Non–muscle invasive bladder cancer (NMIBC) represents 70% of all initial bladder cancer diagnoses [1,2]. Some high-risk patients (high-grade [HG] pT1 or carcinoma in situ [Cis]) [3] respond to bladder-conservative treatments and behave in a non-lethal fashion, while others are aggressive and tend to progress to muscle invasion and even metastatic disease. pT1 tumors are mostly HG and progress in >50% of cases; deaths occur in 25% of patients during the first five years [3]. Cis is mostly concomitant, with increasing recurrence rates from 43% to 73% [4]. Even if Cis is primary or concomitant, 50% of patients progress to muscle invasion and 20% will ultimately die of metastases if a radical cystectomy is not performed [5]. Intravesical administration of live-attenuated Bacillus Calmette-Guérin (BCG) is the primary adjuvant treatment for high-risk NMIBC patients [6]. Nonetheless, the response rate (RR) to BCG is about 60%, with a 5-year recurrence rate of 30%–40% [7]. Moreover, for those patients presenting either pT1 or Cis, or both tumors, who do not respond to BCG, the risk of progression to invasive disease may reach 50% [8]. Thus, there is still a strong need to find appropriate predictive biomarkers of response to BCG that may help the selection of patients with a low chance of response, before or early after treatment starts, to offer them alternative therapies. Although the exact mechanism of BCG antitumor activity is not fully understood, it is recognized that BCG acts as a localized immunomodulatory agent promoting Th1 immune polarization, and its efficacy must be related to the immune response elicited in the bladder [9].

GATA binding protein 3 (GATA-3) is a transcription factor that is critical for T cell development and Th2 differentiation [10]. T-box transcription factor 21 (T-Bet) is mostly expressed in CD4+ Th1 cells, and its expression is induced by a combination of T cell receptors and IFN-γ signaling [11]. In addition, T-Bet promotes Th1 polarization by preventing GATA-3-mediated Th2 cell development [12].

In the past, eosinophils were inaccurately considered destructive end-stage effector cells (helminth infections and allergy/asthma) due to the release of toxic cationic granule proteins and the production of reactive oxygen species. More recently, eosinophils have been shown to play a fundamental role in remodeling/repair activities, and more importantly, to modulate local immunity [13].

An emerging concept relates to the prognostic and/or predictive value of Th1/Th2 polarization of tumor-infiltrating cells (TILs) before treatment, which may affect the response to BCG immunotherapy. Interestingly, in patients with Cis treated with BCG induction alone [14], those who responded to BCG showed an increased density of intratumor Th2 cells and a higher Th2/Th1 ratio prior to treatment, suggesting that BCG would be effective only when the tumor microenvironment (TME) has shifted from Th2 to Th1.

Programmed death ligand 1 (PD-L1) expressed by tumor and immune cells in the TME suppresses antitumor immune responses and promotes tumor progression [15]. However, PD-L1+ TILs have been correlated with improved overall survival in patients with metastatic urothelial cancer [16]. In a recent study, no association of BCG response with peritumoral PD-L1 expression in pretreatment biopsies was reported [17]. On the contrary, high PD-L1 expression in BCG-induced granulomas has been associated with resistance to therapy in urothelial carcinomas [18]. These data suggest that high numbers of PD-L1+ cells in the bladder could suppress the effectiveness of BCG, a hypothesis also supported by the recent demonstration that combined BCG and anti-PD-L1 treatment increased antitumor immunity in an immunocompetent orthotopic rat bladder cancer model [19]. Thus, the PD-1/PD-L1 axis could be involved in the antitumor immune response of NMIBC.

The aim of our study was to identify potential biomarkers to predict the response to BCG in a high-risk population of NMIBC patients, including those with HG pTa, pT1, and Cis tumors, focusing on Th1/Th2 lymphocyte polarization in the TME. We also explored a possible association of PD-1/PD-L1 expression in NMIBC biopsies with BCG response.

There is a real need to identify patients with NMIBC that will not benefit from BCG immunotherapy, in order to offer them alternative treatments or clinical trials. In vitro and in vivo evidence supports that BCG acts as a potent Th1 inducer in the TME and is presumably responsible for long-lasting disease control [9,22]. However, the nature of the tumor antigens that are targets of the cytotoxic immune response or whether the anti-BCG immune response is involved in tumor elimination is still unknown. Additionally, experimental evidence has shown a direct effect of BCG in killing bladder cancer cells [23].

