Novel Microbiome Signatures for Non-invasive Diagnosis of Adenoma Recurrence After Colonoscopic Polypectomy

Jessie Qiaoyi Liang; Yao Zeng; Grace Kwok; Chun Pan Cheung; Bing Yee Suen; Jessica Y. L. Ching; Ka Fai To; Jun Yu; Francis K. L. Chan; Siew Chien Ng

Disclosures

Aliment Pharmacol Ther. 2022;55(7):847-855. 

In This Article

Results

Clinical Characteristics

A total of 222 consecutive eligible subjects who had adenomas at baseline colonoscopy were included in this study: Group I: 118 subjects had stool samples collected before index colonoscopy; Group II: 61 subjects had follow-up stool samples collected before surveillance colonoscopy; and Group III: 43 subjects had both baseline and follow-up stool samples. Stool samples collected from 154 subjects with normal index colonoscopy were included for marker comparison. A total of 161 baseline stool samples were collected before index colonoscopy from Group I and Group III, whereas 104 follow-up stools were collected before surveillance colonoscopy from Group II and Group III.

In all, 48 of the 104 post-polypectomy subjects were found to have adenomas at follow-up colonoscopy, seven of which were advanced adenomas. Detailed clinical characteristics are shown in Table 1 and Table 2. In all, 42 of the 104 (40.4%) patients underwent surveillance colonoscopy in less than 3 years. Among them, 7 had endoscopic submucosal resection/endoscopic submucosal dissection for large or laterally spreading adenomas (surveillance interval: 0.8–2.4 years), 3 had sessile serrated adenomas (surveillance interval: 1–1.6 years), 1 had multiple (>10) adenomas (surveillance interval: 1 year), 17 had a family history of first-degree relatives with colorectal neoplasia, 3 had a family history of other cancers (surveillance interval: 1 year) and 11 requested shorter surveillance interval due to anxiety or other miscellaneous reasons (surveillance interval: 1–2.3 years). None of the patients had repeated colonoscopy due to incomplete polyp removal and all patients had clear resection margin at index colonoscopy. There was no difference in the mean surveillance intervals between patients with and without recurrent adenoma in the overall cohort (2.3 ± 1.2 years versus 2.5 ± 1.2 years; P = 0.26), or in the subgroup with surveillance interval < 3 years (1.3 ± 0.4 years versus 1.3 ± 0.5 years; P = 0.57).

Stool Bacterial Gene Markers Increased With Adenoma Recurrence

Compared with normal controls, the levels of Fn, m3 and Ch were significantly higher while that of Bc was significantly lower in patients with adenoma at baseline (Figure 1A). We first compared the levels of four bacterial gene markers Fn, m3, Ch and Bc in baseline stool samples of subjects with adenomas and in follow-up stool samples with and without recurrent adenomas after polypectomy. In group III with paired samples, Fn (P < 0.05) and m3 (P < 0.0001) were significantly increased in follow-up samples with recurrent adenomas but not in samples without recurrence compared with baseline samples. Ch showed a non-significant decrease in follow-up samples with no recurrent adenoma (P = 0.066) but no change in those with recurrence, whereas Bc showed no change in follow-up stool samples regardless of adenoma recurrence status (Figure 1B). These findings were further validated in an enlarged dataset involving all samples from Groups I to III (Figure 1C).

Figure 1.

Changes in faecal bacterial markers in post-polypectomy subjects as compared to patients with baseline advanced adenoma. A, Comparison of the levels of four faecal bacterial markers in baseline stool samples collected before index colonoscopy between patients diagnosed with advanced adenoma and subjects confirmed with normal colon. B, Comparison of the levels of four faecal bacterial markers between baseline stool samples collected before index colonoscopic diagnosis of advanced adenoma and follow-up stools before surveillance colonoscopy from subjects who developed recurrent adenoma and those without recurrence in group III subjects with paired baseline and follow-up stools. (C) Comparison of the levels of four faecal bacterial markers between baseline stool samples and follow-up samples from all subjects recruited in this study. Fn, fusobacterium nucleatum; m3, Lachnoclostridium marker m3; Ch, Clostridium hathewayi; Bc, Bacteroides clarus; no-R, no-recurrence; R, recurrence

