Challenges in the Diagnosis of Urothelial Carcinoma Variants: Can Emerging Molecular Data Complement Pathology Review?

Accepted Manuscript Title: Challenges in the Diagnosis of Urothelial Carcinoma Variants: Can Emerging Molecular Data Complement Pathology Review? Author: James P. Solomon, Brett M. Lowenthal, A. Karim Kader, J. Kellogg Parsons, Thomas W. Flaig, Arlene O. Siefker-Radtke, Lars Dyrskjøt, Donna E. Hansel PII: DOI: Reference:

S0090-4295(16)30721-X http://dx.doi.org/doi: 10.1016/j.urology.2016.10.014 URL 20082

To appear in:

Urology

Received date: Accepted date:

12-9-2016 7-10-2016

Please cite this article as: James P. Solomon, Brett M. Lowenthal, A. Karim Kader, J. Kellogg Parsons, Thomas W. Flaig, Arlene O. Siefker-Radtke, Lars Dyrskjøt, Donna E. Hansel, Challenges in the Diagnosis of Urothelial Carcinoma Variants: Can Emerging Molecular Data Complement Pathology Review?, Urology (2016), http://dx.doi.org/doi: 10.1016/j.urology.2016.10.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Challenges in the Diagnosis of Urothelial Carcinoma Variants: Can Emerging Molecular Data Complement Pathology Review? James P. Solomon MD PhD1, Brett M. Lowenthal MD1, A. Karim Kader MD PhD2, J. Kellogg Parsons MD MHS2, Thomas W. Flaig MD3, Arlene O. Siefker-Radtke MD4, Lars Dyrskjøt PhD5, and Donna E. Hansel MD PhD1,2,*

1. Department of Pathology, University of California San Diego, San Diego, California 2. Department of Urology, University of California San Diego, San Diego, California 3. Associate Dean for Clinical Research, University of Colorado School of Medicine, Aurora, Colorado 4. Department of Genitourinary Medical Oncology, U.T. M.D. Anderson Cancer Center, Houston, Texas 5. Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark

*Correspondence to: Donna E. Hansel, MD PhD Professor of Pathology Chief, Division of Anatomic Pathology Department of Pathology University of California, San Diego 9500 Gilman Drive, MC 0612 La Jolla, CA 92093 Research Phone: 858-534-1716 Clinical Phone: 619-543-5764 Fax: 619-543-5249 Email: [email protected]; [email protected]

Keywords: Urinary bladder; urinary bladder neoplasms; bladder cancer; pathology; diagnosis, differential; prognosis

Abstract Urothelial carcinoma can exhibit a wide variety of histopathological phenotypes or variant morphologies, classifications of which have recently been revised in the 2016 WHO Classification of Tumours of the Urinary System and Male Genital Organs. Many of these variants not only present diagnostic challenges, but also have clinical implications that affect patient prognosis and treatment strategies. This review will discuss these variant morphologies and their relationship to current understanding of the underlying biology of urothelial carcinoma and molecular classification paradigms.

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Introduction Urothelial carcinoma (UC) has a proclivity for exhibiting distinctive histopathological phenotypes, or “variants”, that likely reflect underlying diversity in molecular composition. Recognition and improved clinical understanding of these variants has impacted therapeutic approaches for a subset of bladder cancer patients and informed our understanding of how distinct variant categories can affect patient prognosis. Their correct identification thus has important clinical implications, since misdiagnosis may result in improper or unnecessarily aggressive treatment. Despite morphological criteria being described for many of these variants, the diagnosis may be somewhat subjective and challenging. Literature from several academic institutions shows that up to 30% of bladder cancer diagnoses may be changed upon re-review by an expert genitourinary pathologist, with the presence of variant morphologies being a major source of discrepancy.[1] Many variants may be under-recognized or misclassified due to evolving criteria for diagnostic inclusion, high interobserver variability, lack of ancillary tests to confirm variant diagnosis, and sampling limitations.[1] In a subset of patients, multiple variants may occur concurrently and at differing proportions, with some suggesting that the amount of each variant be reported as a percentage of the total lesion.[2] Molecular classification of bladder tumors using genomic platforms is increasing in prevalence. The new 2016 WHO Classification of Tumours of the Urinary System and Male Genital Organs revised several categories of UC variants and also incorporated emerging molecular data for many of these entities (Table 1).[3] Genomic characterization of UC has identified a high rate of somatic mutation, but understanding of the clinical implications, including recognition of the key driver mutations and clinically relevant gene alterations, is still evolving.[4] Moreover, the relationship of these alterations to histopathological variants is often unclear. While several molecular classifications have been proposed, many have either directly excluded variant morphologies or not analyzed them when present.[5]

