Fig. 1: Overall CD20⁺ B cell density is not clinically significant, but the density of TILs within cancer cell islands is significant.

a Sample image of TNBC tissue illustrating the heterogeneity of the tumor microenvironment. Color code: T cells (CD3⁺; green), B cells (CD20⁺; cyan), cytotoxic T cells (CD8⁺; bright red), regulatory T cells (FoxP3⁺, magenta), cancer cells (Pan-cytokeratin cells; dark brownish red), and nuclei (DAPI; dark blue). Cancer cell islands correspond to clusters of cancer cells (dark brownish red). Scale bar = 100 μm. b Example of cancer cell islands interspersed with stroma. Color code: T cells (CD3⁺; green), B cells (CD20⁺; cyan), cancer cells (Pan-cytokeratin cells; red), and nuclei (DAPI; dark blue). Scale bar = 50 μm. c, d The density of B cells overall in tumor does not differentiate between good vs. poor outcome. c Box and whisker plot showing CD20⁺ B cell density in the entire tumor is not significantly different between good (n = 24) vs. poor (n = 12) outcome. Center line represents the median, box limits represent the upper and lower quartiles, whiskers indicate maximum and minimum values within 1.5 interquartile ranges of the upper and lower quartiles; individual points are outliers. d Relapse-free survival (RFS) plot showing that CD20⁺ B cell density in the entire tumor is not a significant factor in TNBC clinical outcome, using a threshold value of 55 cells/mm² as described in “Methods”. e–g Density of B and T cells within cancer cell islands is clinically significant. e Plot showing density of CD20⁺ B cells within cancer cell islands is higher for good (n = 24) vs. poor (n = 12) outcome (p = 0.02). Center line represents the median, box limits represent the upper and lower quartiles, whiskers indicate maximum and minimum values within 1.5 interquartile ranges of the upper and lower quartiles; individual points are outliers. f RFS plot showing the clinical significance of density of CD20⁺ B cells within cancer cell islands using a threshold value of 0.974 cells/mm² (see “Methods”). g Plot showing density of CD3⁺ T cells within cancer cell islands is higher for good (n = 24) vs. poor (n = 12) outcome (p = 0.01). Center line represents the median, box limits represent the upper and lower quartiles, whiskers indicate maximum and minimum values within 1.5 interquartile ranges of the upper and lower quartiles; individual points are outliers. h RFS plot showing the clinical significance of density of CD3⁺ T cells within cancer cell islands using a threshold value of 34.8 cells/mm² (see “Methods”). P-values in box plots are determined by unpaired, two-tailed Student t-tests. ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 1 × 10⁻⁴.