Multimarker Assessment of B-Cell and Plasma Cell Subsets and Their Prognostic Role in the Colorectal Cancer Microenvironment

Abstract

Purpose: Although the association between cytotoxic T lymphocytes and favorable prognosis in colorectal cancer is well established, the prognostic significance of B lymphocytes remains more ambiguous. This study aimed to assess the characteristics and significance of various B-cell and plasma cell subsets in colorectal tumors.

Experimental design: We designed a seven-plex IHC assay, combined with machine learning-based image analysis, to identify various B-cell and plasma cell populations and applied it to study a cohort of 912 colorectal tumors. We assessed the prognostic significance of B-cell and plasma cell densities using Kaplan-Meier estimators and Cox regression models. Additionally, we designed a more clinically applicable three-plex assay, which we used to study B-cell and plasma cell densities in a separate validation cohort of 737 patients.

Results: High plasma cell density in the center of the tumor was associated with longer cancer-specific survival independent of disease stage, mismatch repair status, T-cell densities, and other covariates. In the study cohort, the multivariable HR for high (vs. low) plasma cell density was 0.48 (95% confidence interval, 0.32-0.72; Ptrend = 0.0005), whereas the corresponding HR in the validation cohort was 0.37 (95% confidence interval, 0.21-0.65; Ptrend = 0.0003). Of the specific subsets, IgG1-IgG2- plasma cells showed the strongest association with improved survival. High B-cell densities were not independently associated with a better prognosis.

Conclusions: Plasma cell densities in the center of the tumor represent a relevant tumor-immune biomarker in colorectal cancer, complementing T-cell density measurements.

Figure 1.

mIHC to identify various B-cell and plasma cell populations and their distribution in the colorectal cancer microenvironment. A and D, mIHC image of a representative example tumor. B and E, Image analysis result output showing the main cell types identified in the mIHC image. C and F, NND plot showing NNDs from individual B and plasma cells to the closest tumor cell in the example image. G, Box plot showing the distribution of NNDs from each B cell and plasma cell to the closest tumor cell across all tumors of the main study cohort (n = 912). The box plot is based on 501,460 plasma cells and 184,653 B cells. The P value was calculated using the Wilcoxon rank-sum test.

Figure 2.

Kaplan–Meier estimates of CSS in the main cohort. The curves visualize CSS according to the ordinal quartile categories (Q1–Q4, from low to high) of overall plasma cell densities in the IM (A) and CT (B) and overall B-cell densities in the IM (C) and CT (D). P values were calculated using the log-rank test.

Figure 3.

Three-plex IHC assay and Kaplan–Meier estimates of CSS in the validation cohort. The mIHC images (A and C) show representative example tumors, and the image analysis result images (B and D) show the main cell types identified. Kaplan–Meier curves visualize CSS according to the ordinal quartile categories (Q1–Q4, from low to high) of overall plasma cell densities in the IM (E) and CT (F) and overall B-cell densities in the IM (G) and CT (H). P values were calculated using the log-rank test.