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Supplementary Figures 1-3 from Fecal Bacteria Act as Novel Biomarkers for Noninvasive Diagnosis of Colorectal Cancer

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posted on 2023-03-31, 20:12 authored by Qiaoyi Liang, Jonathan Chiu, Yingxuan Chen, Yanqin Huang, Akira Higashimori, Jingyuan Fang, Hassan Brim, Hassan Ashktorab, Siew Chien Ng, Simon Siu Man Ng, Shu Zheng, Francis Ka Leung Chan, Joseph Jao Yiu Sung, Jun Yu

Supplementary Figure S1. Good consistency in quantification of nusG/F. nucleatum (Fn) was achieved by two experimenters using our convenient platform. Two samples with outlier highlevels of Fn were further excluded so as to show more clearly the good consistency even in samples with relatively low levels of Fn. Abundances of Fn were shown in arbitrary unit; Supplementary Figure S2. Contaminant human DNA had little effect on the new qPCR platform. (A) A representative example of qPCR evaluation of the internal control on a mixture of 10 randomly selected fecal samples added with different concentrations of human DNA. (B) A representative example of duplex qPCR evaluation of the internal control and F. nucleatum on a randomly selected fecal sample added with different concentrations of human DNA; Supplementary Figure S3. Metagenome sequencing data showed that combination with other markers improved the diagnostic ability of Fusobacterium nucleatum (Fn) for colorectal cancer. ROC curves for Fn alone and simple linear combination of all the five selected bacterial marker candidates including Fn, Bacteroides clarus (Bc), Clostridium hathewayi (Ch), one undefined species (labeled as m7), and Roseburia intestinalis (Ri). The best cutoff values that maximize sensitivity and specificity were determined for Fn and 5-markers individually in the metagenome sequencing cohort with 74 CRC patients and 54 controls. AUROC, area under the receiver operating characteristic curve; PPV, positive predictive value; NPV, negative predictive value.

Funding

China 863 program

China 973 program

National Nature and Science Foundation of China

The National Key Technology R&D Program

Shenzhen Municipal Science and Technology R&D funding

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ARTICLE ABSTRACT

Purpose: Gut microbiota have been implicated in the development of colorectal cancer. We evaluated the utility of fecal bacterial marker candidates identified by our metagenome sequencing analysis for colorectal cancer diagnosis.Experimental Design: Subjects (total 439; 203 colorectal cancer and 236 healthy subjects) from two independent Asian cohorts were included. Probe-based duplex quantitative PCR (qPCR) assays were established for the quantification of bacterial marker candidates.Results: Candidates identified by metagenome sequencing, including Fusobacterium nucleatum (Fn), Bacteroides clarus (Bc), Roseburia intestinalis (Ri), Clostridium hathewayi (Ch), and one undefined species (labeled as m7), were examined in fecal samples of 203 colorectal cancer patients and 236 healthy controls by duplex-qPCR. Strong positive correlations were demonstrated between the quantification of each candidate by our qPCR assays and metagenomics approach (r = 0.801–0.934, all P < 0.0001). Fn was significantly more abundant in colorectal cancer than controls (P < 0.0001), with AUROC of 0.868 (P < 0.0001). At the best cut-off value maximizing sum of sensitivity and specificity, Fn discriminated colorectal cancer from controls with a sensitivity of 77.7%, and specificity of 79.5% in cohort I. A simple linear combination of four bacteria (Fn + Ch + m7-Bc) showed an improved diagnostic ability compared with Fn alone (AUROC = 0.886, P < 0.0001) in cohort I. These findings were further confirmed in an independent cohort II. In particular, improved diagnostic performances of Fn alone (sensitivity 92.8%, specificity 79.8%) and four bacteria (sensitivity 92.8%, specificity 81.5%) were achieved in combination with fecal immunochemical testing for the detection of colorectal cancer.Conclusions: Stool-based colorectal cancer–associated bacteria can serve as novel noninvasive diagnostic biomarkers for colorectal cancer. Clin Cancer Res; 23(8); 2061–70. ©2016 AACR.

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