LBPO.CL02 · 临床研究 · Late-Breaking
结直肠癌中ctDNA假阴性反映CMS3表型和DNASE1L3阳性巨噬细胞，识别出易复发亚组
ctDNA false negativity in colorectal cancer reflects CMS3 phenotype and DNASE1L3 positive macrophages, identifying recurrence prone subgroup
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
循环肿瘤DNA（ctDNA）检测越来越多地用于结直肠癌（CRC）的微小残留病（MRD）检测，但假阴性结果仍是一项关键局限。我们旨在识别ctDNA假阴性的生物学决定因素并评估其预后意义。
从一项全国性前瞻性登记研究（CIRCULATE-JAPAN）中，我们首先分析了1,727例具有术前ctDNA结果的II-III期CRC患者；其中82例（4.7%）在基线时ctDNA阴性。我们对1,290例有可用组织的肿瘤进行测序，并采用分层抽样将其细分为发现队列（n=737；48例ctDNA阴性）和验证队列（n=553；36例ctDNA阴性）。多组学整合包括全外显子组测序、bulk RNA-seq和ctDNA检测数据。对ctDNA阴性肿瘤进行了单细胞RNA测序。在920例可评估患者中评估了无病生存期（DFS）。
术前ctDNA阴性肿瘤在右侧CRC中富集，并表现出低增殖表型，伴G2/M检查点和MYC信号通路的负富集。转录组分类显示以共识分子亚型（CMS）3为主。突变分析显示RAS/RAF通路改变（KRAS/BRAF）在ctDNA阴性肿瘤中显著更为常见。ctDNA阴性肿瘤显示出显著更高的DNASE1L3表达——一种巨噬细胞分泌的核酸酶——定位于常驻/C1QC型巨噬细胞，且与促肿瘤的SPP1阳性肿瘤相关巨噬细胞（其与CAF丰度共变化）相互排斥；这一模式支持肿瘤微环境（TME）中增强的细胞外DNA消化是导致ctDNA可检测性降低的一个因素。在临床上，ctDNA阴性相比ctDNA阳性趋向于更好的DFS（HR 2.01，P=0.087）。然而，在ctDNA阴性患者中，高DNASE1L3表达识别出一个复发风险显著更高的亚组（P=0.03）。在复发病例中，DNASE1L3表达在复发时ctDNA假阴性的患者中更高。
ctDNA可检测性反映了肿瘤细胞状态和TME介导的DNA清除两者。DNASE1L3成为ctDNA假阴性的一个生物学贡献因素，也是一种预后标志物，可揭示ctDNA阴性CRC患者中易复发的亚组。这些发现凸显了需要基于DNASE1L3的MRD解读和量身定制的监测策略。
查看英文原文 English 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.
利益披露 Disclosure
H. Ebi,
AMGEN Independent Contractor.
Chugai Pharmaceutical Independent Contractor.
AstraZeneca Independent Contractor.
Guardant Independent Contractor.
Konica Minolta Independent Contractor.
Riken Genesis Independent Contractor.
Amoy Diagnostics Independent Contractor, ).
Astellas Pharmaceutical Independent Contractor, ).
Incyte Independent Contractor.
Ono Pharmaceutical Independent Contractor.
Merck Biopharma Independent Contractor.
Boehringer Ingelheim Independent Contractor.
Otsuka Pharmaceutical Independent Contractor.
Bristol-Myers Squibb Independent Contractor.
Cyberomix Independent Contractor.
R. Yamaguchi, None.
Y. Nakamura,
Becton, Dickinson and Company, CareNet, Inc., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eisai Co., Ltd., Exact Sciences Corporation, Genomedia Inc., Gilead Sciences, Inc. Independent Contractor.
Hisamitsu Pharmaceutical Co., Inc., Merck Biopharma Co., Ltd., Miyarisan Pharmaceutical Co., Ltd., MSD K.K., Natera, Inc., Premo Partners, Inc., Roche Diagnostics K.K., Roche Ltd., Seagen, Inc. Independent Contractor.
Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Tempus Labs, Inc., Zeria Pharmaceutical Co., Ltd. Independent Contractor.
Tempus Labs, Inc., Roche Diagnostics K.K., Genomedia Inc., ).
Daiichi Sankyo Co., Ltd, Chugai Pharmaceutical Co., Ltd., Guardant Health Independent Contractor, ).
S. Kisoda, None.
J. Watanabe,
Johnson and Johnson, Medtronic, Eli Lilly, Takeda Pharmaceuticals Independent Contractor.
Medtronic, AMCO, TERUMO, Stryker Japan ).
O. Muto, None..
H. Yukami, None.
S. Mishima,
Taiho Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., Eli Lilly CO, Ltd. Independent Contractor.
H. Bando,
Ono Pharmaceutical Independent Contractor, ).
Eli Lilly Japan, and Taiho Pharmaceutical. Independent Contractor.
H. Taniguchi,
MSD K.K, Merck Biopharma, Taiho, Lilly Japan, Bristol-Myers Squibb Japan, Chugai Pharmaceutical, Ono Yakuhin, Amgen Independent Contractor.
Takeda Phamaceutical Independent Contractor, ).
Daiichi Sankyo ).
I. Takemasa,
Johnson &Johnson, Intuitive, Medicaroid, Eli Lilly Independent Contractor.
Medtronic Independent Contractor, ).
Sysmex ).
T. Kato, None.
A. Aleshin,
Natera, Inc. Stock.
D. Kotani,
Takeda, Chugai, Lilly, Seagen, Guardant Health, Eisai, Taiho, Bristol Myers Squibb, Daiichi-Sankyo, Pfizer, Merck biopharma, and Sysmex Independent Contractor.
Novartis, Servier, Janssen, IQVIA, Syneoshealth, CIMIC, Cimicshiftzero ).
Ono, MSD, Independent Contractor, ).
I. Imoto, None.
E. Oki,
Guardant Health, Inc.; ).
Chugai Pharmaceutical, Bristol Meyers, Ono Pharmaceutical., Eli Lilly, Takeda Pharmaceutical, Glaxosmithkline plc Independent Contractor.
M. Aoki, None.
T. Yoshino,
Chugai Pharmaceutical, Ono Pharmaceutical, Takeda Pharmaceutical Independent Contractor, ).
Merck Biopharma, Bayer Yakuhin, and MSD K.K, Sumitomo Corp Independent Contractor.
Amgen, Bristol-Myers Squibb, Daiichi Sankyo, Eisai, FALCO biosystems, Genomedia, Medical & Biological Laboratories, Merus N.V., Molecular Health GmbH, MSD, Nippon Boehringer Ingelheim, Pfizer Japan ).
Roche Diagnostics, Sanofi, Sysmex, Taiho Pharmaceutical ).