Use of circulating tumor DNA (ctDNA) to monitor patients undergoing total neoadjuvant treatment (TNT) for locally advanced rectal adenocarcinoma (LARC).

30 Background: While many patients with LARC experience durable responses after TNT, relapse risk remains a concern and is not reliably predicted by standard clinicopathologic measures. ctDNA is a promising biomarker for minimal residual disease (MRD) that could improve post-TNT risk-stratification and guide postoperative surveillance; however, reduced sensitivity demonstrated in previous studies has limited its clinical utility in this setting. Methods: In an ongoing prospective study of patients with LARC receiving TNT, plasma was collected pre-treatment, after TNT but before surgery, post-surgery (“landmark”), and every 3 months for 1 year. ctDNA analysis was performed using a tumor-informed MRD assay interrogating up to 50 personalized variants (Haystack MRD), and results were evaluated alongside clinical outcomes. Results: Forty patients treated with TNT followed by surgery were included in this analysis. Pre-treatment ctDNA was detected in 13/13 (100%) patients. Among 34 patients with ctDNA results post-TNT, 32/34 (94%) had residual disease and 2/34 (6%) achieved pathologic complete response (pCR). ctDNA was detected post-TNT in 22/32 (69%) patients with residual disease, while both patients with pCR were ctDNA negative. Post-TNT sensitivity was higher in patients with pathologic stage III/IV versus I/II disease (15/18 [83%] vs 7/14 [50%]). Out of 20 patients with ctDNA results at the landmark post-surgical time point, all 5 (100%) patients with positive ctDNA relapsed, while 1/15 (7%) ctDNA-negative patients relapsed (this patient had detectable ctDNA at 6-months post-surgery), yielding 83% sensitivity and 93% negative predictive value for disease relapse at the landmark timepoint, and 100% sensitivity within 6 months of surgery. Testing of additional patients and time points is ongoing. Conclusions: A next-generation tumor-informed MRD assay shows excellent sensitivity for ctDNA detection in LARC patients pre-treatment (100%) and post-surgery (83% at landmark, 100% at 6 months). Post-TNT sensitivity was 69% overall and 83% in stage III/IV disease, representing an improvement over previous reports. These initial findings support integrating ctDNA into post-TNT risk stratification and postoperative surveillance in LARC; testing of additional patients/time points and further follow up is ongoing to validate these results.