In the search for a possible biomarker of response to BCG, we evaluated lymphocyte polarization in the TME, along with the quantitation of eosinophils (associated with a Th2 immune microenvironment), at the maximum focus of infiltration, by IHC in pre-BCG biopsies of patients who had high-risk NMIBC, including HG pTa, LG/HG pT, and Cis tumors, and received adequate BCG. Our goal was to define a Th2-score associated with BCG response and set a cutoff to predict true BCG-R patients with >90% sensitivity. Patients with higher Th2-scores showed significantly higher RFS after BCG treatment. Nunez-Nateras et al. [14] reported that a Th2-score obtained after quantitation of the Th2/Th1 lymphocyte ratio plus eosinophilic infiltration of pre-BCG biopsies was associated with BCG response in 38 NMIBC patients with Cis. The authors hypothesized that a pre-existing Th2 TME would be susceptible to Th1 polarization, and therefore, Th2 status would correlate with the response to BCG treatment. However, in that study, patients received only one course of induction ×6, which is inadequate today. Of note, BCG response was assessed only after a short follow-up (6–8 weeks after BCG induction). Another study confirmed Nunez-Nateras’s findings [24]. However, the small number of patients included (19 BCG-R and 4 BCG-NR) may limit the conclusions, and the fact that only 25% of the patients had Cis makes comparison difficult. Another report on 22 HG pT1 patients (none of them had Cis tumors) showed that BCG-R patients had a significantly lower absolute number of peritumoral T-Bet+ cells and a higher G/T ratio [17]. However, the BCG strain and the treatment scheme were not specified. Furthermore, TILs G/T ratio adjusted for the systemic inflammation (neutrophil-to-lymphocyte ratio) showed a significantly higher association with the BCG response, a prolonged RFS in patients with a lower T-Bet+/Lymphocyte ratio, and higher GTR/NLR. In a later study [25], which included only 19 BCG-R and 4 BCG-NR patients the authors analyzed differences in the expression of GATA-3+ and T-Bet+ TILs (only evaluated in the lamina propria) between BCG-R vs. BCG-NR and found a clear tendency toward increased GATA-3+ T cell counts and GATA-3/T-Bet ratio.

Taken together, our results provide strong evidence that, after surgery, BCG can eradicate NMIBC cells only when the TME converts from Th2 to Th1.

Interestingly, we observed an increase in Th2 TILs in post-BCG biopsies from relapsed patients, probably reflecting the failure of BCG to induce a pro-inflammatory status. Presumably, the residual tumor cells that established strong immunosuppression sustained Th2 polarization.

Emerging data highlight that eosinophils infiltrate multiple tumors and display pleiotropic and even opposing roles (pro- vs. anti-tumorigenic activities) [26]. Furthermore, crosstalk between activated eosinophils and T cells increases CD8+ T cell migration to TME [27]. In addition, in patients treated with immune checkpoint inhibitors, increased eosinophilia is associated with responsiveness to therapy [27]. Thus, a higher pre-BCG Eo count in R patients could represent a Th2 polarized TME that can be shifted to Th1 by BCG administration, and eosinophils may contribute to Th1 cell recruitment. However, it was reported that blood eosinophils in patients with NMIBC could predict disease recurrence during BCG immunotherapy [28]. More data are needed to clarify this point.

Immune checkpoint upregulation is associated with a lack of antitumor cytotoxicity [29]. The upregulation of PD-L1 allows tumor cells to escape immune surveillance; thus, PD-L1 expression in the pretreatment TME could be a mechanism of BCG resistance. In our study, PD-L1+ tumor cells in pre-BCG biopsies were not associated with BCG response, in accordance with a previous report [17]. In our exploratory analysis, however, a higher proportion of BCG-R patients had >5% PD-L1+TILs than those from BCG-NR patients. Our findings are in disagreement with another report that found elevated PD-L1 expression in CD8+ TILs in baseline biopsies of BCG-NR patients [30]. However, we used a different anti-PD-L1 antibody (Ventana clone SP263 instead of Spring Biosciences clone SP142 or DAKO clone 22C3) and analyzed fewer patients. We observed >50% PD-1+TILs in a higher proportion of BCG-R than of BCG-NR patients, although this difference was not statistically significant, probably due to the small number of cases analyzed. In the post-BCG biopsies of relapsed patients, no exhausted PD-1+ TILs were detected. A possible explanation is that prior to BCG, an antitumor immune response was generated in some patients, accounting for the detection of exhausted T cells. However, an induced Th2 TME provides a cytokine balance that precludes the cytotoxic activity of TILs. In addition, PD-L1+ cells in the TME could contribute to the further abrogation of the antitumor response. Although our results suggest that the PD-1/PD-L1 pathway was not involved in BCG failure, this finding must be confirmed in a larger cohort of patients. Treatment with BCG shifts this balance toward Th1 polarization in BCG-R, patients increasing the affluence of innate and adaptive immune cells. Th1 cytokine production (i.e., IFN-γ) may fuel the elimination of residual tumor cells by immune effectors. Instead, in pre-BCG tumors with Th1-profiled TILs, other immunosuppressive mechanisms, such as secretion of TGF-β, PGE2, IL-10, and IL-6, as well as accumulation of myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells, could contribute to creating a highly tolerogenic TME [31]. Due to the small number of tumors available, we were not able to evaluate these immune populations in pre-BCG biopsies.

The limitations of this study are primarily associated with its retrospective design since only FFPE tumor biopsies were available for IHC analyses. In addition, the small number of patients reduced its statistical power. Recently, in the search for a biomarker profile associated with NMIBC patients’ response to BCG, we have initiated a prospective research study to validate the Th2-score combined with the analysis of immune cells and cytokines from urine and blood samples, obtained before and during treatment. Our results may have the potential to translate into a better selection of NMIBC patients to receive BCG treatment.