Part 1:parallel Profiling Of Mutations And Methylation Changes From Bile Sample Detects Malignancies In Pancreatobiliary System Part 2:the Commensal Consortium Of The Gut Microbiome Is Associated With Favourable Responses To Anti-PD-1 Therapy In Patients

Objective:Based on the parallel profiling of mutations and methylation changes from bile samples,a screening model of malignancies in the pancreaticobiliary system was established.Then it can provide an auxiliary diagnosis method for patients with indefinite results during ERCP in clinical,so as to improve the diagnostic yield for pancreaticobiliary diseases.Moreover,some patients can have timely treatment and better prognosis.Content:In this study,259 bile samples were collected from 5 hospitals and used for mutation and methylation analysis.A diagnostic model(Bilescreen)was constructed based on the sequencing results and clinicopathological characteristics.The sensitivity and specificity of Bilescreen was analyzed in the test set,which included the undefined cases during ERCP.The serum CA19-9 were also analyzed,comparing with the performance of mutation and methylation changes in the bile.In addition,we also compared the microbiome in the benign and malignant bile fluid.Method:Bile samples were collected and then divided into training set,validation set and test set.DNA was extracted from bile samples.Quantitative real-time PCR was performed for the detection of the proportion of human-derived DNA and bacterial DNA.For the high level of human-derived DNA,libraries were constructed by the"Mutation Capsule" method.Bilescreen was constructed based on mutation and methylation results in the training set,and was optimized in the validation set.The test set was provided to verify the model.The characteristics of patients,including serum CA 19-9,were collected from medical records.According to the clinical information,the sensitivity and specificity of serum CA19-9,mutation and methylation in bile were also analyzed.For the high level of bacterial DNA in the bile,the V4 region of bacterial DNA was amplified and sequenced by 16S rDNA V4 amplicon sequencing.The distribution of microbiome in the bile of pancreaticobiliary diseases was analyzed,in order to compare the differences between benign and malignant diseases.Results:In the training set,the sensitivity of serum CA19-9 and Bilescreen was comparable.It was 88.10%and 90.48%respectivily.The specificity of mutation achieved 100%.The specificity of mutation and methylation was 97.56%.However,the specificity of serum CA19-9 declined to 68.85%.In the undetermined cases by ERCP,the sensitivity and specificity of Bilescreen were 83.87%and 85.71%respectively,while the sensitivity of serum CA19-9 was 83.87%and the specificity was only 14.29%.In biliary microbiome,Firmicutes was the main phylum in benign group and Prot.eobacteria was the main phylum in malignant group.There was no significant difference in the relative abundance of these two phyla(p=0.267 and p=0.215).Moreover,six bacterial families were significantly enriched in the malignant group.They were Aeromonadaceae(p=0.019).Lachnospiraceae(p=0.030),Microbacteriaceae(p=0.045),Porphyromonadaceaea(p=0.030).Prevotellaceae(p=0.045)and Veillonellaceae(p=0.045).Conclusions:In the current study,DNA in the bile samples was subjected to next generation sequencing and then a diagnostic model,Bilescreen,was constructed based on the sequencing results.The diagnostic yield for Bilescreen through the two dimensions of mutation and methylation was comparable with clinical conventional diagnosis.Bilescreen increased the detection of malignancies in the undetermined cases by ERCP,which is expected to be applied in the clinical diagnosis.In the aspect of microbiome,specific bacterial families were overrepresented in the bile of patients with malignancies,indicating that there were differences in the distribution of biliary microorganisms between benign and malignant groups.It could be another biomarker to distinguish benign and malignant patients and improve the diagnostic yield of pancreaticobiliary diseases.Objective:In this study,we analyzed the differences of gut microbiome in the baseline fecal samples of patients with advanced thoracic neoplasms during anti-programmed death protein 1(PD-1)therapy.The relationship between the different gut microbiome and the efficacy of immunotherapy was also explored.It is expected that the significant different bacterial taxa can be used as biomarkers to predict the efficacy of anti-PD1 treatment,so that more patients can benefit from immunotherapy.Content:Herein,we determined the associations between baseline gut commensal microbes and the clinical treatment efficacy of patients with advanced thoracic neoplasms during anti-PD-1 therapy.First,fecal samples were collected prior to and during anti-PD1 treatment.DNA was extracted from stool samples and was applied to 16S ribosomal RNA gene sequencing.Alpha diversities,beta diversities and the distribution of gut microbiome were analyzed.The different microbiome between the responders and nonresponders was compared at the bacterial family level.Combined with the clinical data,the correlation between the efficacy and different bacterial families was also analyzed.Methods:Forty-two patients with advanced thoracic carcinoma who received anti-PD-1 treatment were enrolled in the study.Baseline and time-serial stool samples were analyzed using 16S ribosomal RNA gene sequencing.The alpha and beta diversities of gut microbiome at baseline and during the treatment were compared between the responders and nonresponders group.The compositions of the gut microbiome were also analyzed at the bacterial phylum level and family level respectively.Tumor responses and disease control rate were defined as short-term effect.Patient progression-free survival and overall survival were defined as long-term effect.They were used to measure the association between gut microbiome and clinical outcomes.Univariate and multivariate logistic regression analyses were used to determine the risk factors for gut microbiome.Results:The diversities of the baseline gut microbiota were similar between responders(n=23)and nonresponders(n=19).Firmicutes,Bacteroidetes.Proteobacteria and Actinobacteria were the main bacteria phyla at baseline and there were no significant distributions between the responder and nonresponder groups.The relative abundances of the Akkermansiaceae,Enterococcaceae,Enterobacteriaceae,Carnobacteriaceae and Clostridiales Family XI bacterial families were significantly higher in the responder group.These 5 bacterial families acted as a commensal consortium and better stratified patients according to clinical responses(p=0.014).Patients with a higher abundance of commensal microbes had prolonged PFS(p=0.00016).Using multivariable analysis,the abundance of the commensal consortium was identified as an independent predictor of anti-PD-1 immunotherapy in thoracic neoplasms(HR:0.17;95%CI:0.05-0.55;p=0.003).Moreover,the abundance of the commensal microbiome slightly fluctuated and was not significantly altered over the course of anti-PD-1 treatment(p=0.878).Conclusions:Baseline gut microbiota may have a critical impact on anti-PD-1 treatment in advanced thoracic neoplasms.The abundance of gut commensal microbes,including Akkermansiaceae,Enterococcaceae,Enterobacteriaceae.Carnobacteriaceae and Clostridiales Family XI.at diagnosis might be useful for the early prediction of anti-PD-1 immunotherapy responses.