Urothelial carcinoma (UCa) represents the most common form of bladder cancer in the United States (>90%) and is characterized by frequent mutations in TP53, RB and PTEN[1]. Morphologically, UCa consists of invasive nests of carcinoma cells with variable atypia and frequent surrounding retraction artifact (Figure 1A), although this appearance can vary significantly. Less common forms of bladder cancer in the United States include squamous cell carcinoma (SCCa; Figure 1B), adenocarcinoma and small cell carcinoma, which are defined as pure morphologic entities that lack a typical urothelial component [2]. Our understanding of the molecular relationships between these other forms of bladder cancer that arise from the urothelial lining has been limited and may be based on the low number of cases available for study and/or lack of significant attention paid to this topic. It appears, however, that despite a similar origin from the surface urothelium, these various forms of bladder cancer show differential clinical behavior, morphologic appearances, immunohistochemical markers and response to chemotherapy.

SCCa represents the second most common form of bladder cancer in the United States (5-8% of bladder cancer cases) and is distinguished by invasive squamous carcinoma cells containing desmosomes and often keratin formation [3]. A higher incidence of bladder SCCa has been reported in parts of the Middle East and Africa, however, where chronic infection with the water-bourne parasite Schistosoma haematobium, represents a major risk factor in the development of this disease [4]. Other potential risk factors in the development of SCCa include long-term catheterization, calculi that form in the urinary tract, and a nonfunctioning bladder, amongst others, and these risk factors may account for many instances of SCCa identified in the United States and Europe.

Despite the distinct appearance of UCa and SCCa, the cellular origin of these two forms of bladder cancer has been debated. Whereas UCa can arise in association with surface high-grade changes of the urothelium (e.g., carcinoma in situ) and SCCa can arise in association with squamous dysplasia and squamous carcinoma in situ of the bladder, there are numerous cases that show overlap of surface changes. For example, SCCa can occur in the absence of any surface squamous metaplasia and may only be associated with urothelial carcinoma in situ [3]. Furthermore, UCa itself has been shown to demonstrate “divergent” differentiation with the ability of UCa tumor cells to develop squamous or glandular features. These findings suggest that 1) the molecular relationship between historically distinct subtypes of bladder cancer may be more similar than previously hypothesized and 2) the surface urothelium in a bladder undergoing neoplastic alterations may be unusually suited to give rise to divergent phenotypes in the setting of both in situ and invasive disease.

To date, much of the molecular analysis on bladder SCCa has been limited due to a predominant focus on infectious, Schistosomal-derived cases as well as use of immortalized bladder cancer cell lines in a number of studies [5–8]. In this setting, the understanding of the relationship between UCa and SCCa, as well as the distinction between primary molecular changes and those secondarily induced by infection-specific responses, becomes difficult. We sought to compare the two most common forms of pure bladder cancer in the US (UCa and non-infectious SCCa) using multi-level gene expression analysis to determine the relationship and possible hierarchy of these two examples of bladder cancer. The results from our study suggest a closer relationship between these neoplastic entities than previously proposed; a shared evolution of these cancers may represent an opportunity for targeting bladder cancer along common pathways early in the disease process.

Current pathological classification distinguishes UCa and SCCa as distinct diagnostic entities [4]. This has resulted in numerous publications that have evaluated the differences in clinical outcomes, treatment response and molecular profiles that distinguish these two bladder cancer types, with mixed results [29]. Although some studies have suggested that when compared stage-for-stage, the outcomes are similar for patients with bladder UCa and SCCa [3, 29, 30], other studies have implicated that SCCa and/or UCa with squamous differentiation may present at a higher stage and behave more aggressively [31–33]. One consistency amongst studies is the limited response of bladder SCCa of the bladder to conventional chemotherapy and/or radiation therapy that is administered in the setting of UCa and may relate to the squamous phenotype [34, 35]. To date, however, the relationship between these two forms of bladder cancer that arise from the urothelial lining of the bladder has not been clearly delineated.

