A novel faecal Lachnoclostridium marker for the non-invasive diagnosis of colorectal adenoma and cancer

Abstract

Objective: There is a need for early detection of colorectal cancer (CRC) at precancerous-stage adenoma. Here, we identified novel faecal bacterial markers for diagnosing adenoma.

Design: This study included 1012 subjects (274 CRC, 353 adenoma and 385 controls) from two independent Asian groups. Candidate markers were identified by metagenomics and validated by targeted quantitative PCR.

Results: Metagenomic analysis identified 'm3' from a Lachnoclostridium sp., Fusobacterium nucleatum (Fn) and Clostridium hathewayi (Ch) to be significantly enriched in adenoma. Faecal m3 and Fn were significantly increased from normal to adenoma to CRC (p<0.0001, linear trend by one-way ANOVA) in group I (n=698), which was further confirmed in group II (n=313; p<0.0001). Faecal m3 may perform better than Fn in distinguishing adenoma from controls (areas under the receiver operating characteristic curve (AUROCs) m3=0.675 vs Fn=0.620, p=0.09), while Fn performed better in diagnosing CRC (AUROCs Fn=0.862 vs m3=0.741, p<0.0001). At 78.5% specificity, m3 and Fn showed sensitivities of 48.3% and 33.8% for adenoma, and 62.1% and 77.8% for CRC, respectively. In a subgroup tested with faecal immunochemical test (FIT; n=642), m3 performed better than FIT in detecting adenoma (sensitivities for non-advanced and advanced adenomas of 44.2% and 50.8% by m3 (specificity=79.6%) vs 0% and 16.1% by FIT (specificity=98.5%)). Combining with FIT improved sensitivity of m3 for advanced adenoma to 56.8%. The combination of m3 with Fn, Ch, Bacteroides clarus and FIT performed best for diagnosing CRC (specificity=81.2% and sensitivity=93.8%).

Conclusion: This study identifies a novel bacterial marker m3 for the non-invasive diagnosis of colorectal adenoma.

Keywords: colonic bacteria; colorectal adenomas; colorectal cancer screening.

Figure 1

Identification and characterisation of m3. (A) Metagenome sequencing identified m3, as well as Fusobacterium nucleatum (Fn) and Clostridium hathewayi (Ch), to be significantly increased in faecal samples of patients with adenoma. (B) DNA sequence of m3 showed high similarity to Lachnoclostridium sp. YL32. (C) m3 encodes a putative reverse transcriptase (RTase) that maps to a group II intron RTase, lacking the first 60 amino acids but retaining the RTase conserved domain. A, adenoma; CRC, colorectal cancer; N, normal control.

Figure 2

Quantitative detection of faecal m3 in the diagnosis of patients with colorectal cancer (CRC) and adenoma. (A) Relative abundance of m3 in faecal samples differed significantly between healthy control subjects (N, n=288), patients with adenoma (A, n=207) and patients with CRC (n=203). ***p<0.0001 as compared with N; ^##p<0.001 as compared with A. (B) No significant difference in faecal abundance of m3 was observed between non-advanced and advanced adenomas. (C) No difference in faecal abundance of m3 was observed among patients with CRC of different tumour-node-metastasis (TNM) stages. (D) Occurrence rates of m3 was significantly higher in patients with adenoma compared with control subjects, and highest in patients with CRC. (E) Receiver operating characteristic (ROC) curves and diagnostic performance of m3 in discriminating patients with CRC and adenoma from control subjects, respectively. AUROC, area under ROC.

Figure 3

Comparison and combination of bacterial markers for non-invasive diagnosis of colorectal cancer (CRC) and adenoma. (A) Relative abundances of Fusobacterium nucleatum (Fn), Clostridium hathewayi (Ch) in faecal samples of control subjects, patients with adenoma and patients with CRC. N, normal control; A, adenoma; ***p<0.0001 as compared with N; ^###p<0.0001 as compared with A. (B) ROC curve analyses showed Fn could discriminate adenoma and CRC from controls, while Ch could discriminate CRC but not adenoma from controls. (C) Comparison of ROC curves of Fn, m3 and their combination. (D) Diagnostic performances of Fn, m3 and their combination. Fn performed better than m3 in diagnosing CRC, and m3 was superior to Fn in diagnosing adenoma. Combination with Fn improved the diagnostic performance of m3 for CRC but not for adenoma.

Figure 4

Combination of four markers for olorectal cancer (CRC) and m3 alone for adenoma. (A) Receiver operating characteristic (ROC) curve analysis of combination of the five bacterial markers of interest showed that combination of Fn, m3, Ch and Bc by a logistic regression (LR) model worked best for CRC diagnosis. Shown p values are by comparison ROC curves. (B) Level of the combination of Fn, m3, Ch and Bc (LR4) in faecal samples and comparison of its diagnostic performance with m3. N, normal control; A, adenoma; ***p<0.0001 as compared with N; ^###p<0.0001 as compared with A. (C) Proposed strategy for the application of Fn, m3, Ch and Bc in the diagnosis of CRC and adenoma.

Figure 5

Validation of bacterial markers in diagnosing colorectal cancer (CRC) and adenoma in a second independent group of faecal samples. (A) Relative faecal abundances of Fn and m3 and level of the combination of Fn, m3, Ch and Bc (LR4) in patients with CRC and adenoma compared with control subjects of the second group. N, normal control; A, adenoma; *p<0.05 and ***p<0.0001 as compared with N; ^#p<0.05 and ^##p<0.001 as compared with A. (B) Comparison of ROC curves and diagnostic performances of Fn, m3 and LR4.

Figure 6

Comparison and combination of bacterial markers with faecal immunochemical test (FIT). (A) Comparison of sensitivity and specificity of FIT, m3, combination of four makers (Fn, m3, Ch and Bc; LR4) and combination of bacterial markers with FIT in a subgroup of Hong Kong samples. LR4 combined with FIT performed best for colorectal cancer (CRC) detection, while m3 combined with FIT performed best for detecting adenoma. (B) Comparison of the sensitivities of FIT, LR4 and their combination in detecting CRC according to tumour-node-metastasis (TNM) stage subsets. (C) Comparison of the sensitivities of FIT, m3 and their combination in detecting non-advanced and advanced adenomas. All comparison of sensitivities was conducted by χ² tests. A, non-advanced adenoma; AA, advanced adenoma.