Fig 2: Sample cores for every subgroup of The Cancer Genome Atlas. (A,D,G,J) Hematoxylin and eosin. (B,E,H,K) Multiplexed immunofluorescence. (C,F,I,L) After digital image analysis. (A–C) POLEmut. (D–F) MMRdef. (G–I) NSMP. (J–L) p53abn. Note the gradient between POLE mutated tumors with highest infiltrate of both CD3+ (green) and CD8+ (red) lymphocytes to NSMP tumors with lowest CD3+ and CD8+ cells, with MMRdef and p53abn tumors in between.

Fig 3: Boxplots of cell densities for the testing cohort (A,C,E) and validation cohort (B,D,F). (A,B) Total cell densities per mm2 for CD3+ (red) and CD8+ (blue) cells in comparison with the four molecular subgroups according to The Cancer Genome Atlas (TCGA). POLE mutated tumors show the highest immune infiltrate, where NSMP and p53abn tumors show the lowest. (C,D) CD3+ cell densities per mm2 in the three compartments of a core. The compartments are intra-tumoral (blue), close (red), and distant (green). Note the gradient between the highest values for close and the lowest values for intra-tumoral CD3+ lymphocytes. (E,F) CD8+ cell densities per mm2 intra-tumoral (blue), close (red), and distant (green) for every TCGA group. Note the distinct gradients between intra-tumoral, close, and distant regions. In the testing cohort (E), CD8+ lymphocytes are highest intra-tumoral in POLE mutated tumors. In the validation cohort (F), the gradients are different to the testing cohort with highest values close to the tumor, except in p53abn tumors. The red line indicates the cut-off of 50 intra-tumoral CD8+ cells per mm2. Significances are outlined as non-parametric median comparison following Kruskal-Wallis Test marked with asterixes as *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

Fig 4: Validation of multiplex immunohistochemistry with conventional immunohistochemistry using tissue microarray (TMA) cores. TMAs were virtually set in a whole slide image of a colon cancer case stained in duplicate and were used for comparison of multiplex with conventional immunohistochemistry. Comparison was calculated with a Spearman correlation (rs). (A) Multiplexed immunofluorescence after digital image analysis with stromal cells (blue), CD3+ lymphocytes (green) and CD8+ lymphocytes (red). Note the possibility of multiplexed immunohistochemistry to stain double positive CD3+ and CD8+ cells in a single TMA core, leading to a more exact quantification of these cells. (B) Multiplexed immunofluorescence original data. (C) Hematoxylin and eosin staining. (D) Hematoxylin and diaminobenzidine staining showing CD3+ lymphocytes. (E) Hematoxylin and diaminobenzidine staining showing CD8+ lymphocytes. (F) Spearman correlation of CD3+ cell counts with conventional immunohistochemistry on x-axis and multiplex immunofluorescence on y-axis. (G) Spearman correlation of CD8+ cell counts with conventional immunohistochemistry on x-axis and multiplex immunofluorescence on y-axis.