Abstract 6434: Microenvironment of adjacent non-neoplastic regions determines prognostic outcomes in locally advanced colorectal cancer after surgical resection: A multi-center & multi-omics study

Abstract Background: There is an unmet need for additive predictive biomarkers for the recurrence of stage II/III colorectal cancer (CRC) after surgery. We hypothesized that the heterogeneities of tumors present a substantial impediment to finding agnostic biomarkers. To avoid this, we focused on the normal tissue from the margins of resected specimens. Methods: We divide the groups using RNA sequencing derived from 273 tumors and 273 paired normal tissues from 5 Korean cancer centers. Groups were defined as follows: tumor-like microenvironment in normal mucosa (tNME): high level of tumor signature in normal mucosa; healthy normal microenvironment (hNME): low tumor signature in normal tissue. The impact of this classification was further evaluated in TCGA cohort. For the mechanistic explanation, we conducted 16s rRNA metagenomics and single-cell RNA sequencing from 47 patients and validated by spatial transcriptomics. Results: There were no significant differences between subgroups in baseline characteristics, including age, sex, medical centers, sidedness, stage and the distance between tumor and paired normal tissue. With a median follow-up of 58.2 months, the tNME group showed poor 5-year recurrence-free survival (54.7 vs. 73.0%, HR=1.94, P=0.002) and 5-year overall survival (OS; 78.2 vs. 83.0%, HR=1.76, P=0.033). In addition, tNME showed a trend toward poorer 5-year OS (34.5% vs. 68.8%, HR=3.76, P=0.071) in TCGA. The pathway analysis exhibited tNME had decreased maintenance of membrane while increased expression of humoral reaction to bacteria. Only the tNME group showed similar microbiome diversity between tumor and normal tissues, consisting of more proportion of carcinogenic genera, including fusobacterium and bacteroides than hNME. scRNA-seq revealed a positive co-occurrence of Th17, GZMKhighCD8+T cells, and IL1Bhigh neutrophils in the tumor and normal tissues of tNME. A high proportion of EMP1high epithelial cells, were observed in the tNME group, with upregulated epithelial-mesenchymal transition (EMT), bacterial-cellular-invasion and leukocyte signaling pathways. The cell-cell interaction analysis suggest Th17 cells promote IL1Bhigh neutrophil retention, increasing crosstalk with CD8+ T cells, and inducing the production of high levels of GZMK, which in turn decreases E-cadherin in the intestinal epithelium. A weak intestinal barrier may provoke more bacteria-infected cancer cells, promoting cancer cell progression via EMT and recruiting IL1Bhigh neutrophils and GZMKhighCD8+T cells, resulting in a vicious cycle. Conclusions: The microenvironmental status of normal tissues offers a promising biomarker for stage II/III CRC. Microbiota dysbiosis-induced organized microniches with immune and epithelial cells was related to metachronous recurrence after surgical resection. Citation Format: Yeong Hak Bang, Ji Hye Choi, Kyunghee Park, Jinyeong Lim, Dae Hee Pyo, Yong Beom Cho, Tae-You Kim, Kyu Joo Park, Seung-Bum Ryoo, Sung-Bum Kang, Chang Sik Yu, Kil-yong Lee, Jaeim Lee, Jin-Young Lee, Young-Joon Kim, Woong-Yang Park. Microenvironment of adjacent non-neoplastic regions determines prognostic outcomes in locally advanced colorectal cancer after surgical resection: A multi-center & multi-omics study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6434.