A Th2-score in the tumor microenvironment as a predictive biomarker of response to Bacillus Calmette Guérin in patients with non-muscle invasive bladder carcinoma: A retrospective study

Abstract

Intravesical Bacillus Calmette Guerin (BCG) is the gold standard therapy for intermediate/high-risk non-muscle invasive bladder cancer (NMIBC). However, the response rate is ~60%, and 50% of non-responders will progress to muscle-invasive disease. BCG induces massive local infiltration of inflammatory cells (Th1) and ultimately cytotoxic tumor elimination. We searched for predictive biomarker of BCG response by analyzing tumor-infiltrating lymphocyte (TIL) polarization in the tumor microenvironment (TME) in pre-treatment biopsies. Pre-treatment biopsies from patients with NMIBC who received adequate intravesical instillation of BCG (n = 32) were evaluated retrospectively by immunohistochemistry. TME polarization was assessed by quantifying the T-Bet+ (Th1) and GATA-3+ (Th2) lymphocyte ratio (G/T), and the density and degranulation of EPX+ eosinophils. In addition, PD-1/PD-L1 staining was quantified. The results correlated with BCG response. In most non-responders, Th1/Th2 markers were compared in pre-and post-BCG biopsies. ORR was 65.6% in the study population. BCG responders had a higher G/T ratio and a greater number of degranulated EPX+ cells. Variables combined into a Th2-score showed a significant association with higher scores in responders (p = 0.027). A Th2-score cut-off value >48.1 allowed discrimination of responders with 91% sensitivity but lower specificity. Relapse-free survival was significantly associated with the Th2-score (p = 0.007). In post-BCG biopsies from recurring patients, TILs increased Th2-polarization, probably reflecting BCG failure to induce a pro-inflammatory status and, thus, a lack of response. PD-L1/PD-1 expression was not associated with the response to BCG. Our results support the hypothesis that a pre-existing Th2-polarized TME predicts a better response to BCG, assuming a reversion to Th1 polarization and antitumor activity.

Keywords: BCG predictive biomarkers; Lymphocyte polarization; Non-muscle invasive bladder cancer.

Figure 1. Expression of GATA-3, T-Bet, and EPX in all NMIBC pre-BCG biopsies. (A) Representative pictures of immunohistochemistry staining for GATA-3, T-Bet, and EPX in pre-BCG NMIBC biopsies of R and NR patients. Original magnification 400×. Scale bar = 50 μm. In the case of EPX staining, an inset shows Eodgn in detail. (B) Quantitation of GATA-3+, T-Bet+ TILs, and EPX+ eosinophils EPX in all pre-BCG NMIBC biopsies analyzed. ***Highly significant, p = 0.0005, Mann–Whitney test.

Figure 2. Th2-score in the TME can predict response to BCG in NMIBC patients. (A) Th2-score was higher in BCG-Responder than in BCG-NR non-responder patients (*p = 0.027, Mann–Whitney test); (B) ROC curve for the Th2-score allows discrimination of BCG-R patients (≥48.1) with 91% sensitivity and 57% specificity. (C) Kaplan Meier curve of recurrence-free survival (RFS) in months. Patients were stratified by Th2-score as low or high, based on the ROC curve cut-off (<48.1 = low count; ≥48.1 = high count) (p = 0.007, log-rank test (R); (D–F) Comparison of GATA-3 mean counts, G/T, and Th2-score in pre- and post-BCG biopsies (relapses), respectively.

Figure 3. PD-L1/PD-1 expression in pre-BCG TILs of NMIBC biopsies. Quantitation of PD-L1 in tumor cells (A) and TILs (B) performed in BCG R or NR. (C) A positive correlation was found between PD-L1+ tumor cells and PD-L1+ TILs (C, r = 0.68, p = 0.0014, Spearman correlation test). (D) Invasive urothelial carcinoma showing PD-L1+ expression at the plasma membranes (left) and stromal TILs expressing PD-L1 in proximity to tumor cells (right). (E) Representative pictures of immunohistochemical staining of PD-L1 and PD-1 of pre-BCG tumor biopsies of BCG-R and BCG-NR patients. A, B, and D were compared with the Mann–Whitney test. Original magnification 400×. Scale bar = 50 μm.

FIGURE S1. Each variable quantitated by IHQ was plotted for BCG-R and BCG-NR patients (n = 32).

FIGURE S2. Kaplan Meyer curve of recurrence-free survival (RFS) in months. Patients were stratified by GATA-3 (A), T-Bet (B), G/T (C) and Eo counts (D) as low or high, based on the median values of the different variables analyzed (65.5 for age; 21.5 for GATA-3+; 2.9 for T-Bet+; 3.6 for G/T and 5.9 for EPX+). p values are indicated (log rank test, R).