The results from our study suggest that UCa generally shares the majority of its dysregulated genes relative to normal urothelium in common with SCCa, with very few uniquely dysregulated genes; in contrast, SCCa – while sharing many genes in common with UCa – shows a much larger category of dysregulated genes that are often in common with SCCa arising at other sites. When considering the relationship between these two closely related entities, two possibilities emerge. First, invasive UCa may represent a default pathway of bladder cancer development, with clonal change resulting in SCCa development and overgrowth of a pre-existent UCa. This hypothesis is supported by the not infrequent finding of mixed morphology bladder cancers, where a well-documented UCa contains areas of squamous and/or glandular differentiation [4]. Further supporting this hypothesis is a prior paper that has examined the relationship of co-existent small cell carcinoma and UCa of the bladder: the results from this prior study suggest that the small cell carcinoma in this setting represented a clonal outgrowth from the background invasive UCa [36] a finding that might not be dissimilar across all other bladder cancer “subtypes” and which can be supported by the findings in this paper. A second possibility is that an early bladder cancer stem cell exists, either prior to invasion or early in the course of invasion, which gives rise to distinct morphological entities along discrete molecular lineages that are considered pure subtypes. Specifically, early molecular changes define a number of shared alterations between various bladder cancer subtypes that subsequently diverge along different morphologic lines [36]. In such a scenario, the limited number of additional alterations identified in UCa would suggest this to be a “default” pathway in bladder carcinogenesis, with significant additional alterations required to develop the squamous phenotype.

Regardless of the model proposed, the current data supports a close evolution between UCa and SCCa, with gene expression changes in the latter primarily reflecting morphological correlates of the squamous phenotype seen in SCCa arising from different sites. Our data also suggest that proliferative changes, including deregulation (and in some cases amplification) of mitotic spindle checkpoint components may be critical in the early stages of bladder tumorigenesis. Further validation of our findings using other “pure” types of bladder cancer – such as adenocarcinoma and small cell carcinoma – will further strengthen the implications of our results, although the rare nature of these other forms of bladder cancer may make such a study challenging.

Although we have used only one technique to analyze the relationship between UCa and SCCa, our ability to reproducibly segregate the entities in our study using both supervised and unsupervised clustering analysis suggest that our data is robust. A second limitation is the use of a limited number of specimens for analysis, although the use of 10 SCCa samples is relatively high given the rarity of this disease entity. The overall gene expression profiles between our two bladder cancer entities suggest that the development of these bladder cancer forms occurs along similar lines. However, it is clear that the magnitude of expression changes differs in some instances; for example, reduction in ADH1B occurs by a factor of 40-fold in UCa and 70-fold in SCCa. The importance of relative fold change (versus directionality) in these cancer subtypes was not a primary focus of investigation in this study. However, the relative increase or decrease in mRNA expression may have a relevant biological role when studied in a whole cell system.

Our study has identified numerous categories of genes that may be of relevance to the development of UCa, including mitotic spindle regulators, putative stem cell factors, nicotine metabolizing enzymes and inflammatory regulators. The vast majority of these genes appear to be similarly dysregulated in bladder SCCa. One exception is a large category of inflammatory mediators (POU2AF1, IL33, IL6, ITK, CCL19) that are altered and may reflect the administration of BCG, which is frequently given intravesically for superficial UCa but not SCCa. In contrast, SCCa appears to overlap significantly in gene expression differences with UCa and additionally contains a large number of additional up- and down-regulated gene products. Perhaps not surprisingly, many of these gene transcripts have been reported in SCCa from the head and neck region, oral cavity, lung and skin. As SCCa is considered to have limited response to therapies conventionally employed for UCa, the broad list of discrete targets – some of which are currently undergoing clinical trials for targeted therapy in other forms of SCCa – may provide an alternative treatment for patients with either pure or mixed SCCa of the bladder.

In summary, we found that UCa and SCCa of the bladder share a number of differentially regulated genes, suggesting a close evolution of these two major subtypes of bladder cancer. Future studies that seek to further delineate the relationship and, thus, pathogenesis of various forms of bladder cancer will provide additional insight into the development of bladder cancer. Ultimately, the finding of shared molecular changes may allow investigators to develop targeted therapy that may be used either earlier in the course of disease or treat a broader range of cancer morphologies with success.