Intratumoral heterogeneity of surrogate molecular subtypes in urothelial carcinoma in situ of the urinary bladder: implications for prognostic stratification of high-risk non-muscle-invasive bladder cancer

Bladder cancer (BC) is the most common malignancy of the urinary tract: an estimated number of 549,000 new cases and 200,000 deaths were registered in 2018 worldwide [1]. Muscle-invasive bladder cancer (MIBC) has an unfavorable prognosis (5-year survival <50%) and mainly develops from carcinoma in situ (CIS), a flat-growing, high-grade (HG) lesion characterized by frequent TP53 alterations [2].

Based on the current treatment guidelines by the European Association of Urology (EAU) and the American Urological Association (AUA) [3, 4], first-line intravesical bacillus Calmette-Guérin (BCG)-based immunotherapy following transurethral resection (TUR) of the tumor is recommended for treatment of high-risk (HR) non-MIBC (NMIBC), including patients exhibiting CIS lesions. However, BCG therapy fails in a substantial number of patients [5] caused by cessation of the therapy due to resistance or toxicity [6, 7]. Radical cystectomy (RC) is recommended as second-line treatment in case of BCG failure [3, 4]. Although associated with an excellent tumor-specific survival [8], RC is a morbid surgery significantly impacting quality of life and thus not all patients are eligible or refuse RC [7]. Moreover, the RC-related rate of overtreatment is assumed to be high [5] due to a lack of reliable prognostic markers [3]. Obviously, there is a need for both, alternative bladder-preserving therapies and reliable prognostic markers allowing the identification of HR NMIBC patients with worse prognosis that might benefit from timely RC and those profiting most from conservative treatment.

Extensive molecular characterization of MIBC has led to the identification of different subtypes with divergent clinical outcomes. In general, a major luminal subgroup associated with favorable and a basal subtype exhibiting worse survival was discovered [9, 10]. Importantly, immunohistochemistry-based subgrouping by surrogate markers might allow feasible implementation of subtype stratification into clinical routine [11]. Regarding non-muscle-invasive urothelial cancer, preliminary data indicate that especially luminal-like urothelial lesions (of the upper tract and the urinary bladder) might be related with worse prognosis compared to those with basal phenotype [12, 13].

With regard to the lack of reliable prognostic stratification markers for HR NMIBC patients presenting with aggressive CIS lesions [3], we sought to analyze the potential prognostic impact of both clinico-pathological parameters including CIS focality and additionally immunohistochemistry-based surrogate subtypes of bladder cancer in a cohort of HR NMIBC patients with CIS. To the best of our knowledge, this is the first study analyzing the potential prognostic value of the recently described molecular subtypes, defined by immunohistochemical surrogate markers, in CIS lesions of HR NMIBC patients.

Currently, no reliable prognostic factors are available to predict the disease course of CIS patients [3]. Thus, there is an unmet need to identify markers allowing stratification of CIS patients into those cases benefiting most from early radical cystectomy and those profiting from conservative treatment.

Existing prognostic scores and risk tables are based on data from studies that were not specifically focused on CIS patients [21, 23, 24]. The European Organisation for Research and Treatment of Cancer (EORTC) scoring system for instance was developed on the basis of survival data from NMIBC patients mostly without CIS, and predominantly treated by chemotherapy [23]. The basis of the CUETO (Club Urologico Espanol de Tratamiento) model are data from NMIBC patients treated by suboptimal BCG therapy and again, patients with CIS only represented a small fraction (10% of patients) [21]. In a more recent study, potential prognostic factors in intermediate and high-risk NMIBC patients treated with 1–3 years maintenance BCG were investigated, however, without inclusion of CIS patients [24]. Indeed, several smaller studies analyzing the prognostic value of different factors specifically in CIS patients, including established clinico-pathological parameters, have been performed. For instance, exhibiting concurrent CIS and T1 lesions compared to primary CIS [25], having an extended CIS [26] and CIS localized in the prostatic urethra [27] have been associated with worse patient outcome. It may be emphasized here that all studies are limited to mere histological diagnosis of CIS as a variable, without any further analysis of the lesional biology.

Molecular subtypes have been described previously in MIBC, resembling those found in breast cancer patients [10, 20]. Importantly, these subtypes seem promising with regard to prognostic stratification of MIBC patients, with basal tumors being potentially associated with a more aggressive behavior in comparison to luminal cancers while putatively responding better to chemotherapy than luminal carcinomas [10, 20].

Far less is known about the impact of these molecular subtypes in NMIBC patients. In contrast to MIBC, first data in NMIBC and non-muscle-invasive urothelial cancer of the upper urinary tract indicate that especially luminal-like tumors are associated with an unfavorable outcome [12, 13, 28‐30]. The use of a minimal set of immunohistochemical markers (including KRT20 and KRT5/6) has been demonstrated to be a feasible and reliable approach to reflect intrinsic molecular subtypes (at least) in MIBC samples [11].

