Therapeutic implications of PD-L1 expression in bladder cancer with squamous differentiation

The immune system plays an important role in disease protection and cell clearing by orchestrating T-cell mediated immune responses [1]. Several immune checkpoints ensure correct cell recognition. Under normal conditions programmed cell death-1 (PD-1)-receptor is expressed on the surface of activated T-cells and its ligand programmed cell death ligand-1 (PD-L1) on the surface of dendritic cells and macrophages. PD1/PD-L1 interaction induces the activation of Src homology region 2 domain-containing phosphatases modulating the T-cell antigen receptor (TCR) signalling and mediating immune tolerance to self-antigens [2, 3]. However, cancer cells can misuse these checkpoints by overexpressing PD-L1 in tumour cells protecting themselves from cytotoxic T-cell immune detection and elimination [4]. Recently, immunotherapy targeting the PD1/PD-L1 axis has emerged as promising field in anti-cancer therapy for various tumour entities (including non-small lung cancer, renal cell cancer, or head and neck squamous cell cancer) [5]. By blocking theses immune checkpoint proteins, cancer cells’ resistance to immune response can be overcome and effective T-cell response against cancer cells can be restored [4, 6]. Meanwhile ICI treatment is an integral part of first line (in platinum ineligible patients) and second line clinical management of patients with urothelial carcinoma: Five different checkpoint inhibitors, i.e. pembrolizumab, nivolumab, atezolizumab, durvalumab, and avelumab, have been assessed in clinical trials of advanced bladder cancer during the last years [7] and can be used for second line treatment, but only pembrolizumab and atezolizumab are currently approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for first line therapy in urothelial cancer [8, 9]. In this setting treatment with ICIs depends on complementary PD-L1 assessment based on different PD-L1 antibodies and immunohistochemical assays creating a wealth of different scoring algorithms and evaluation criteria. Recent studies revealed substantial inter-assay heterogeneity of PD-L1 expression in different tumour entities including bladder cancer with also some degree of inter-observer diversity as well [10‐12].

The impact of ICI treatment in patients with rare bladder tumours remains poorly studied. Histologically, bladder cancer comprises a heterogeneous group of tumours including those with squamous differentiation (SD-BLCA), i.e. urothelial cancers with squamous differentiation (UC/SCC) and pure squamous cell carcinoma (SCC). SD-BLCA is characterized by poor outcome and lack of effective (neo) adjuvant therapy [13‐15]. Pure SCC can be classified into two subgroups, i.e. SCC associated with schistosomiasis whose incidence rate is increased in regions where schistosomiasis is endemic (e.g. in the Middle East), and non-Schistosomiasis associated SCC [16]. Recently PD-L1 expression was studied in Schistosomiasis-related SCC of the bladder highlighting an association between negative PD-L1 expression and clinico-pathological parameters like tumour stage and unfavourable patients’ outcome [17]. In 2018 Udager and colleagues analysed PD-L1 protein expression in 17 pure SCC samples of the urinary bladder demonstrating frequent PD-L1 positivity (65%) [18]. Reis et al. confirmed strong PD-L1 expression in immune and tumour cells in 16 urothelial cancers with squamous differentiation [19], however, a comprehensive study involving the most prominent diagnostic PD-L1 antibodies and corresponding scoring algorithms (immune cell (IC)-score, tumour proportion (TP)-score and combined positivity score (CPS)) in non-Schistosomiasis SCC is still missing. Therefore, we aim to give insights into the therapeutic implications of PD-L1 expression in non-Schistosomiasis associated SD-BLCA by assessing PD-L1 expression using four different PD-L1 antibodies (DAKO 28–8, DAKO 22C3, Ventana SP263, Ventana SP142) in both a retrospective cohort including 45 mixed UC/SCC / 63 pure SCC and in tissue samples derived from a SD-BLCA index patient who showed excellent response of pulmonary metastasis upon nivolumab treatment.

So far, the clinical management of patients with squamous differentiated bladder cancer is limited by the choice of effective (neo) adjuvant therapies [13‐15]. The five year survival rate is worse varying between 16 and 48% [22, 23]. A ray of hope might be immunotherapy by PD1/PD-L1 checkpoint inhibitors which recently revolutionized the therapeutic landscape of various cancers including urothelial cancer [24]. In urothelial cancers efficacy of different ICIs have been assessed in clinical trials during the last years [7]. For instance, the Keynote-045 trial demonstrated a clinical benefit of pembrolizumab over chemotherapy for efficacy and safety upon treatment of locally advanced/metastatic, platinum-refractory urothelial tumours [25]. Meanwhile two ICI agents, i.e. pembrolizumab and atezolizumab, have been approved by the FDA and EMA for first-line therapy of platinum-ineligible patients with PD-L1 expression as specified by scoring algorithms [8, 9]. Accumulating studies also indicate strong PD-L1 expression in squamous tumors of the urinary bladder [17‐19], but underlying retrospective cohorts are less suitable to assess the additive value of ICI therapies in SCC disease management: The patient cohort analyzed by Owyong and colleagues comprised mainly Schistosomiasis-associated SCC [17], while the publications by both Reis et al. and Udager et al. lack sufficient SCC sample numbers [18, 19]. Moreover, all studies were characterized by the absence of a clinical setting.

