Abstract LB115: ctDNA false negativity in colorectal cancer reflects CMS3 phenotype and DNASE1L3 positive macrophages, identifying recurrence prone subgroup

Abstract Circulating tumor DNA (ctDNA) assays are increasingly used for minimal residual disease (MRD) detection in colorectal cancer (CRC), yet false-negative results remain a critical limitation. We aimed to identify biological determinants of ctDNA false-negativity and evaluate their prognostic significance. From a nationwide prospective registry (CIRCULATE-JAPAN), we first analyzed 1,727 stage II-III CRC patients with preoperative ctDNA results; 82 (4.7%) were ctDNA-negative at baseline. We sequenced 1,290 tumors with available tissue and subdivided them into a discovery cohort (n=737; 48 ctDNA-negative) and a validation cohort (n=553; 36 ctDNA-negative) using stratified sampling. Multi-omics integration included whole-exome sequencing, bulk RNA-seq, and ctDNA assay data. Single-cell RNA sequence was performed on ctDNA-negative tumors. Disease-free survival (DFS) was assessed in 920 evaluable patients. Preoperative ctDNA-negative tumors were enriched in right-sided CRC and exhibited a low-proliferation phenotype with negative enrichment of G2/M checkpoint and MYC signaling pathways. Transcriptomic classification revealed predominant Consensus Molecular Subtype (CMS) 3. Mutation analysis showed RAS/RAF pathway alterations (KRAS/BRAF) were significantly more frequent in ctDNA-negative tumors. ctDNA-negative tumors showed significantly higher DNASE1L3 expression—a macrophage-secreted nuclease—localized to resident/C1QC-type macrophages and mutually exclusive with pro-tumor, SPP1-positive tumor associated macrophage that co-vary with CAF abundance; this pattern supports enhanced extracellular DNA digestion in the tumor microenvironment (TME) as a contributor to reduced ctDNA detectability. Clinically, ctDNA negativity trended toward improved DFS versus ctDNA positivity (HR 2.01, P=0.087). However, within ctDNA-negative patients, high DNASE1L3 expression identified a subgroup with significantly higher recurrence risk (P=0.03). Among relapse cases, DNASE1L3 expression was higher in patients with false-negative ctDNA at recurrence. ctDNA detectability reflects both tumor cell state and TME-mediated DNA clearance. DNASE1L3 emerges as a biological contributor to ctDNA false-negativity and a prognostic marker that uncovers a recurrence-prone subgroup among ctDNA-negative CRC patients. These findings highlight the need for DNASE1L3-informed MRD interpretation and tailored surveillance strategies. Citation Format: Hiromichi Ebi, Rui Yamaguchi, Yoshiaki Nakamura, Satoru Kisoda, Jun Watanabe, Osamu Muto, Hiroki Yukami, Saori Mishima, Hideaki Bando, Hiroya Taniguchi, Ichiro Takemasa, Takeshi Kato, Alexey Aleshin, Daisuke Kotani, Issei Imoto, Eiji Oki, Masahiro Aoki, Takayuki Yoshino. ctDNA false negativity in colorectal cancer reflects CMS3 phenotype and DNASE1L3 positive macrophages, identifying recurrence prone subgroup [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB115.