Introduction

Colorectal cancer (CRC) often develops from precancerous lesions such as tubular adenomas (TA) and sessile serrated lesions (SSL). The gut microbiome has been implicated in colorectal carcinogenesis, but specific microbial signatures associated with different adenoma subtypes remain incompletely characterized. This study aimed to identify microbial biomarkers associated with TA and SSL, and explore their correlations with dietary macronutrients.

Aims & Methods

Stool samples were collected from 801 patients prior to colonoscopy from National University Hospital and Tan Tock Seng Hospital, Singapore. After colonoscopy, the patients were classified as having TA (n=315), SSL (n=24), both (n=8), or neither (controls, n=454). Shotgun metagenomic sequencing was performed using Novaseq6000, and microbial taxonomy was annotated with MetaPhlAn4. Multivariate linear regression adjusted for age, gender, and BMI was used to identify species associated with polyp subtypes. A subset of 172 participants completed a validated food frequency questionnaire (FFQ) assessing 19 macronutrients, enabling correlation analysis between diet and adenoma-associated microbes.

Results

Patients with TA were older (mean age 64.0±7.8 vs 62.3±7.5 years in controls, p=0.02) and more likely to be male (69.5% vs 52.4%, p<0.01). After adjustment for confounders, we identified 38 microbial species significantly associated with polyp subtypes; 20 were associated with SSL and 18 with TA, with minimal overlap between the two polyp subtypes. Only two species showed associations with both polyp types: Holdemanella biformis and GGB3277_SGB4327, with the latter showing opposing directional relationships (negative for TA, positive for SSL). Of note, SSL were associated with increased abundance of Faecalibacterium SGB15346 and Bifidobacterium adolescentis. In the dietary analysis, we identified significant correlations between SSL-associated microbes and sugar intake, particularly Faecalibacterium SGB15346 (r=0.170, p=0.03) and Clostridium sp_AM49_4BH (r=0.156, p=0.04). E. hominis, positively associated with SSL, showed the strongest dietary associations overall, correlating positively with carbohydrate intake (r=0.243, p<0.01) and starch consumption (r=0.246, p<0.01).

Conclusion

Our study identified distinct microbial signatures associated with TA and SSL, with minimal overlap, suggesting separate host-microbial biological pathways in their development. The positive correlation between SSL-associated microbes and sugar intake provides a potential mechanistic link between dietary habits and SSL formation, which may be particularly relevant given the increasing incidence of early-onset colorectal neoplasia. These findings offer potential microbial biomarkers for early detection of colorectal polyps and suggest targeted dietary interventions as a strategy for precision chemoprevention.

Introduction

Serum assays to measure biologic drugs, especially TNF inhibitors, and their anti-drug antibodies (ADAb) are utilized to manage suboptimal response and to proactively titrate doses and dosing intervals. Sensitive, drug-tolerant and high-resolution anti-drug antibody assays are an increasingly important tool necessary for detection and management of immunogenicity, especially when low-intermediate in titer and potentially reversible.¹

Aims & Methods

Serum samples from 72,819, 53,131, and 2587 patients were analyzed for infliximab (IFX), adalimumab (ADL), and certolizumab pegol (CTZ) drug (in μg/mL) and corresponding anti-drug antibodies (in ng/mL) as measured by drug-specific lab developed chemiluminescent immunoassays. Drug assays measure the free ADAb-unbound fraction of drug when serum ADAb are present. All ADAb assays detect total antibodies (including IgM & IgG subtypes), and their drug tolerance has been validated. Drug-specificity of ADAbs is verified by a confirmatory test step. An extensive blinded method comparison of our IFX and Anti-IFX assays has been published.²

Results

Drug and ADAb results are summarized in Table 1. Immungenicity rates were 31%, 29%, and 68% for IFX, ADL, and CTZ, respectively. ADAb-free samples were used to evaluate distribution of drug levels. Even in the absence of immunogenicity, many patients are subtherapeutic, 25%, 45% and 40%, for IFX, ADL, and CTZ, respectively, when using proposed therapeutic targets of 5 μg/mL for IFX, 7.5 for ADL, and 20 for CTZ.³ Therapeutic levels were observed in 27% for IFX, 37% for ADL and 32% for CTZ. Higher drug concentrations in the remaining samples may represent non-trough collected samples.

A wide range of immunogenicity titers was observed. Analyses of the inverse relationship between ADAb and drug led to determination of cut-points for low/high titer designations where in the setting of high ADAb, almost invariably, concomitant free drug is very low or nil. As a good rule, anti-drug antibodies should be interpreted in the context of the concomitant free drug trough concentration.

Table 1. Analysis of Patient Samples for IFX, ADL, CTZ and Respective Anti-Drug Antibodies

Conclusion

Here, we report concentrations of TNF-inhibitors used to treat IBD and their anti-drug antibodies in >128,000 patient samples. Though clinical histories and blood collection timing are not known, we observed that many patients may have insufficient drug levels. Immunogenicity to TNF inhibitors was frequent where a wide numeric range of ADAb was observed. Numeric ADAb antibody results should be interpreted with the help of titer designations by the laboratory because low to intermediate titers may be transient or reversible whereas high titers with nil or very low drug indicate refractory immunogenicity. High resolution ADAb assays have enabled the early detection and successful reversal of intermediate titers of Anti-IFX and Anti-ADL with dose escalation +/- co-immunommodulator.¹ Thus, biologic TDM aims to optimize drug efficacy and longevity by informing and expediting adjustments to medication.

References

1. Colman RJ et al. Antibodies-to-infliximab accelerate clearance while dose intensification reverses immunogenicity and recaptures clinical response in paediatric Crohn’s disease. Aliment Pharmacol Ther. 2022;55:593–603.

2. Marini J et al. Comparisons of Serum Infliximab and Antibodies-to-Infliximab Tests Used in Inflammatory Bowel Disease Clinical Trials of Remicade. American Association of Pharmaceutical Scientists Journal 2016 DOI: 10.1208/s12248-016-9981-3.

3. Feuerstein JD et al. American Gastroenterological Association Institute Guideline on Therapeutic Drug Monitoring in Inflammatory Bowel Disease. Gastroenterology 2017;153:827–834.