Analysis And Clinical Significance Of Intestinal Microbiota In Colorectal Neoplasms

Part Ⅰ:A Weighted Dysbiosis Index for Colorectal Cancer Analysis Based on Gut MicrobiomeObjective:Occurrence of gut microbial dysbiosis in colorectal cancer(CRC)is a complex process involving compositional alterations of multiple bacterial species or taxa.Condensing this complex changes into a simple index would facilitate clinical application of gut microbiome data.The study aim was to build a single numeric index to measure gut dysbiosis associated with CRC based on 16S rRNA sequencing data.Methods:Stool samples were collected froml29 patients belonging to CRC group(70 cases)and healthy group(59 cases)prior to colon resection surgery or endoscopic examination.Microbial composition and diversity were analyzed by 16S rRNA sequencing to compare the composition of gut microbiota between the control and CRC group,as well as among the patients with different pathological lesions and types of CRC.Based on the relative abundance of CRC enriched or depleted taxa,a weighted dysbiosis index of cancer(WDIc)model was built,tested with 123 samples from a separate cohort of patients,and further validated with 396 samples from three datasets published by various authors.Results:A signature of 7 taxa was identified:Fusobacterium nucleatum,Parvimonas,Peptostreptococcus,Clostridiales Family Ⅺ and Porphyromonas were significantly enriched in CRC group while Pseudobutyrivibrio and Streptococcus salivarius were depleted in CRC group compared to healthy group.At the same time,it was found that there were characteristic differences in the composition of gut microbiome among patients with CRC of different tumor location and pathological types.On average the WDIc scores of these 7 taxa was significantly higher in the CRC group than in the healthy group and positively correlated with tumor size and TNM staging.WDIc could differentiate CRC from the healthy samples with an AUC of 0.83 of the ROC curve,and with a sensitivity of 71.4%and a specificity of 83.1%.It had sensitivity of 71.0%and specificity of 87.0%when tested in the second cohort of patients and reasonable accuracy when further validated in the three public datasets.Conclusion:We developed an index to measure the gut microbiota dysbiosis in CRC patients,which had a good discriminatory power to distinguish CRC from the healthy samples,and may facilitate the potential clinical application of gut microbiota data in CRC detection.Part Ⅱ:The Correlation Between Changes of Gut Microbiome and Colorectal AdenomaObjective:Colorectal adenoma(CRA)is a classic precancerous disease of CRC and early diagnosis and treatment will greatly improve the prognosis of thus patients.Clinical screening indicators such as fecal occlusive blood test(FOBT)and serum carcinoma embryonic antigen(CEA)have low sensitivity to CRA,and there is still lack of non-invasive and efficient early detection methods.With the development of sequencing technology,changes in the abundance of gut microbiota in CRA patients have been found.This study aims to find a new marker to screen early colorectal lesions based on 16S rRNA sequencing.Methods:Stool samples were collected from 155 participants belonging to CRA group(96 cases,including advanced adenoma group 52 cases and nonadvanced adenoma group 44 cases)and healthy group(59 cases).FOBT and serum CEA were tested.Microbial compositions of the samples were assessed by 16S rRNA sequencing.The differences of intestinal microflora composition between control and CRA,advanced and non advanced adenoma patients were compared,and the changes of intestinal metagenome functions along the adenoma-carcinoma sequence were analyzed.Based on the relative abundance of CRA enriched or depleted taxa,a weighted dysbiosis index of adenoma(WDIa)model was built.The efficacy of WDIa,serum CEA and fecal OB were compared and analyzed.Results:Body mass index(BMI)of patients with adenoma was higher than healthy controls(P=0.014).And the size of intestinal polypus was much larger in patients with advanced adenoma than that of nonadvanced adenoma(P=9.15*10-18).Compared with control group,the abundance of Klebsiella Pneumoniae,Parabacteroides Merdae and Fusobacterium were significantly increased in the adenoma group.However,the abundance of Lachnospiraceae,Blautia,Actinobacteria,Bifidobacterium,Roseburia,Streptococcus,Pseudobutyrivibrio,Fusobacterium Ulcerans were significantly decreased.Prevotella and Bacteroides were significantly increased in nonadvanced adenoma group while Peptostreptococcus Anaerobius and Lachnoclostridium were significantly increased in advanced adenoma group.At the same time,30 Kyoto encyclopedia of genes and genomes(KEGG)pathways,such as amino acid metabolism,cardiovascular、nervous and endocrine system related diseases,peroxisome proliferators activated receptors(PPARs)and p53 pathways,were found to be differentially expressed in different stages of colorectal tumorigenesis.For FOBT,the sensitivity to CRA was 45.5%,the specificity was 91.5%,false positive rate was 8.5%and false negalive rate was 54.5%.For CEA,the sensitivity was 52.3%,the specificity was 70.6%,and the area under the ROC curve was 0.614.For WDIa,the sensitivity was 88.1%,the specificity was 54.2%,and the area under the ROC curve was 0.756.Conclusion:There were characteristic changes of intestinal microbial composition and metagenomic functions during the CRA stage of colorectal tumorigenesis.The sensitivity to CRA of WDIa in this study was significantly higher than that of FOBT and serum CEA.WDIa could identify early CRA more quickly and conveniently,and had the potential to be a noval marker of early colon lesions.Part Ⅲ:Alteration of the abundance of Parvimonas micra in the gut along the adenoma-carcinoma sequenceObjective:Parvimonas micra(P.micra)is reported to be associated with CRC.However,its association with CRA and its role in the initiation of colorectal tumors remain unknown,The present study aimed to clarify the relationship between P.micra and CRA and CRC by exploring the changes of P.micra abundance in an adenoma-carcinoma sequence in a SuZhou cohort and 4 public sequencing datasets.Methods:To investigate the alterations of P.micra abundance in the gut along the adenoma-carcinoma sequence,quantitative polymerase chain reaction(qPCR)was conducted to measure the relative abundance of P.micra in fecal samples from 277 subjects(128 patients with CRA,66 patients with CRC and 83 healthy individuals as controls)who underwent colonoscopy as outpatients.Then,the relative abundance of P.micra was analyzed in fecal samples from 596 subjects(185 healthy controls,158 CRC,253 CRA)in four public 16S rRNA sequencing datasets.Results:The qPCR results demonstrated that the CRA group had an abundance of P.micra(P=0.2)similar to that of the healthy group,while the CRC group had a significantly increased abundance(P=8.2×10-11).The level of P.micra effectively discriminated patients with CRC from healthy controls,while it poorly discriminated patients with CRA from healthy controls;with an area under the receiver operating characteristic curve of 0.867 for patients with CRC and 0.554 for patients with CRA.The same pattern of the alteration of P.micra abundance,which was low in healthy controls and patients with CRA but elevated in patients with CRC,was found in all four public sequencing datasets.Conclusion:These results suggested that P.micra was closely associated with CRC,and may serve as a diagnostic marker for CRC but not CRA.Moreover,it was indicated that P.micra,as an opportunistic pathogen of CRC,may promote CRC development but serve a limited role in CRA tumorigenesis.