TIME (Tumor Immunity in the MicroEnvironment) classification based on tumor CD274 (PD-L1) expression status and tumor-infiltrating lymphocytes in colorectal carcinomas
Related Experiment Video
Updated: Feb 9, 2026

Establishment of a Co-culture System of Patient-Derived Colorectal Tumor Organoids and Tumor-Infiltrating Lymphocytes (TILs)
Published on: June 27, 2025
Tsuyoshi Hamada1, Thing Rinda Soong2, Yohei Masugi1
1Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA.
Abstract:
Inhibitors targeting the PDCD1 (programmed cell death 1, PD-1) immune checkpoint pathway have revolutionized cancer treatment strategies. The TIME (Tumor Immunity in the MicroEnvironment) classification based on tumor CD274 (PDCD1 ligand 1, PD-L1) expression and tumor-infiltrating lymphocytes (TIL) has been proposed to predict response to immunotherapy. It remains to be determined clinical, pathological, and molecular features of TIME subtypes of colorectal cancer. Using 812 colon and rectal carcinoma cases from the Nurses' Health Study and Health Professionals Follow-up Study, we examined the association of tumor characteristics and survival outcomes with four TIME subtypes (TIME 1, CD274low/TILabsent; TIME 2, CD274high/TILpresent; TIME 3, CD274low/TILpresent; and TIME 4, CD274high/TILabsent). In survival analyses, Cox proportional hazards models were adjusted for potential confounders, including microsatellite instability (MSI) status, CpG island methylator phenotype (CIMP) status, LINE-1 methylation level, and KRAS, BRAF, and PIK3CA mutation status. TIME subtypes 1, 2, 3 and 4 had 218 (27%), 117 (14%), 103 (13%), and 374 (46%) colorectal cancer cases, respectively. Compared with TIL-absent subtypes (TIME 1 and 4), TIL-present subtypes (TIME 2 and 3) were associated with high-level MSI, high-degree CIMP, BRAF mutation, and higher amounts of neoantigens (p < 0.001). TIME subtypes were not significantly associated with colorectal cancer-specific or overall survival. In conclusion, TIL-present TIME subtypes of colorectal cancer are associated with high levels of MSI and neoantigen load, supporting better responsiveness to cancer immunotherapy. Further studies examining tumor molecular alterations and additional factors in the tumor microenvironment may inform development of immunoprevention and immunotherapy strategies.
Insights
Tumor Immunity in the MicroEnvironment (TIME) subtypes in colorectal cancer were analyzed. TIL-present TIME subtypes correlate with high MSI and neoantigen load, suggesting better immunotherapy response.
Area of Science:
- Oncology
- Immunology
- Genetics
Background:
- Immune checkpoint inhibitors targeting PD-1/PD-L1 have transformed cancer therapy.
- The TIME classification, based on PD-L1 expression and TILs, aims to predict immunotherapy response.
- Clinical, pathological, and molecular features of TIME subtypes in colorectal cancer require further elucidation.
Purpose of the Study:
- To investigate the clinical, pathological, and molecular characteristics of TIME subtypes in colorectal cancer.
- To assess the association of TIME subtypes with survival outcomes.
- To explore the relationship between TIME subtypes and tumor molecular features relevant to immunotherapy.
Main Methods:
- Analysis of 812 colon and rectal carcinoma cases from the Nurses' Health Study and Health Professionals Follow-up Study.
- Classification of tumors into four TIME subtypes based on CD274 (PD-L1) expression and TILs.
- Cox proportional hazards models adjusted for MSI, CIMP, LINE-1 methylation, and KRAS, BRAF, PIK3CA mutations.
Main Results:
- TIME subtypes 1, 2, 3, and 4 comprised 27%, 14%, 13%, and 46% of cases, respectively.
- TIL-present subtypes (TIME 2 and 3) showed associations with high MSI, high CIMP, BRAF mutation, and increased neoantigen load (p < 0.001).
- No significant association was found between TIME subtypes and colorectal cancer-specific or overall survival.
Conclusions:
- TIL-present TIME subtypes in colorectal cancer are linked to high MSI and neoantigen load, indicating potential for improved immunotherapy response.
- Further research into tumor molecular alterations and microenvironmental factors is crucial for advancing immunoprevention and immunotherapy strategies.
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