| BackgroundsPancreatic cancer is a devastating digestive system malignancy.It is the third most common cause of cancer related death.Since its morbidity has been increasing year by year,and the mortality rate has remained relatively stable.It is expected to become the second most common cause of cancer related death by 2030,causing a huge global health and economic burden.The disease is characterized by late onset of symptoms,very limited treatment,with a 5-year survival rate of<9%,so it is known as the "king of carcinoma".At present,radical surgery is still the only hope for curing pancreatic cancer.However,most patients manifested are in the advanced stage when surgical resection offers little benefit.Early detection is the best way to improve their survival rate.The research progress in the etiology,diagnosis,and treatment of pancreatic cancer lags behind other tumors.The etiology is not completely clear at present.It is speculated that the interaction of genetics and environment results in pancreatic cancer.The diagnosis mainly relies on pancreatic imaging while lacks effective early diagnosis tools.It is not sensitive to Radiochemotherapy and immunotherapy,surgery is only available to a limited number of patients.Pancreatic cancer is estimated to develop over a decade,so there is indeed a window of opportunity for early intervention.As an important environmental factor,commensal microbiota has been proven to play a complicated role in the pathogenesis of various cancers,and the interaction between them is greater than expected.The prevalence of dysbiosis in pancreatic cancer patients is well established,but the specific characteristics of the disorder have not been agreed.There are many differences among published studies on microbiota in pancreatic cancer with research methods,experimental design and population selection,which may affect the analysis results of microbial characteristics,thus achieving inconsistent or even contradictory conclusions.Considering the limitations of the present study,in order to better understand characteristics of microbiota in Chinese patients with pancreatic cancer,this study enrolled a relatively large clinical cohort to investigated their oral microbiota as well as gut microbiota.We attempted to conduct a comprehensive description of microbial characteristics in pancreatic cancer patients,and evaluate the potential of developing microbial biomarkers as diagnostic tools for pancreatic cancer.The pathogenesis of pancreatic cancer with possible microbiota involvement was initially discussed,which may provide new ideas for the diagnosis and treatment of pancreatic cancer.ObjectivesWe aim to describe characteristics of the oral and gut microbiota in a Chinese pancreatic cancer cohort,and to evaluate the potential of microbial biomarkers as diagnostic tools for pancreatic cancer,and initially explore the role and related mechanisms of microbiota in the pathogenesis of pancreatic cancer.Methods1.There are 50 patients with pancreatic cancer admitted to Pancreatic Surgery of Changhai Hospital,50 healthy volunteers undergoing physical examination at the Health Management Center of Air Force Hospital of Eastern Theater,15 patients with chronic pancreatitis admitted to Department of Gastroenterology of Changhai Hospital enrolled in our cohort,and collected their saliva and feces over the same period.The samples were quickly aliquoted and stored for future use.At the same time,its basic demographic and clinical data are recorded in detail,including:age,gender,BMI,ethnicity,eating habits,history of basic diseases,history of medication,digestive system surgery history.2.Based on the previous study results of our team and literature research,we select 18 clinical indexes(UA,CA19-9,CEA,CA125,RBC,WBC,LYMP%,NEU%,EOS.abs,HGB,HCT,P-LCR,TBiL,DBiL,GLU,Amy,FIB,and CRP)that may be associated with pancreatic cancer.The values of these indexes during the period of sample collection for patients with pancreatic cancer were recorded.3.Total DNA extraction of all samples were performed simultaneously,16S rRNA gene V3-V4 region was amplified by PCR,then conducted library construction with quality control,after quantification and sample mixing,we utilized Illumina MiSeq PE250 high throughput sequencing platform to analyze the processed samples.The original sequencing data was spliced for quality control to obtain the operational taxnomic unit(OTU),and a series of analyses were performed on saliva OTU and fecal OTU in parallel:including OTU clustering,flattening,Core microbiome analysis,Venn map analysis,OTU PCA analysis,Specaccum analysis.Obtain the OTU abundance table,and then conduct species classification and abundance analysis,alpha diversity analysis,and beta diversity analysis.4.Parallel analysis of data from saliva and stool samples.Each group was analyzed for significant differences in phylum and genus such as LEfSe difference analysis,rank sum test analysis,and based on this,a random forest algorithm was used to construct the diagnostic model,ROC analysis was performed to evaluate the diagnostic efficacy of traditional tumor markers on PC,and Spearman correlation coefficient analysis was performed on the identified differential genera in pancreatic cancer group.Spearman correlation analysis was performed on the genera and clinical indexes;16S function prediction analysis was performed on the differential microbiota.Results1.After clinical diagnosis and quality control,16S rRNA sequencing data of 88 saliva samples(37 PC,36 HC,15 CP)and 92 stool samples(38 PC,39 HC,15 CP)were finally included for subsequent analysis.2.The alpha diversity of oral microbiota in the pancreatic cancer group was higher than that in the chronic pancreatitis group,but there was no significant difference with the healthy control group.The microbial composition of pancreatic cancer has altered significantly.Specifically,compared with the other two groups,the average relative abundance of the two phylums,Firmicutes and Verrucomicrobia,were significantly higher