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


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

In This Article

Materials and Methods

Subject Recruitment and Stool Sample Collection

This retrospective study included subjects who were enrolled in a polyp surveillance programme conducted in a tertiary referral centre with a catchment population of 1.2 million between 2009 and 2019 (CREC Ref No: 2010.198). Subjects found to have adenoma on index colonoscopy underwent polypectomy and had regular surveillance colonoscopy according to international guidelines.[2,18,19] Consecutive subjects were eligible in this study if they provided stool samples within 1 month before bowel preparation of index colonoscopy (baseline) and/or surveillance colonoscopy (follow-up). Stool samples were excluded if subjects had taken antibiotics within 3 months before stool collection. Recurrence was defined as at least one adenoma found on surveillance colonoscopy. To minimise the possibility that recurrent adenomas were due to missed lesions at index colonoscopy, we included only colonoscopy with satisfactory bowel preparation, adenomas identified more than 6 months after the index colonoscopy and colonoscopy performed by experienced colonoscopists with an adenoma detection rate of >30%. All polyps were evaluated by an experienced pathologist (TKF). Advanced adenomas were defined as adenomas with a diameter of ≥1 cm, with a tubulovillous or villous component, or with high-grade dysplasia. Subjects were asked to collect stool samples in standardised containers at home, and delivered to the hospitals in insulating polystyrene foam containers. Stool samples were then stored at −80°C immediately for further analysis. The study was approved by the Joint NTEC-CUHK Clinical Research Ethics Committee (CREC Ref No: 2021.104). All subjects provided written informed consent.

Stool DNA Extraction and Quantification of Bacterial Gene Markers

Stool DNA extraction was performed using Norgen Stool DNA Isolation Kit (Norgen Biotek Corp) manually following the manufacturer's instructions. DNA quality and quantity were determined using gel electrophoresis and a NanoDrop spectrophotometer. Faecal levels of four bacterial gene markers (Fn, m3, Bc and Ch) were quantified by qPCR. These markers have previously been shown to be enriched in CRC alone (Fn and Ch), both adenoma and CRC (m3), and healthy subjects (Bc). Primer and probe sequences targeting the markers and 16 s rDNA internal control have been verified for target specificity in our previous studies.[15,16] Each probe carried a 5' reporter dye FAM (6-carboxy fluorescein) or VIC (4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein) and a 3' quencher dye TAMRA (6-carboxytetramethyl-rhodamine). Primers and hydrolysis probes were synthesised by Invitrogen. qPCR amplifications were performed on an ABI QuantStudio sequence detection system as previously described, with thermal cycler parameters of 95°C 10 minutes and (95°C 15 seconds, 60°C 1 minute) × 45 cycles.[15,16] Positive controls of the markers and a negative control (H2O as template) were included in every experiment. Measurements were performed in triplicates for each sample. Relative level of each marker was calculated using delta Cq method as compared to internal 16 s rDNA control (Power (2, −[Cqtarget-Cqcontrol]) and shown as log value of "*10e6 + 1." All samples were processed together regardless of baseline/follow-up samples and the status of adenoma recurrence. The technicians involved were blinded to the identity of stool samples. Quantification of microbiome markers using our qPCR platform has been verified to be stable on a batch of 20 randomly selected stool samples between different laboratories in our institution (all P > 0.1 by paired t-tests). We included positive control samples in each experiment to monitor if deviation in quantification results occurred. Intra-assay coefficients of variability (CVs) for Cq values were all <0.7%. Intra-assay and inter-assay CVs for each marker were <5% and <9.5%, respectively.

Faecal Immunochemical Test

Quantitative OC-Sensor test was performed on an automatic OCsensor instrument (Eiken Chemical) according to the manufacturer's instructions, using a positive cut-off value equivalent to a concentration of 100 ng of haemoglobin per millilitre (ng Hb/ml).

Sample Size Calculation

In our pilot study of 40 subjects, the mean levels of m3 upon follow-up at 1–5 years were 5.924 (log of relative level of m3/16s rDNA control) and 2.569 in patients with and without recurrent adenomas, respectively. We estimated that a total of 70 subjects would be required to detect a difference of 2.7 with a SD of 4.0 to achieve 80% power at a two-sided significance level of 5%. To adjust for possible confounding effects of covariates, we further increased the total sample size by a variance inflation factor of 1.25 to a total of 88 subjects (44 subjects per group) with follow-up stools.

Scoring Algorithms and cut-off Values

The combined score of four bacterial gene markers (4Bac) using a logistic regression model (4Bac score = I1 + β1*Fn + β2*m3 + β3*Bc + β4*Ch) was determined in our previous study.[16] To discriminate patients with recurrent adenoma from those without recurrence as determined by colonoscopy and histological examination, the combined score of follow-up markers is: I2 + βx*m3followup + βy*Chfollowup + βz*Fnfollowup, whereas the combined score of baseline and follow-up markers is: I3 + (βi*Fnfollowup - βii*FnBaseline) + (βiii*m3followup-βiv*m3Baseline) + (βiv*Chfollowup-βvi*ChBaseline). I represents the intercept, β represents the regression coefficient and markers represent the corresponding relative levels. Cut-off values were determined by receiver operating characteristic (ROC) analyses that maximised the Youden index (J = Sensitivity + Specificity −1).[20]

Statistical Analysis

Values were expressed as median (IQR, interquartile range) or mean ± SD where appropriate. The differences in levels of bacterial gene markers were determined by Mann-Whitney U test or paired t-test. Mann-Whitney U test was used to assess trends of changes at follow-up compared with baseline whereby groups at different time points and different status were considered independent of each other. Paired t-test was performed for comparison between paired baseline and follow-up in the same patient. Continuous clinical and pathological variables were compared by t-test or one-way ANOVA. ROC curves were used to evaluate the diagnostic values of bacterial gene markers or models in distinguishing between patients with and without recurrent adenomas. Pairwise comparison of ROC curves was performed using a non-parametric approach.[21] All tests were done by Graphpad Prism 5.0 (Graphpad Software Inc.) or MedCalc Statistical Software V.18.5 (MedCalc Software bvba, Ostend, Belgium;; 2018). P < 0.05 was taken as statistical significance.