chinese and western medicine group(n=46).2.Tumor gene mutation in peripheral blood cells.Among the 83 patients,54 cases(65.06%)had tumor-related mutations in plasma,and 29 cases(34.94%)had no tumor-related mutations.According to the mutation frequency,the genes of TOP20 were screened as APC,TP53,KRAS,PIK3CA,FBXW7,SMAD4,LRP1B,PTPN13,EGFR,CTNNB1,ROS1,PKHD1,EPHA3,SMARCA4,FAT1,HDAC9,PREX2,NOTCH1,SMAD3,SF3B1.In addition,6 patients had germline gene mutations,which were APC(46.49%),RAD51C(42.93%),PDE11A,ABCB1 and TTF1.APC and RAD51C were known pathogenic genes.3.Bioinformatics analysis of mutant genes.The mutual functions of mutant proteins focus on protein autophosphorylation,FLT3 signal,interleukin signal,peptide tyrosine phosphorylation,peptide tyrosine modification,DNA integrity checkpoint,EGFR tyrosine kinase inhibitor resistance pathway,cell response to toxic substances and protein kinase B signal transduction pathway.4.The relationship between ctDNA mutation and prognosis.The prognosis of ctDNA high mutation group was better than that of low mutation group(17.47monthsvs8.57months,P=0.033).There was a significant difference in the median OS between the two groups in the tumor tissue mutation load((tumor mutational burden,TMB)(16.63 months vs 10.00 months P=0.007).The prognosis of patients with),KRAS mutant type was worse than that of KRAS wild type(8.57 monthsvsl6.63 months P=0.047),and the survival prognosis of patients with PIK3CA mutant type was worse(6.97 monthsvs15.80 months,P=0.045).No correlation was found between the gene status of APC and TP53 and prognosis.5.Analysis of gene mutation and clinical factors.ctDNA allele mutation frequency was significantly correlated with liver metastasis(P=0.014)and TCM syndrome type(P=0.037),and tumor mutation load TMB was significantly correlated with liver metastasis(P=0.007),lung metastasis(P=0.049),TCM syndrome type(P=0.012)and eight-class syndrome differentiation(P=0.044).KRAS mutation was significantly correlated with liver metastasis and lymph node metastasis(P<0.05),and PIK3CA gene mutation was significantly correlated with age and lymph node metastasis(P<0.05).TP53 gene mutation was significantly correlated with liver metastasis and TCM syndrome type of deficiency of spleen and kidney(P<0.05),but no significant clinical factors were found for APC gene mutation.Conclusions1.The biological pathways and pathways related to proteases such as tyrosine kinase and serine/threonine kinase may be associated with the poor prognosis of the failure of routine treatment for advanced colorectal cancer,which needs to be verified in the later stage.2.The frequency of ctDNA mutation,TMB KRAS and PIK3CA genes in patients with advanced colorectal cancer after the failure of standard treatment with traditional Chinese medicine are related to the prognosis,and liver metastasis is related to bad gene mutation.3.The treatment of invigorating the spleen and tonifying the kidney with traditional Chinese medicine is related to the reduction of ctDNA mutation frequency and tumor suppressor gene mutation;The TMB load of patients with Yin syndrome is lower,so it may be difficult to benefit from immunotherapy.PART 3 Bioinformatics analysis of differentially expressed genes in patirents with advanced colorectal cancer who failed conventional therapyObjectiveTo study the mechanism of difference in efficacy of conventional therapy for advanced colorectal cancer,and to explore the possible molecular targets and related pathway mechanisms that may affect the prognosis,which will provide new ideas and new angles for its clinical treatment.MethodsIn this study,GO(Gene Ontology gene ontology analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes,Kyoto gene and genome encyclopedia analysis were used to screen differential genes in advanced colorectal cancer patients who failed conventional therapy.