in patients with pancreatic cancer;There are 7 genera enriched in patients with pancreatic cancer,namely Veillonella,Peptostreptococcus,Akkermansia,Parvimonas,Solobacterium,Olsenella,Actinomyces and Escherichia_Shigella.Compared with the healthy control group,the average relative abundance of Firmicutes in the pancreatic disease group was significantly higher;The average relative abundance of the four genera(Alloprevotella,Granulicatella,Solobacterium,and Streptococcus)were significantly increased.Differential oral genera are mostly symbiotic.Only Veillonella,Stomatobaculum,and Eubacterium are competing with other genera.3.Oral microbiota of patients with pancreatic cancer is mainly characterized by significant enrichment of oral pathogenic bacteria represented by Granulicatella,Solobacterium,Alloprevotella and Peptostreptococcus.The 16S function analysis predicts that the oral microbiota of pancreatic cancer has little effect on metabolism.It mainly strengthens the pro-inflammatory pathway and immunosuppressive pathway,specifically bacterial toxin,Staphylococcus aureus infection and alanine metabolism.Pathway in antigens processing and presentation as well as cell migration and secretion are significantly weakened.4.Alpha diversity of gut microbiota in pancreatic cancer group has not changed significantly,but the microbiota composition has altered strongly.Specifically,compared with the other two groups,Prevotella and Coprobacter was significantly enriched;compared with the healthy control group,there were four genera enriched in the pancreatic disease group namely Prevotella,Peptostreptococcus,Actinomyces,and Aeromonas.The average relative abundance of 9 genera,including Aeromonas,Bifidobacterium,Campylobacter,Coprobacillus,Escherichia_Shigella,and Gordonibacter,were relatively high.The competition and synergy between different gut genera are not seemingly strong.The five genera,including Butyricicoccus,Clostrdium?,Fusicatenibacterium,Faecalibacterium,and Gemmiger,exhibit a core symbiotic relationship.5.The characteristics of gut microbiota in patients with pancreatic cancer are mainly manifested in significantly reduced probiotic abundance,while Prevotella,Bifidobacterium,Escherichia_Shigella,Peptostreptococcus and Actinomyces were the most abundant pathogenic bacteria.The 16S function prediction of differential genus mainly found obvious activation of inflammatory pathways and disturbances of fatty acid metabolism,specifically arachidonic acid metabolism,?-linolenic acid metabolism,fatty acid extension,cytochrome P450 metabolism,LPS biosynthesis,and pathogenic E.coli infection,and systemic lupus erythematosus pathways are enhanced,while cytoskeletal protein pathway is weakened.6.The AUC values of diagnostic models based on oral/gut microbiota characteristics in patients with pancreatic cancer are 0.916(95%CI:0.832-1)and 0.856(95%CI:0.74-0.972),respectively,which shows good classification ability in our cohort.In addition,the classification ability of traditional tumor markers on PC group was also evaluated in the cohort,CA19-9 combined with CEA had the best diagnostic effect on PC compared with other combination,and the AUC value was up to 0.878(95%CI:0.805-0.952).The capability of classification suggests that microbial markers have the potential to develop into non-invasive diagnostic tools for pancreatic cancer.Based on the analysis of differential genus and differential function,candidate genera that may be involved in the course of pancreatic cancer were screened,namely Granulicatella,Peptostreptococcus,Prevotella,Bifidobacterium,and Escherichia_Shigella;pathways involved in the development of pancreatic cancer were analyzed as bacterial toxin,alanine metabolism,arachidonic acid metabolism,lipopolysaccharide biosynthesis,systemic lupus erythematosus pathway,and cytoskeleton pathway.ConclusionsThis study comprehensively described the microbiota characteristics of a Chinese pancreatic cancer cohort,and found that the microbial diversity was not significantly reduced,but the microbiota composition had changed signifi cantly.Oral pathogenic genera such as Granulicatella,Peptostreptococcus,Alloprevotella,Veillonella,Solobacterum,and Streptococcus showed a significant enrichment in patients with pancreatic cancer;Probiotics abundance is significantly reduced.Opportunistic genera such as Prevotella,Bifidobacterium,Escherichia_Shigella,Peptostreptococcus,and Actinomyces were enriched significantly.Diagnostic models based on the characteristics of the oral or gut microbiota can well discriminate the pancreatic cancer samples in our cohort,indicating that microbial biomarkers have the potential to develop into diagnostic tools for pancreatic cancer.In addition,candidate genera and related pathways that may be involved in the course of pancreatic cancer were identified.Our results help to further understand the role of microbiota in the course of pancreatic cancer and provide new insights for its diagnosis and treatment.It should be noted that we have confirmed the association between microbiota and pancreatic cancer in a Chinese pancreatic cancer cohort,but this study can not provide direct evidence as to whether there is a causal link between the two.Based on our research analysis,combined with published research results on pancreatic cancer,we propose that harmful bacteria in the oral cavity translocated and colonized in the pancreas,evading immune surveillance,inducing tissue inflammation through local effects directly.Inflammation leads to an imbalance of oxidative stress in the microenvironment and changes in energy dynamics,thus damages DNA,eventually leading to molecular mutations and tumor transformation,promoting the development of pancreatic cancer;while the gut microbiota affects the tumor microenvironment through metabolic and pro-inflammatory pathways and interacts with other known risk factors of pancreatic cancer,such as smoking,alcoholism,etc.,which promote tumorigenesis remotely. |
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