Introduction
Cancer arising from the urothelium of the bladder is estimated to be the fourth most common malignancy in American men, with an estimated 80,000 new cases in 2017 in the USA [
1]. Most bladder carcinomas present as non-muscle-invasive, low-grade papillary carcinomas and are associated with an excellent prognosis. These tumors recur frequently but rarely progress to muscle-invasive disease. In contrast, muscle-invasive bladder cancer (MIBC) is associated with an unfavorable prognosis (5-year survival < 50%) due to a high risk of local and systemic disease progression [
2]. Most MIBCs arise from carcinoma in situ (CIS), a flat, high-grade, superficial urothelial lesion that is characterized by
TP53 mutations and accounts for approximately 10% of all diagnosed bladder tumors [
3]. CIS is considered to be an obligate precancerous lesion, while concomitant CIS is associated with a markedly worse prognosis in patients with bladder cancer, a fact that is mirrored in multiple clinical risk scores (Spanish Urological Club for Oncological Treatment (CUETO), European Organization for Research and Treatment of Cancer (EORTC)) [
4].
Therapeutic options for urothelial CIS are limited and based on a moderate to low level of evidence. To inhibit disease progression and recurrence, current guidelines recommend transurethral resection, followed by intravesical instillation of Bacillus Calmette-Guérin (BCG) as first-line therapy [
5,
6]. BCG instillation, however, has numerous side effects, and a high rate of tumors persist or recur irrespective of treatment [
7]. In such cases, a radical cystectomy is usually performed, a procedure with high surgical morbidity [
8]. Therefore, the need for new and efficient local therapies as well as reliable prognostic and predictive markers are of upmost clinical importance.
A recent stratification of bladder cancer into two main molecular subtypes with prognostic significance—“luminal” and “basal”—has furthered our understanding of urothelial carcinoma biology [
9‐
12]. Luminal tumors are characterized by the same markers as the differentiated layer of the urothelium, while basal tumors are enriched with squamous markers like CK5 and CK14, typical for undifferentiated basal cells [
13]. In MIBC, luminal tumors have a favorable prognosis, while basal carcinomas are associated with advanced cancer stages at diagnosis, with metastatic disease and shorter overall survival [
9‐
12]. Intriguingly, molecular stratification of a large, heterogeneous cohort of early high-grade bladder cancer revealed an inverse correlation between prognosis and molecular subtype, defining the luminal subgroup as the more aggressive one in non-muscle-invasive bladder cancer (NMIBC) [
14]. However, only three cases of CIS were included in this study and it remains unclear whether this classification is also applicable in CIS.
Aiming to investigate the role of the recently discovered molecular subtypes of bladder cancer in urothelial CIS, we analyzed luminal and basal marker expression using an established surrogate immunohistochemistry (IHC) panel comprising the luminal markers CK20, GATA3, human epidermal growth factor receptor type 2 (Her2), and estrogen receptor (ER) β as well as the basal markers CK5/6 and CK14 in 156 CIS tissue samples from 132 patients. Furthermore, we compared protein expression of these markers in CIS and corresponding invasive tumor parts of the same patient, in an attempt to better understand the stability of a potential subgroup affiliation in the process of stromal invasion.
Moreover, we included two predictive markers and potential treatment targets in our IHC panel, ERβ and Her2, aiming to evaluate new therapeutic options for urothelial CIS. The significance of ERβ signaling in urothelial cancer proliferation and the feasibility of ER targeting therapies have been recognized in numerous studies [
15‐
17]. Anti-Her2 therapies are established treatment options in cases of
Her2-amplified breast and gastric cancer and have been investigated and proposed as a therapeutic strategy for MIBC [
18‐
20]. To clarify the molecular background of Her2 expression in urothelial CIS, we performed fluorescence in situ hybridization (FISH) in a large fraction of our CIS cohort. We aimed to examine whether protein expression of Her2 and ERβ may yield conclusive findings on potential therapeutic targets for urothelial CIS.
