Frequencies and correlations among immune cells
Densities of CD8+, GZMB+, CD68+ and CD163+ cells were calculated for stroma areas at different areas of interest. This included tumour proximity (TP) zones with different radii around the tumour (TP10mm, TP25mm, TP50mm, TP100mm) each for CT, FR, ME and NE (where TP zones were measured from the normal epithelium).
Distributions of immune cell type densities in the total stroma area for the different tumour regions and normal epithelium are shown in Figure 3. Comparison of densities between TP25mm zone and the complementary stroma area more distant from the tumour is shown in Figure S1 for the tumour centre. Whereas the density of GZMB and CD68 did not differ between these areas, CD8 (p = 0.003) and CD163 (p < 0.001) were significantly lower in the TP25mm zone.
Interrelationships between immune cell types are shown in Table S1. Strong correlations were observed between various immune cell types at different tumour regions, including between all examined immune cell types at the tumour centre (for all p < 0.01).
(Figure 3, Figure S1, Table S1)
Correlation of immune cells with clinicopathological features
Of all examined regions of interest (tissue regions and TP zones) the strongest effects from immune cell density on survival were observed at the tumour centre in the TP25mm zone for CD8, GZMB and CD68 (Table 3). Therefore, we used these measurements for the following analyses. Patients were classified as either high or low for each of these three markers by using the median density as the cut-off.
Overall distributions of patients’ clinicopathological features and their associations with immune cell density in the TP25mm zone at the tumour centre are displayed for both cohorts in Table 1 and Table 2. In cohort 1, notably high CD8, high GZMB and high CD68 were significantly associated with lower TNM stage and higher Klintrup-Mäkinen score. GZMB was the sole variable that showed significant associations of high density with lower pT stage (p = 0.036), absent venous (p = 0.004) and absent perineural invasion (p = 0.006). High CD8 was the only variable significantly associated with less tumour budding (p = 0.041). In cohort 2, the only significant association was observed between high GZMB and lower pT stage (p = 0.036).
(Table 1, Table 2)
Survival data was obtained for all patients in cohort 1 (n = 107) and for all patients except one (unclear recurrence status) in cohort 2 (n = 150). Overall survival was assessed in cohort 1 and disease-free survival in cohort 2. Median follow-up time was 49 months (range 1-183 months) in cohort 1 and 65 months (range 45-130 months) in cohort 2. To achieve maximum comparability between immune cell types, patients with missing data for at least one stained antigen (missing or non-eligible TMA cores) were excluded from survival analyses of the corresponding tissue region. Effect size of immune cell type density on survival was examined in a univariate analysis for different areas of interest in cohort 1 (Table 3) and cohort 2 (Table S2). In cohort 1, the strongest prognostic effects were generally observed at the tumour centre, followed by the tumour front and the tumour microenvironment. Comparing TP zones with varying radii around the tumour, lowest hazard ratios and the largest number of highly significant associations (p < 0.001) were observed at the TP25mm zone, closely followed by the TP50mm zone. CD163 was inferior to the other markers in every region of interest, however significant associations of a high CD163 with longer survival (p = 0.012) were still observed in the total stroma area at the tumour centre. As expected, no significant correlations were observed for any immune cell type and TP zone upon examination of the normal colorectal epithelium.
Due to these findings, further analyses were based on CD8, GZMB and CD68 densities divided into two groups by the median in the TP25mm zone at the tumour centre. Additionally, patients were allocated to two groups based on the combined GZMB and CD68 density where patients with both densities above the median were classified as GZMB/CD68 high and patients with at least one below the median as low. This combination was chosen to be compared with the current “benchmark” of the CD8 density and its thoroughly described prognostic effect.3,11
Kaplan-Meier curves are displayed in Figure 4. In cohort 1, highly significant associations (all p < 0.001) with longer overall survival were observed for high TP25mm density of CD8, GZMB and CD68 as well as for a high combined GZMB/CD68 score. In cohort 2, longer disease-free survival was associated with high GZMB (p = 0.035), high CD68 (p = 0.004) and a high combined GZMB/CD68 score (p < 0.001). However, CD8, despite a trend, (p = 0.072) did not show a significant association with disease-free survival in the TNM stage II cohort. No statistically significant association was observed in any of the two cohorts neither for GZMB:CD8 ratio nor for CD68:CD163 ratio nor for subtracting CD163 from CD68.
In a multivariate survival analysis, prognostic effect of CD8 was compared with the combined GZMB/CD68 score in both cohorts (Table 4). In cohort 1 both high CD8 (p = 0.002) and high combined GZMB/CD68 score (p = 0.007) remained significant predictors of longer overall survival with roughly similar hazard ratios upon inclusion of TNM stage and post-operative chemotherapy as additional variables. In cohort 2, prognostic effect on disease-free survival of the combined GZMB/CD68 score (p = 0.005) was superior to CD8 (p = 0.142) with pT stage and venous invasion as additional variables.
(Table 3, Table S2, Figure 4, Table 4)