(Gene Set Enrichment Analysis GSEA)all genes in the sample were used to analyze the function and biological process of genes more comprehensively.The key Hub genes were screened by protein-protein interaction(Protein-protein Interaction PPI)network.The relationship between Hub gene and the prognosis of colorectal cancer was analyzed in order to explore the mechanism of failure in conventional treatment of advanced colorectal cancer.Results1.General information.Search the GEO public genetic database(Gene Expression Omnibus GEO)and obtain the GSE72970 data set,which contains 12 samples of advanced colorectal cancer with failed conventional treatment and 7 samples of advanced colorectal cancer with complete remission after conventional treatment.2.DEGs in patients with advanced colorectal cancer who failed to receive standard chemotherapy.The online tool of GEO2R platform was used to screen the DEGs of patients with advanced colorectal cancer who failed conventional treatment compared with complete remission in GSE72970 data set.Screening results:a total of 323 DEGs,were obtained from failed CRC compared with complete remission gene samples,of which 97 were up-regulated and 226 were down-regulated.3.GO analysis and KEGG analysis of DEGs.GO analysis and KEGG analysis were used to compare the DEGs of failed mCRC with complete remission of mCRC.The results of GO analysis showed that the cytological components of Top6 enriched by DEGs were endoplasmic reticulum,presynaptic active region,chromatin accessibility complex,Epsilon DNA polymerase complex,RNA polymerase I transcription factor complex and peri-chromatin fibers.The molecular function(Molecular function MF)of Top 6 enriched by DEGs are:protease inhibitor activity,molecular function unknown,S-transferase activity,galactosyltransferase activity,antigen binding,protein serine/threonine phosphatase activity.The biological processes(Biological process BP)of Top 6 enriched by DEGs include metabolism,energy pathway,unknown biological process,apoptosis,cell cycle regulation and neurotransmitter transport.The results of KEGG analysis showed that the Top 6 biological pathways significantly enriched by DEGs were as follows:Prophase and metaphase of mitosis,ab initio biosynthesis of guanosine nucleotides,M phase,deposition of new CENPA nucleosomes at centromere,nucleosome assembly,mitotic M-M/G1 phase.4.GSEA analysis.The functional set of enriched Top 6:chaperone-mediated autophagy,catecholamine metabolism,T cell-mediated cytotoxic regulation,selection of intrathymic T cells,membrane targeting of cotranscriptional proteins,and positive regulation of antigen receptor-mediated signal pathways.5.Construction of PPI network and screening of Hub gene.The screened DEGs was used to construct PPI network with the help of STRING tool,and the cyto Hubba plug-in of Cytoscape was used to screen Top20 Hub gene.They are:SPC25,CENPH,CENPK,SPDL1,CENPQ,PPP2CA,DSN1,KIF14,CDC6,UBE2V2,CDC23,HACE1,SMURF2,KBTBD6,KBTBD7,TRIP13,SHCBP1,CLCA1,NIP7,CLCA4.6.Prognostic analysis of Hub geneGEPIA website was used to analyze the effect of Hub gene on the prognosis of mCRC patients with failed chemotherapy.The results showed that the expression of CLCA1 gene was correlated with OS(paired 0.012),and the survival prognosis of patients with high expression of CLCA1 was better than that of patients with low expression.At the same time,the expression of CLCA1 and TRIP13 genes was closely related to the DFS of CRC patients.Conclusions1.The enrichment of protease including serine/threonine protein kinase(PKB/AKT),tyrosine kinase,apoptosis autophagy,cell cycle regulation,protein transcriptional modification,target gene TP53,P53 pathway activation and other biological processes are consistent with the second part of peripheral blood ctDNA mutant gene enrichment pathway.The mechanism of the pathway related to the failure of conventional treatment of refractory colorectal cancer has been preliminarily verified.2.The expression of CLCA1 and TRIP13 is related to the prognosis of advanced colorectal cancer and may be a potential biomarker of prognosis in the future. |
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