In the presented study, we now provide evidence for suitable ICI treatment of squamous bladder cancer by analysing both PD-L1 staining of a larger retrospective cohort of 108 SD-BLCA samples and of a SD-BLCA index patient whose pulmonary metastasis showed complete response upon nivolumab treatment. In concordance with the current FDA/EMA guidelines of urothelial cancers calling for PD-L1 positivity to protect from side effects [8, 9, 26], we revealed frequent PD-L1 expression in squamous bladder tumours up to 62% for immune and up to 52% for tumour cells. These findings, based on four different antibodies (DAKO 28–8, DAKO 22C3, Ventana SP263 and Ventana SP142), confirmed the data of the recent publications [17‐19] and are comparable with studies of urothelial cancer [10]. So far, ICI treatment is an integral part of the therapy of squamous cancers in other organs like the lung and head and neck: The Checkmate-017 study revealed an improved overall survival (OS) and a favourable safety profile for nivolumab compared to docetaxel in patients with pre-treated squamous NSCLC [27]. The Keynote-407 study showed clinical significance of combined pembrolizumab treatment with chemotherapy in patients with metastatic squamous NSCLC [28]. In HNSCC the Keynote-012 study demonstrated clinically significant activity in patients with pre-treated tumors for pembrolizumab irrespective of human papillomavirus (HPV) status [29].

However, different PD-L1 antibodies, associated immunohistochemical assays and scoring algorithms, still challenge a robust selection of patients who will benefit from ICI treatment. In line with previous studies in urothelial cancer [10, 12], we also confirmed a substantial inter-assay heterogeneity of PD-L1 expression in squamous bladder cancer. Scheel et al. reported, that the four PD-L1 assays do not show comparable staining patterns in NSCLC [11]. The Blueprint PD-L1 Immunohistochemistry (IHC) Assay Comparison Project also studied the performance of the four PD-L1 IHC assays (22C3, 28–8, SP142, and SP263) in NSCLC, and found -very similar to our results- an analytical comparability of 22C3, 28–8, and SP263 whereas the SP142 assay showed lowest levels of correlation [30]. Hirsch and colleagues concluded that despite similar analytical performance of PD-L1 expression, interchanging assays and cut-offs would lead to “misclassification” of PD-L1 status for a substantial amount of patients. As a consequence, different patient numbers would be eligible for first line therapy with PD-L1 checkpoint inhibitors but still without clear evidence which staining results and cut-off levels really predict therapy response. For urothelial bladder cancer, the clinical consequences with substantial amounts of discordant classifications due to inter-assay and especially inter-algorithm variability, i.e. nearly 50% discordances between eligibility for first line treatment with atezolizumab or pembrolizumab, have been reported previously [10]. In clinical trials, for instance, the objective response rate (ORR) of urothelial cancer patients with nivolumab treatment did not significantly differ between PD-L1 positive (> 1%) and PD-L1 negative tumors (< 1%) (Checkmate-032 study) [31]. Beyond that the reliability of PD-L1 assays to predict ICI response is reduced by various aspects such as non-immunity dependent upregulation of PD-L1 expression (e.g. via PTEN) [32] or intratumoral heterogeneity and dynamic alteration by treatment and cancer progression [24]. In turn, higher ORR have been shown to be associated with increased PD-L1 expression also in urothelial cancer [33]. In this Keynote phase 2 study the subgroup of bladder cancer patients with PD-L1 expression above a cut-off ≥10% showed highest ORR upon pembrolizumab treatment. PD-L1 expression, as revealed in our cohort of squamous bladder cancers, may thereof be suitable to facilitate patient selection for ICI therapies.

This notion is supported by the here presented index patient with a squamous bladder cancer demonstrating partial therapy success upon ICI treatment. Prior to nivolumab treatment, the primary tumour exhibited a substantial percentage of squamous differentiation (80%) with strong PD-L1 expression. Upon ICI treatment the primary tumour showed clinically only short response but thereafter local progress was observed. Interestingly, after nivolumab therapy completion the progressive tumour lesion (< 50% squamous component) was characterized by reduced PD-L1 positivity. Of clinical significance, the pulmonary metastasis showed long-lasting response without any evidence of harmful side effects.

Our data reveal strong PD-L1 expression in squamous differentiated bladder cancers comparable with urothelial cancer whose disease management has been successfully improved by ICI therapy. Considering the encouraging clinical data of our index patient we propose to consider treatment of ICI also for both mixed and pure SCC of the urinary bladder. However, according to the tumour and inter-assay heterogeneity of PD-L1 expression, the utility of given scoring algorithms for robust patients’ therapy selection remains questionable and should be considered in future study designs.