Abstract Background: Microbiome-derived metabolites have demonstrated significant impact on immunotherapy response, particularly in the context of immune checkpoint blockade (ICB). Trimethylamine-N-oxide (TMAO), an amine oxide generated when gut bacteria metabolize dietary choline, has been identified in preclinical studies to enhance anti-tumor immune responses. Here, we evaluated circulating choline metabolites with respect to clinical outcomes and circulating tumor DNA (ctDNA) residual disease in patients with resectable cancers receiving neoadjuvant ICB. Methods: Pre-treatment choline, trimethylamine (TMA) and TMAO plasma concentrations from 51 patients with stage I-III diffuse pleural mesothelioma (DPM; n = 25 patients; NCT03918252) or stage II/III esophageal/gastro-esophageal junction cancer (E/GEJ; n = 26 patients; NCT03044613) on neoadjuvant ICB, were quantified via liquid chromatography-mass spectrometry (SCIEX Triple Quad 6500+). Clinical outcomes were compared across metabolite thresholds, as were ctDNA residual disease through tumor-informed cell-free DNA (cfDNA) whole-genome sequencing (WGS). ctDNA residual disease in the DPM cohort was measured by intersecting tumor (n = 28), white blood cell (n = 28), and plasma (n = 97) WGS. Results: In the DPM cohort, median baseline choline, TMA and TMAO concentrations were 3970, 2840 and 256 ng/mL, respectively. Higher baseline TMAO levels were associated with lower ctDNA residual disease at C2D1 (Fisher’s exact, p = 0.028), C3D1 (Fisher’s exact, p = 0.057) and pre-surgery (Fisher’s exact, p = 0.01). Further, patients with higher TMAO had either undetectable ctDNA throughout the neoadjuvant window or ≥95% reduction in cfDNA tumor fraction from baseline to pre-surgery (Fisher’s exact, p = 0.028). In this cohort, ctDNA residual disease strongly correlated with progression-free and overall survival (log-rank, p 0.05). In the E/GEJ cohort, median baseline choline, TMA and TMAO concentrations were 279, 2945 and 4175 ng/mL, respectively. Patients who attained a major pathologic response had numerically higher TMAO levels (Fisher’s exact, p = 0.078). In line with the findings in the DPM cohort, patients in the E/GEJ cohort with lower TMAO (lowest quintile; 197 ng/mL) had shorter overall survival (log-rank, p = 0.034) compared to those with higher TMAO concentration ( ≥197 ng/mL), an association that remained significant after adjusting for clinicopathological variables (Cox multivariable, p = 0.04). Conclusion: Our findings indicate that higher plasma TMAO levels may be linked to molecular ctDNA response and better clinical outcomes with neoadjuvant ICB, further supporting a potential immunomodulatory role of choline metabolites in enhancing immunotherapy response. Citation Format: Rachel J. Keogh, Paul K. Lee, N.V. Rajeshkumar, Blair V. Landon, Joshua E. Reuss, Jaime Wehr, Gavin Pereira, Amna Jamali, Noushin Niknafs, Ronan J. Kelly, Ali H. Zaidi, Josephine L. Feliciano, Julie R. Brahmer, Vincent K. Lam, Patrick M. Forde, Chi V. Dang, Valsamo Anagnostou. Elevated plasma trimethylamine-N-oxide (TMAO) levels correlate with better clinical and molecular responses in immunotherapy-treated resectable gastroesophageal cancer and pleural mesothelioma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4724.
Keogh et al. (Fri,) studied this question.