Extended Data Fig. 6: Supporting information for the characterization of immune hot pMMR/MSS LARC tumors with favorable outcomes from NAT.

(a) Quantification of intra-tumoral TILs from H&E slides for 20 patients, including cases from IG1 (n = 6), IG2 (n = 6), IG3 (n = 5) and IG4 (n = 3). Statistical significance was assessed using a two-sided Mann–Whitney U-test. P values were corrected using the Bonferroni method. Boxplots’ center lines indicate medians, edges indicate the interquartile range, and the whiskers extend to the highest and lowest values not considered outliers. Right panel shows correlation between estimated fractions of intra-tumoral and inter-tumoral TILs. Statistical significance was assessed using a two-sided Spearman correlation. Error bands represent 95% confidence intervals. (b) ssGSEA scores for immune cell signatures from Bindea et al.³². Displayed cell types are the ones with an adjusted p-value < 0.10 after Bonferroni correction, based on a Kruskal-Wallis test. (c) Comparison of ssGSEA scores for specific oncogenic pathway signatures from the Hallmark set³⁵ across the four immune clusters. Displayed cell types are the ones with an adjusted p-value < 0.10 after Bonferroni correction, based on a Kruskal-Wallis test. In panels (B) and (C), sample sizes are: IG1 (n = 52), IG2 (n = 37), IG3 (n = 7), and IG4 (n = 5). (d) Correlation plot showing gene signatures for 27 selected oncogenic pathways (yellow diamonds) and immune cell infiltrates (green diamonds). Right panels show illustrative scatter plots for pairs of variables with strong positive and negative correlations. White dots in the correlation heatmap highlight pairs of variables with significant two-sided Spearman correlation after Bonferroni correction. Error bands represent 95% confidence intervals. In panels (B) and (C), statistical significance was assessed using a two-sided Mann–Whitney U-test. P values were corrected using the Bonferroni method and significant results are denoted as *q < 0.05, **q < 0.01, ***q < 0.005 and ****q < 0.001. Boxplots’ center lines indicate medians, edges indicate the interquartile range, and the whiskers extend to the highest and lowest values not considered outliers.