Discussion
In this study, we aimed to elaborate on the affiliation of urothelial CIS to a specific molecular subgroup and to clarify whether molecular subtyping of CIS may also be suitable for prognostic stratification. In this regard, we investigated the immunoreactivity of proteins characteristically expressed in the two intrinsic molecular subsets of bladder cancer [
10] and observed that the majority of 156 CIS cases were characterized by the expression of luminal markers and the absence of basal protein expression. In a cohort of 48 patients, we further investigated whether luminal and basal markers remain stable in the course of progression from CIS to invasive disease. Remarkably, we observed that while urothelial CIS strongly and consistently expressed luminal markers, affiliating it to the luminal subtype, this was not the case in the corresponding invasive cells. A significant loss of luminal and a gain of basal marker expression were detected in the invasive compartment.
Data supporting our observation can be extrapolated from recent molecular studies. A strong CIS signature gene expression was found mainly in the basal subgroup of MIBC in the 2017 TCGA dataset, suggesting that these basal tumors evolved from CIS lesions [
27]. It was furthermore observed that MIBCs harboring multiple
TP53 and
RB1 pathway alterations, which are also characteristic for the CIS pathway, are found predominantly in the basal subgroup [
10,
13].
The hypothesis of CIS as a precursor lesion of basal muscle-invasive tumors is substantiated by the observation that CIS cells parallel the biology of basal urothelial stem cells. Since urothelial stem cells are capable of sustaining long-term growth and proliferation, it is probable that their longevity makes them the most susceptible to the accumulation of oncogenic hits and therefore multistep carcinogenesis [
28].
At the same time, molecular evidence underlines the affiliation of CIS to a luminal subgroup. Hedegaard et al. conducted expression profiling of NMIBC, discovering that cases overexpressing the above-mentioned CIS gene signature belonged to the luminal-like group (“class 2”), with high levels of luminal (CK20) and low levels of basal (CK5) markers. As the study only included three CIS cases, we consider our significantly larger cohort a possible endorsement of these data [
14]. The underlying mechanisms causing CIS cells to consistently express luminal markers, as well as the stimuli inducing the basal protein expression in invasive cells of CIS origin, remain to be explored.
Only minor advances towards more efficient and targeted intravesical therapies for urothelial CIS have been made in the last decades [
29]. At the time of initial diagnosis, an organ-sparing approach of transurethral resection and BCG instillation is usually chosen [
30]. The latter in particular is associated with severe morbidity and a high probability of so-called BCG failure, including disease progression and recurrence [
31]. The risk of tumor progression to a life-threatening muscle-invasive situation encourages clinicians to recommend radical cystectomy in patients with extensive or recurrent CIS. This procedure is on the one hand associated with an excellent tumor-specific survival [
32], but on the other hand bears a significant risk of overtreatment and associated surgical morbidity [
8,
33]. Clinical and preclinical work on new therapeutic targets in bladder cancer has mainly focused on MIBC [
34], and few authors have examined new therapeutic targets in urothelial CIS [
29].
Two markers from our luminal panel, Her2 and ER, are well-established predictive markers and therapeutic targets in other tumor entities [
35,
36]. We aimed to evaluate the therapeutic potential of Her2 and ERβ in urothelial CIS by assessing protein expression of both markers, as well as the molecular background of Her2 expression by FISH analysis.
Two ERs exist in the human body, ERα and β, which mediate ligand-dependent transactivation of gene expression [
37]. Not only is ERβ the predominant ER in the bladder with a potential prognostic role in NMIBC, but it is also postulated to promote cancer growth and progression [
16]. Multiple studies have investigated ERβ protein expression in both normal urothelium as well as urothelial carcinoma [
16,
17,
38]; however, to the best of our knowledge, none of these included CIS. Our analyses showed ERβ positivity in 88% (138/156) of CIS cases.
ERβ has previously been identified as a druggable target in bladder cancer using the selective ER modulator Raloxifene or ERβ-specific antagonists such as PHTPP [
16,
35]. Furthermore, current evidence suggests that ERβ plays a central role in the proliferative and invasive potential of bladder cancer cells and therefore may present a promising target for selective ERβ inhibitors [
39]. As such, the observed ERβ positivity in the majority of CIS cases in our cohort warrants further investigation of ERβ as a potential target in early, flat, high-grade bladder cancer.