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Although this may enhance the generalizability of data to the more prevalent conventional UC, it has in many ways limited understanding of variants that emerge in the context of UC. Despite this potential limitation, several studies have taken a targeted approach to identify and further characterize molecular alterations in a subset of UC variants. Here, we describe recent changes in the diagnosis of UC variants and highlight critical features of each that are relevant for the differential diagnosis, patient prognosis, and therapy. We discuss known molecular alterations and emphasize their potential role in generating a more objective classification schema for bladder cancer in the future. Non-urothelial bladder cancer subtypes— including primary bladder adenocarcinoma, urachal carcinoma, pure squamous cell carcinoma, and pure small cell carcinoma—are beyond the scope of this review.

Variants of Urothelial Carcinoma Urothelial carcinoma with divergent differentiation The most common UC variant is urothelial carcinoma with divergent differentiation, which includes squamous, glandular or trophoblastic differentiation occurring in a background of conventional UC (Fig. 1A-D). Squamous differentiation is seen in up to 40% of UC, although this percentage varies substantially by study.[6] This may be due in part to subjectivity in defining squamous features, with different criteria used by individual pathologists. For example, some require clear-cut keratinization for a diagnosis of squamous differentiation, whereas others may be satisfied with dense pink cytoplasm and the appearance of intercellular desmosomes. Another challenge specific to squamous differentiation is the distinction from pure “squamous cell carcinoma”, which has significant clinical implication as a pure squamous cell primary would be treated more aggressively. No specific markers exist to help with this distinction and the diagnosis is

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often dependent on the clinical history and the absence of a clear-cut conventional UC component prior to or contemporary with the carcinoma, although there can be some overlap in appearance.[6] Unfortunately, immunhistochemical stains for squamous and urothelial differentiation overlap in most cases, with both entities frequently being immunoreactive for cytokeratin 5/6 and p63 – findings that generally cluster squamous differentiation with the “basal” molecular subtype of UC, as discussed later.[7] Although human papillomavirus has been identified in a subset of these cases, it is generally not considered to be causative to the development of this variant or associated with the presence of viralrelated changes in the genital tract.[8] Glandular differentiation occurs in up to 18% of UC, although the percentage is variable in case series likely due to the subjectivity inherent in the diagnosis.[6] Many different “patterns” of glandular differentiation exist, including lesions morphologically similar to colonic adenocarcinoma, those that express mucin, or a variety of mixed types. While patients with squamous and/or glandular differentiation are more likely to present with advanced disease,[6, 9] stage-for-stage comparisons between squamous and glandular differentiation versus conventional UC have shown no significant survival differences.[9] Light microscopic identification of trophoblastic cells is rare and, when present, is identical to syncytiotrophoblasts present within choriocarcinoma. However, use of beta-human chorionic gonadotropin (-hCG) immunostaining can show trophoblastic differentiation in up to 35% of UC, which also correlates with higher pathologic stage.[10] Serum hCG elevation occurs commonly in patients with metastatic disease and inversely correlates with chemoresponsiveness.[11] Immunohistochemical stain and quantification of the extent of divergent differentiation is currently not recommended for final diagnosis, although some studies have suggested that extensive divergent differentiation may be associated with diminished therapeutic response.[12]

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Overall, most clinicians treat UC with divergent differentiation in the same manner as conventional UC, as prognosis is generally thought to be similar, and retrospective analysis of clinical trial data have supported this approach.[13] The inclusion of cases with syncytiotrophoblastic cells is a recent addition to the divergent differentiation category, and many sites have not historically included this as part of standard reporting.

