Novel Computational Analysis Identifies Cytotoxic Lymphocyte-to-Monocyte Balance in Tumors as a Predictor of Recurrence-Free Survival in Colorectal Carcinoma

Abstract

The microenvironment and immune infiltrate population of colorectal tumors can serve as a stronger predictor of patient survival than microsatellite-status or traditional T- or N-staging. This study aimed to leverage transcriptomic techniques to identify specific immune cell populations and their ratios associated with cancer recurrence in colorectal cancer patients. The goal was to identify patients who could benefit from early adjuvant interventions, identify those at higher risk of recurrence for surveillance, and identify potential combinatorial immunotherapy strategies tailored to this disease. We found that a lower ratio of cytotoxic lymphocyte: monocytic lineage cells, and not microsatellite-status, was associated with cancer recurrence. Additional differential gene expression analysis of the monocytic lineage demonstrated that genes specifically associated with tumor associated macrophages and a protumoral phenotype were overexpressed in the tumor microenvironment in patients that went on to have recurrent disease. Gene Ontology analysis revealed that pathways associated with pro-tumoral extracellular matrix remodeling were suppressed in tumors exhibiting a high cytotoxic lymphocyte: monocytic lineage ratio, suggesting a diminished propensity for tumor progression. The development of these prognostic markers not only associates with colorectal cancer recurrence, aiding in risk stratification and guiding adjuvant therapy decisions for resected early-stage patients, but also suggests that effective colon cancer treatments will likely require a combination of cytotoxic T-cell-directed immunomodulation and targeted inhibition of tumor-associated macrophages.

FIG. 1

Disease recurrence after primary colon cancer resection and genetic evidence of isolated immunocyte populations. Microenvironment Cell Populations-counter was utilized to generate estimates of immune and stromal cell populations in the tumor microenvironment. Comparisons were made between tumors isolated from patients with recurrent and nonrecurrent disease. A Tumor-infiltrating cell types were correlated with recurrence (n = 253). B Identification of the optimal cutoff value for the T-cell MCP score (6.334). C Optimal cutoff value for the CTLs-MCP score (6.518). No significant correlation was observed between isolated T-cell MCP scores or CTLs MCP scores and recurrence-free survival (RFS). CTLs cytotoxic T-lymphocytes

FIG. 2

Increased cytotoxic lymphocyte: monocytic lineage ratio is associated with improved recurrence-free survival. Microenvironment Cell Populations-counter was utilized to compare cell ratios in tumors isolated from patients with recurrent and non-recurrent disease after colorectal cancer resection. A Decreased T cells:monocytic lineage and CTL:monocytic lineage ratios were evident in recurrent tumors of all stages (p = 0.044 and p < 0.001, respectively), B as well as in early-stage tumors alone (p < 0.001 and p = 0.002, respectively). The optimal cutoff values for the C T-cells:monocytic lineage ratio (0.8233), and D CTLs:monocytic lineage ratio (0.8417) were established, and an association of these ratios with recurrence was then confirmed through Kaplan-Meier analysis. Improved RFS was also observed in E T cell:monocytic lineage ratio^high and F CTL: monocytic lineage ratio^high groups in patients without metastatic disease G Additionally, the CTL:monocytic lineage ratio represented as the CD8A:CD14 gene expression ratio was examined using the KMP tool, validating that a higher ratio associates with improved recurrence-free survival (n = 1,336). *p < 0.05; **p < 0.005. CTLs cytotoxic T-lymphocytes

FIG. 3

Patients with a tumor microenvironment characterized by high cytotoxic lymphocyte scores and low monocytic lineage scores exhibited improved recurrence-free survival. CTLs MCP-counter score cutoff (6.518) and monocytic lineage cutoff (7.72) were calculated from the respective MCP scores, and a superior RFS was observed in patients in the CTL^high and monocytic lineage^low group compared with the CTL^low and monocytic lineage^high group. MCP Microenvironment Cell Populations; CTLs cytotoxic T-lymphocytes; RFS recurrence-free survival

FIG. 4

Increased cytotoxic lymphocyte: monocytic lineage ratio is associated with reduced ECM remodeling. A Differentially expressed gene analysis (DGE) was conducted to evaluate genes that are significantly overexpressed or underexpressed in patients demonstrating a high CTL:monocytic lineage ratio. B Gene ontology analysis revealed the suppression of pathways related to extracellular matrix remodeling. ECM extracellular matrix; CTLs cytotoxic T-lymphocytes

FIG. 5

Gene profiles of tumor associated macrophages (TAM) of the M2 phenotype were overexpressed in the tumor microenvironment of patients with recurrent disease. A Recurrence analysis of 187 colorectal patients and B validation on 249 TCGA colorectal patients stratified by M2-macrophage MCP scores revealed decreased RFS in the M2-macrophage^high group. The association of M2 macrophage gene markers, such as C TIE-1, D TEK, and E VEGFA, with recurrence was validated using the KMP tool. RFS recurrence-free survival