Biologic correlates of circulating tumor DNA (ctDNA) shedding in the INTERCEPT colorectal cancer (CRC) study.

3591 Background: ctDNA is a promising tool for early cancer detection and monitoring of minimal residual disease (MRD). The relationship between vesicle trafficking of ctDNA-laden exosomes and shedding of detectable ctDNA in patients is poorly understood and was therefore explored in a large prospective patient cohort. Methods: The INTERCEPT program prospectively enrolled patients undergoing curative intent surgery for stage I-IV CRC at MD Anderson Cancer Center. Tumor informed MRD assays (Signatera) were drawn postoperatively and every three months according to reimbursement guidelines. RNA analyses were done from FFPE. Gene set enrichment analyses (GSEA) and Z-Scores comparing ctDNA+ and ctDNA– were analyzed using log2 normalized RNA expression values. Results: The cohort included 579 patients with RNA and post-operative ctDNA analyses; median age 56 years, 56% male and 47% stage IV. Of these, 122 (20%) were ctDNA+ in their first draw (32% of stage IV vs. 11% of stage I-III). In GSEA analyses of Hallmark gene sets between ctDNA+ and ctDNA–, an upregulation was seen for 15/50 gene sets among ctDNA+, with p-value < 0.05 and false discovery rate < 0.25. Two of the top signatures (UV response up and Unfolded protein response) were significant also in analyses stratified by stage. Analysis of the leading-edge genes in these gene set identified several members of the vacuolar ATPase (V-ATPase) family of genes which were highly enriched in ctDNA+. The full V-ATPase gene set substantially differed between ctDNA+ and ctDNA– (mean Z-score 0.48 vs. -0.13, p < 0.001), including when stratified by stage (I-III: 0.46 vs. -0.28, p = 0.002; IV: 0.49 vs. 0.10, p = 0.051). Sixty percent of patients (n = 346) had relapse event data with sufficient follow up. V-ATPases had higher mean Z-scores in those with relapses than those without (0.33 vs. –0.91, p = 0.012), also seen in the ctDNA– group (0.30 vs. -0.20, p = 0.021). Conclusions: V-ATPase genes are differentially expressed in patients with ctDNA+ regardless of tumor stage, a result also mirrored in relapse events. V-ATPases may play a significant role in ctDNA release through regulating intracellular multivesicular bodies to exosome release, thereby providing a potential mechanistic link between tumor biology and ctDNA shedding. This finding may explain the clinical limitations of ctDNA in selected patients and provide personalization of ctDNA testing performance in the future.