In a previous study without clinical follow-up, we have shown that isolated CIS lesions are characterized by the expression of luminal markers including KRT20 and GATA3, whereas lacking the expression of the basal cytokeratins KRT5/6 and KRT14 in the majority of samples [14]. In the current study, we were able to validate our previous observations in a large, independent cohort of HR NMIBC patients with CIS. In addition to our previous analyses, we considered the inter-lesional CIS-heterogeneity of marker expression in CIS patients by analyzing marker expression in different CIS localizations in the same urinary bladders and observed a significant degree of heterogeneity. Using KRT20 and KRT5/6 as luminal and basal surrogate marker respectively, CIS patients were categorized into the three major groups “luminal,” “null,” and “mixed,” while the latter comprised different subgroups due to heterogenous KRT20 and KRT5/6 expression in different CIS localizations. Of note, we did not observe a clear “basal” phenotype in our CIS cohort (KRT20 negative, KRT5/6 positive). In contrast to two recent studies in NMIBC [13, 29], we did not observe a significant association of surrogate CIS subtype with patient outcome. This observation remained stable when using additional cutoffs for KRT20 and KRT5/6 positivity (30% and 80% respectively) (data not shown). These putatively conflicting observations might be explained by significant differences between these studies: First, using RNA data, Breyer et al. focused on surrogate subtypes solely in T1 tumors of NMIBC patients [13]. The second study included stage Ta and T1 NMIBC patients, with a substantial fraction of LG tumors (65%), analyzing surrogate subtypes in Ta and T1 samples [29]. Second, neither of the aforementioned studies considered potential intratumoral heterogeneity (ITH) in Ta and T1 tumors investigated, even though known to be relevant in bladder tumors including heterogeneity with regard to molecular subtypes [31, 32]. While intra-lesional heterogeneity in bladder cancer of different stages and grade, except for CIS, has been studied before [33] it has remained unclear thus far if surrogate subtypes differ when considering distinct tumor localizations present in the same urinary bladder and neither is validated which tumor locus is prognostically informative with regard to molecular subtypes in case of heterogeneity. Data from our previous work suggest that there might be considerable ITH in surrogate molecular subtypes, as a switch from a luminal-like to a more basal-like phenotype was observed during the course of stroma-invasion of CIS lesions [14]. We hypothesize that the surrogate molecular subtype of the highest stage and grade lesion in NMIBC might be prognostically informative and this hypothesis will be analyzed in upcoming studies.

Bladder cancer frequently presents as a multifocal disease, potentially representing different tumor clones of a monoclonal origin [31, 34]. In the current study cohort, 59% and 76% of patients had multifocal CIS lesions and multifocal HG tumors (including papillary HG and pT1 lesions), respectively. It was hypothesized that CIS/HG multifocality is related to worse outcome compared to patients with unifocal CIS/HG tumors. This hypothesis is based on the assumption that a more diverse tumor is able to adapt more efficiently to changing environmental conditions resulting in faster tumor (re-)growth and progression [35]. To our knowledge, the potential prognostic significance of CIS/HG focality in CIS patients is unclear and understudied to date. Surprisingly, neither CIS focality nor HG tumor focality in general were significantly associated with survival in the present cohort. This observation is an accordance with a smaller previous study noting that the extent of CIS is not predictive of recurrence or progression [36]. In contrast, Takenaka and colleagues observed a worse PFS in CIS patients with extended CIS [26].

Additionally, we analyzed the prognostic potential of a larger set of established clinico-pathological factors. Higher patient age was identified as a potential independent prognostic parameter of an unfavorable recurrence-free (RFS), progression-free (PFS) and overall survival (OS) of HR NMIBC patients with CIS. Due to a limited number of events, multivariate analysis for urothelial cancer-specific survival has not been performed. However, a significant association of higher age with worse urothelial cancer-specific survival has been noted in univariate analysis as well. An association of increasing age with worse RFS, PFS, OS, and bladder cancer-specific survival has been observed previously in studies considering NMIBC including patients with CIS [21, 22]. Concerning studies specifically focusing on CIS patients, conflicting observations have been made [26, 37]. Moreover, we noted that current smokers exhibited a significantly worse PFS compared to never smokers and former smokers. Interestingly, no significant difference in PFS was observed between never and former smokers. The value of smoking status as a prognostic stratification marker in non-muscle-invasive bladder is controversial [38, 39]. Focusing specifically on patients with CIS, smoking status has to our knowledge so far not been identified as an independent prognostic factor for PFS and needs further validation [37, 40].

The current study is limited by its retrospective character. Even though the study includes a high number of cases with CIS related to its overall frequency in the bladder, we are aware of case numbers being still fairly small for the observations stated. Even though an obvious degree of inter-lesional CIS heterogeneity with regard to surrogate molecular subtypes was identified in this study, we are aware that the use of tissue microarrays instead of whole tissue slides is a limitation.

In summary, we observed that only patient age and smoking status information were independently associated with outcome of HR NMIBC with CIS. Neither the surrogate molecular subtype of CIS lesions in HR NMIBC patients, nor CIS/HG tumor focality were significantly related to prognosis. Importantly, we identified a considerable degree of inter-lesional CIS heterogeneity with regard to surrogate molecular subtypes and conclude that further clarification of potential heterogeneity in HR NMIBC patients is of high value with regard to potential implementation of molecular subtyping into clinical routine. Moreover, due to the putative transient/heterogenous nature of expression-based molecular subtypes in NMIBC, the prognostic impact of mutational profiles should be considered in upcoming studies.