Paired Stool Samples Showed a High Accuracy in Detecting Adenoma Recurrence

We found a significant increase in the level of m3 and combined levels of four markers (Fn, Ch, m3 and Bc) in the follow-up samples compared with baseline samples in subjects who developed adenoma recurrence (P < 0.05 by matched-pair tests) (Figure 2A). In contrast, there was no significant change in levels of bacterial gene markers in follow-up samples in subjects with no recurrence (Figure 2B). Using a logistic regression model that included changes in the markers at follow-up compared with baseline stools, we found that m3 alone showed a good performance for detecting adenoma recurrence with an AUROC of 0.843 (95% CI: 0.700–0.936) (P < 0.0001), sensitivity of 85.0% and specificity of 87.0%. Using logistic regression to combine all the bacterial gene markers, the combination of m3, Fn and Ch achieved an AUROC of 0.950 (95%CI: 0.837–0.993) with sensitivity of 90.0% and specificity of 87.0% for detecting recurrent adenoma (Figure 3). FIT showed limited sensitivity (8.3%) in detecting recurrent adenomas, most of which were non-advanced adenomas. Adding FIT to this panel of combined bacterial gene markers did not improve the diagnostic performance.

Figure 2.

Changes in bacterial markers at follow-up (FU) versus baseline for diagnosis of recurrent adenoma. A, The four bacterial markers and their combined score 4Bac showed no significant difference between baseline and FU stools in patients without recurrence. B, Significant increases in m3 and 4Bac were detected in FU stools as compared to baseline stools in patients with recurrence. Fn, fusobacterium nucleatum; m3, Lachnoclostridium marker m3; Ch, Clostridium hathewayi; Bc, Bacteroides clarus; 4Bac: Combined score of Fn, m3, Ch and Bc previously devised for diagnosis of CRC and adenoma

Figure 3.

Performances of logistic regression models involving changes in FU stools versus baseline stools in discriminating patients with recurrence from those without recurrence. The model combining changes in Fn, m3 and Ch showed the best performance. Fn, fusobacterium nucleatum; m3, Lachnoclostridium marker m3; Ch, Clostridium hathewayi; Bc, Bacteroides clarus; 4Bac: Combined score of Fn, m3, Ch and Bc

Faecal Bacterial Gene Markers as a Standalone Test for Diagnosing Adenoma Recurrence

As shown in Figure 1B,C, the levels of Fn, m3 and Ch were significantly increased in follow-up stools with recurrence as compared with those without recurrence (P < 0.05), whereas the level of Bc showed no difference. Using follow-up stool samples without baseline samples for comparison, combining three markers (Fn, m3 and Ch) could detect recurrent adenoma with an AUROC of 0.741 (95%CI: 0.646–0.822) and sensitivity of 81.3% (Figure 4A). In addition, faecal levels of Fn, m3 and Ch showed no difference in subjects with baseline or recurrent adenomas in the proximal colon versus distal colon (Figure 4B1 and Figure 4B2). Adding FIT to this panel of combined bacterial gene markers did not further improve the diagnostic performance. Correlation analysis showed no significant correlations between age and individual markers or the combined score of m3, Fn and Ch for recurrent adenoma in the follow-up cohort (P > 0.4). On multivariate analysis, the levels of m3 and Ch (P < 0.01), and the combined score of m3, Fn and Ch (P < 0.0001) were independent risk factors for adenoma recurrence. As there were significant imbalances in some baseline characteristics including male gender, co-morbidity, number and size of adenomas between subjects with and without recurrent adenomas (Table 2), we further performed logistic regression analysis and found that none of these factors was a significant predictor of adenoma recurrence. m3ChFn appeared to be the only significant factor in the logistic regression model for recurrent adenomas. Pairwise comparison of ROC curves showed no significant difference between the model consisting of m3ChFn alone and the model combining the four clinical characteristics and m3ChFn (P = 0.135; Figure 4C).

Figure 4.

A, Performance of the logistic regression model involving follow-up levels of m3, Ch and Fn (m3ChFn) in discriminating patients with recurrence from those without recurrence. B, In faecal samples of patients with baseline adenoma (B1) and those with recurrent adenoma (B2), levels of m3, Fn and Ch showed no difference between patients with proximal lesions and those with distal lesions. C, Pairwise comparison of ROC curves showed no significant difference between m3ChFn and the logistic regression model adding clinical characteristics (number and size of adenoma, gender, and co-morbidity at baseline). In the logistic regression involving clinical characteristics and m3ChFn, only m3ChFn was a significant factor in the model. Fn, fusobacterium nucleatum; m3, Lachnoclostridium marker m3; Ch, Clostridium hathewayi; Bc, Bacteroides clarus; no-R, no-recurrence; R, recurrence; AUROC, area under ROC; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value

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