The other marker with therapeutic potential included in our IHC panel is Her2, a member of the epidermal growth factor receptor family, which, when overexpressed, enhances proliferation, cell survival, and the invasive capacity of tumor cells [
40,
41]. Anti-Her2 therapy has emerged as the mainstay of treatment in Her2-amplified breast and gastric cancer [
18,
20]. Conflicting findings have been reported on the rate and the mechanism of Her2 overexpression in MIBC, which, after breast and gastric cancer, shows the highest rate of
Her2 amplification [
13]. A recent investigation of the mechanism of Her2 expression in MIBC showed that Her2 protein overexpression arises from various mechanisms, including gene amplification [
42]. Though Her2 immunostaining has previously been investigated as a diagnostic tool for urothelial CIS [
43], our study is the first to examine Her2 expression in a significantly larger patient cohort and the first to investigate the possible genetic background of Her2 expression in CIS. IHC characterized 52 cases (33%) as Her2 moderate (2+) and 50 cases (32%) as Her2 positive (3+).
FISH analysis detected only a low rate of
Her2 amplifications in 8/126 cases (6%). At the same time, we observed polysomy 17, indicated by an elevated CEP17 count, in 37/126 (29%) of cases. We observed a significantly higher Her2 expression in cases with polysomy 17 or Her2 amplification, compared to CIS cases without these alterations. Chromosome 17 polysomy has been previously described as one of the mechanisms driving Her2 expression in breast cancer, especially in tumors with IHC 2+ scores [
44]. Interestingly, various studies support the effectivity of trastuzumab, a humanized anti-HER2 monoclonal antibody, in polysomic, non-amplified breast cancer [
45,
46].
Although none are currently in clinical use for bladder cancer, there is accumulating evidence that anti-Her2-targeted therapies are promising novel treatment strategies in urothelial carcinoma [
42]. As the driver status of Her2, even in Her2-overexpressing or Her2-amplified bladder cancer cases, is difficult to predict without detailed genome-wide analyses [
42], we propose the use of agents whose impact is independent of Her2-signaling inhibition. Targeting Her2-overexpressing cells via antibody-mediated cytotoxicity of clinically established monoclonal antibodies like trastuzumab or the cytotoxic effects of the emerging antibody-drug conjugate trastuzumab-DM1 (T-DM1) may be a viable option [
36,
47]. We see particular potential for clinical studies investigating the response rate of an intravesical combination therapy of BCG and T-DM1. Furthermore, the large number of patients with BCG-refractory CIS may benefit from a Her2 targeting, which would provide a bladder-sparing approach for patients otherwise subjected to cystectomy.
We are aware of the limitations of this retrospective study. Firstly, the observations from this study need to be validated in independent patient cohorts, possibly with an expansion to other superficial urothelial cancers, such as non-invasive papillary high-grade tumors. Secondly, further molecular studies are needed to explain the phenomenon of the observed marker switch from luminal to basal in the course of invasion. Thirdly, the possible targets identified in this study, ERβ and Her2, belong to the luminal marker panel, which is downregulated in the process of invasion, possibly limiting the suitability of ERβ and Her2 targeting to CIS and should encourage combined therapy modalities.
None withstanding the aforementioned limitations, we have for the first time reported on a shift from luminal to basal marker expression in urothelial carcinoma and distinguished two potential therapeutic targets in CIS. In summary, positivity for either ERβ, Her2, or both proteins was observed in 91% (142/156) of CIS cases, while normal urothelium showed significantly lower expression of both markers, highlighting their potential for clinical use. The protein targets identified in this study, Her2 and ERβ, may be amenable to targeted intravesical therapies in early-stage, high-grade bladder cancer. As mentioned above, validation in an independent cohort of patients and clinical trials are required to confirm our preliminary findings.