Fig. 4.

Analysis of the tumor microenvironments (TME) using immunohistochemistry (IHC) in 21 randomly selected patients with anal squamous cell carcinoma whose primary treatment was either surgery (n = 4), chemoradiotherapy (CRT) (n = 9), or chemoradiotherapy, before and after treatment. (A-C) IHC analyses of intratumoural tissue of 21 patients with anal squamous cell carcinoma. (A) Correlation analysis between intratumoural expression of PD-L1 and infiltration of different immunocytes of patients before treatment. (B) Bar graph of comparison of intratumoural expression of PD-L1 between patients in the CRT and CRT-I groups before treatment (P = 0.414). (C) Representative IHC staining images of intratumoural expression of PD-L1 before treatment of (left) CRT patient 98 and (right) CRT-I patient 99. (D-G) IHC analyses of tissue of the 9 patients treated with CRT and the 8 patients treated with CRT-Ion was evaluated, comparing it between 2 treatment groups, and within each treatment group, both before and after treatment. (D) Bar graphs of comparison between CRT and CRT-I groups of peritumoural stromal infiltration of CD8+ T cells (left) before treatment (P = 0.573) and (right) after treatment (P = 0.031). (E) Representative IHC staining images of infiltration of CD8+ T cells in peritumoral stroma before treatment of (left) CRT patient 98 and (right) CRT-I patient 84. (F) Bar graphs of comparison before and after treatment of infiltration of CD8+ T cells in the peritumoral stroma within the (left) CRT group (P = 0.963) and (right) CRT-I group (P = 0.008). (G) Representative IHC staining images of infiltration of CD8+ T cells in peritumoral stroma after treatment of (left) CRT patient 98 and (right) CRT-I patient 84. Correlation analysis done using linear regression. Comparisons done using Mann-Whitney Test. In Bar graphs, bars represent medians (50th percentile), horizontal lines represent interquartile ranges (75th and 25th percentiles), ns indicates not significant, ‘*’ is P < 0.05 and ‘**’ is P < 0.01.