Microbiome-Derived Cobalamin and Succinyl-CoA are Powerful Biomarkers for Improved Screening of Anal Cancer

Abstract

Background. People with HIV (PWH) exhibit a markedly increased risk of anal cancer, especially men who have sex with men (MSM), with specific microbiota signatures possibly contributing to greater human papillomavirus (HPV) oncogenic potential. Low specificity of the current screening strategy for detecting high-grade squamous intraepithelial lesions (HSIL) hinders anal cancer prevention. We investigated microbiome derivatives associated with HSIL in anal cytology samples. Methods. We recruited a discovery and a validation cohort at four clinical sites in Spain and Italy. Study participants were mostly MSM undergoing HSIL screening with high-resolution anoscopy and anal biopsies to confirm HSIL. We extracted the bacterial DNA, proteins, and metabolites from anal cytology samples, and we performed 16SrRNA gene sequencing, mass spectrometry, and targeted metabolite quantification. Results. We included 213 participants, 167 in the discovery cohort (70 with confirmed HSIL) and 46 in the validation cohort (25 with confirmed HSIL). While we did not find clear microbiome composition signatures associated with HSIL, the microbiome associated with HSIL overexpressed proteins involved in the production of succinyl-CoA and cobalamin, with levels consistently increased in subjects with HSIL in the discovery and validation cohorts. Combined measurement of succinyl-CoA and cobalamin overperformed anal cytology, improving sensitivity from 91.2% to 96.6%, specificity from 34.1% to 81.8%, positive predictive value from 48.1% to 77.8%, and negative predictive value from 85.3% to 97.3%. While anal cytology correctly classified only 59.9% of individuals, combined measurement of both metabolites improved the classification to 87.7%. This test overcame internal (adjusted AUC 0.877) and external validation. From 98 false-positive cytologic results, succinyl-CoA and cobalamin level measurement reclassified 49 (50%) to true negatives. Finally, we demonstrate greater in vitro production of succinyl-CoA and cobalamin in bacteria associated with HSIL or cancer vs. those presumably protective. Conclusions. Cobalamin and succinyl-CoA are overexpressed in the anal microbiome of patients with HSIL and show excellent diagnostic capacity. Their measurement overperforms anal cytology to screen for patients with HSIL. Hence, we discovered two powerful biomarkers for which detection methods can readily be established, that could improve the current strategy of anal cancer screening.

The microbiota as a modulator of mucosal inflammation and HIV/HPV pathogenesis: From association to causation

“…We discovered that cobalamin and succinyl-CoA were increased in the anal microbiome of patients with anal precancer and overperformed the reference test—anal cytology—. Furthermore, we validated the findings in an external validation cohort, and we demonstrated greater in vitro production of succinyl-CoA and cobalamin in bacteria associated with HSIL or cancer vs. those presumably protective ( 172 ). Therefore, starting from a clinical question and integrating data from different omic levels we were able to define a new microbiome-based tool that could help in the prevention of a common cancer in PLWH by discovering two powerful biomarkers of anal precancer that could improve anal cancer prevention.…”

The microbiome as a biomarker of anal precancerous lesions in people with HIV

“…A study by Su Shen et al [25] identified an increased abundance of Atopobium and Sneathia linked to cervical lesions, along with several metabolites and metabolic pathways, including taurine, guanine, and uric acid, suggesting high consumption of amino acids, purines, sugars, and esters in the cervicovaginal microenvironment associated with HR-HPV infection and HSIL. A recent investigation of the microbiome functions linked with HSIL, analyzing the composition, proteins, and metabolites derived from the anal microbiome in two cohorts including 213 MSM with HIV, did not find bacterial taxa robustly associated with HSIL, but higher levels of microbiome-encoded proteins producing succinyl coenzyme A and cobalamin were significantly associated with HSIL, thereby improving diagnostic accuracy [26 ▪▪ ].…”

“…Various bacterial biomarkers and derived metabolic pathways have demonstrated high discriminative ability, often outperforming the low specificity of anal cytology. Specifically, cobalamin and succinyl-CoA microbiome-derived metabolites measured in anal cytobrushes have been validated as HSIL biomarkers in parallel cohorts [26 ▪▪ ]. However, there are several limitations to the applicability of these results, such as the need for prospective validation in larger, diverse populations due to factors like interindividual and ethnic microbiome diversity, country of origin, diet, BMI, and coinfections.…”

“…1). For example, our group recently described mechanisms related to the proteins glycerate kinase 1 (PGK1), Enolase (previously related to cancer and the Warburg effect), and Cobalamin and succinyl-CoA (key components of the TCA cycle), which were overexpressed in the microbiome of individuals with HSIL [26 ▪▪ ].…”

“…There is still work to be done before clinical implementation regarding the use of cobalamin and succinyl-CoA as potential microbiome-associated biomarkers for HSIL screening [26 ▪▪ ]. First, we discovered these biomarkers in a single study with a somewhat limited sample size.…”