High Stroma T-Cell Infiltration is Associated with Better Survival in Stage pT1 Bladder Cancer

Abstract

Stage pT1 bladder cancer (BC) shows highly diverse outcomes. Predictive markers are required to stratify patients for personalized treatment. The present study aimed to validate immune response quantification as a prognostic marker. Patients with pT1 BC (n = 167) treated by transurethral resection of the bladder (TURB) were enrolled. Formaldehyde-fixed paraffin-embedded material was stained for CD3 and CD8. Corresponding T cells were counted in three regions with the highest immune response. Numbers of tertiary lymphoid structures (TLS) and lymphocyte aggregates (LA) were quantified. High CD3⁺ stroma T-cell infiltration was associated with improved survival (p = 0.045), especially in the G3 subgroup (p = 0.01). Cluster with higher immune response showed less recurrence (p = 0.034) and favorable overall survival (OS) (p = 0.019). In contrast, higher CD3⁺ and CD8⁺ tumor T-cell infiltration seemed to have a negative impact on prognosis. TLS and LA were more frequently observed in G3 tumors, indicating an increased anti-tumoral immune response. We proved the role of immune cell infiltration and showed that higher infiltration numbers of CD3⁺ (not CD8⁺) lymphocytes in the stroma are associated with favorable outcome. Immune cell quantification could be used as a marker to help stratify patients' risk and therefore, to optimize patients' management and follow-up examination as well as possible therapies.

Keywords: CD8+ lymphocytes; immunoscore; pT1 bladder cancer; progression; survival; tumor-infiltrating lymphocytes (TILs), CD3+ lymphocytes.

Figure 1

Kaplan–Meier curves for (a) stromal infiltration of CD3⁺ immune cells (all patients) using the median (2882 cells/mm²) as threshold to divide patients into two groups (p = 0.045). (b) Stromal infiltration of CD3⁺ immune cells (only in G3 subgroup) using the k-means clustering approach (p = 0.01). C1 with low and C2 with high immune cell infiltration. In each graph, Kaplan–Meier curves present a significant difference between the two groups, showing an increased survival in patients with higher immune cell infiltration. p-Values are calculated using the log-rank test; x-axis, survival in months; y-axis, estimated survival (fraction of individuals).

Figure 2

Box plots (a,b) comparing the median of tumor T-cell infiltration by using different percentiles to separate patients into two groups. Only the lowest p-values are shown in this composite figure. (a) CD3⁺ immune cell infiltration grouped by the 45th percentile (cut-off: 176 cells/mm²) (p = 0.011). (b) CD8⁺ immune cell infiltration grouped by the 50th percentile (cut-off: 118 cells/mm²) (p = 0.014). Box plots comparing (c,d) the median of tumor T-cell infiltration by using G2 versus G3 to separate patients into two groups. (c) CD3⁺ immune cell infiltration (p = 0.043) and (d) CD8⁺ immune cell infiltration (p = 0.028).

Figure 3

Box plots (a,b) comparing the numbers of (a) lymphocyte aggregates (LA) and (b) tertiary lymphoid structures (TLS) per mm² when separating the patients into two groups by using G2 versus G3 subgroup in CD3⁺ stained slides. Significant higher values of LA (p = 0.0014) and TLS (p = 0.0003) were found in the G3 subgroup. Please note the different scaling of the graphs.

Figure 4

Three different CD3-stained slides in non-muscle-invasive bladder cancer (NMIBC) viewed in Panoramic Viewer 1.15.4. Selection of one high-power field (HPF) each for stroma and for tumor infiltration. Invasion front was between these two corresponding HPFs. The images were scanned with digital slide scanner (camera: CIS VCC, Panoramic MIDI Scan; 3DHistech Ltd., Budapest, Hungary). (a) Example for moderate stroma infiltration and low tumor infiltration, (b) example for low stroma infiltration and low tumor infiltration, (c) example for high stroma infiltration and high tumor infiltration.

Figure 5

(a–d) Very high immune reaction in CD3⁺ stained slides. Each picture shows one HPF in the stroma. Cells were displayed superimposed. Exact differentiation of single cells was not possible for the program. Scanned with digital slide scanner (camera: CIS VCC, Panoramic MIDI Scan; 3DHistech Ltd., Budapest, Hungary).

Figure 6

(a–c) Lymphocyte aggregates and (d–f) tertiary lymphoid structures. CD3⁺ immune-stained slides. LA consist of clustered immune cells. TLS show organized structures such as an active center with surrounding immune cells. Scanned with digital slide scanner (camera: CIS VCC, Panoramic MIDI Scan; 3DHistech Ltd., Budapest, Hungary).