Nested urothelial carcinoma The definition of nested variant of UC has been expanded to include the recently described large nested variant and UC with small tubules, which prior to now have been considered separate categories.[14] These entities share the feature of deceptively bland nests of invasive carcinoma that lack significant atypia (Fig. 2A), making diagnosis challenging. Given their unremarkable appearance, these entities may be erroneously misidentified as von Brunn nest proliferations, nephrogenic adenoma, or other benign processes on biopsy or transurethral resection (Fig. 2B). Conversely, florid benign processes may be concerning for nested UC. In many instances, nested UC may be flagged as an atypical proliferation multiple times until definitive invasion can be proven microscopically, which is often established upon clear-cut invasion into the detrusor muscle.[14] This variant has been associated with advanced tumor stage and nodal invasion,[14] which may reflect in part a bias of late diagnosis. Nested UC appears to be similar in immunohistochemical features and clinical outcomes to conventional UC, with no significant difference in recurrence rate or survival.[15] On occasion, marked nuclear overexpression of p53 may be demonstrated by immunostaining, which can help in the diagnosis.[14]

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Microcystic urothelial carcinoma Microcystic UC is another uncommon and bland form of invasive UC that is characterized by multiple small cystic structures up to 2 mm in diameter (Fig. 2C).[16] Again, because of the bland appearance, the major challenge in the differential diagnosis includes a number of benign entities that include cystitis cystica et glandularis or dilated von Brunn nests (Fig. 2D). In many cases, however, foci of conventional UC can be identified in the background, which can help formulate the correct diagnosis. Although data are limited, univariate survival analysis has shown no significant differences between microcystic variant and conventional UC. The association of this variant with worse outcomes may again be due to the bias of advanced stage at diagnosis due to the deceptively bland morphology.[16]

Micropapillary urothelial carcinoma While increasingly appreciated, the diagnosis of the micropapillary variant of UC remains marked by a high degree of inter-observer variability, a problematic issue since many clinicians advise early cystectomy.[17] While initial prevalence estimates varied between 0.7 and 2.2%, recent studies suggest a prevalence as high 8%, which may depend on the diagnostic cut-off used to identify this variant.[18] The “classic” form is characterized by small clusters of cells that lack true vascular cores present within prominent retraction spaces.[19] These cells show “reverse polarization” of basal and luminal sides of the cells by electron microscopy and MUC1 expression,[20] which may influence the lack of cohesion between tumor and stromal cells. The stromal retraction that is characteristic of this variant can also occur to varying degrees in conventional UC, and this overlap can present significant diagnostic challenges (Fig. 3A-D). It does appear, however, that higher percentages of the micropapillary component within an invasive UC correlate with poor prognosis and suboptimal response to chemotherapy.[21] Furthermore, when micropapillary features are more prominent and comprise a

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higher percentage of the tumor, the identification and diagnosis of this variant is more straightforward. One important caveat, however, is that the use of “micropapillary” terminology to describe non-invasive lesions is particularly problematic, since cases of “micropapillary carcinoma in situ” are exceedingly rare and not necessarily associated with worse outcomes compared to conventional non-invasive UC.[22] Therefore, caution is warranted in applying and interpreting a diagnosis of non-invasive or in situ micropapillary carcinoma, and additional discussion with the pathologist should be considered to avoid mismanagement in such instances. Some studies have observed that intravesical therapy is ineffective for micropapillary variant disease and suggested a lower threshold for cystectomy, even in T1 patients with bacillus CalmetteGuérin-responsive disease. However, other data suggest that a more standard bladder-sparing approach is reasonable in select non-muscle invasive patients.[23, 24] Although a retrospective report suggests that micropapillary tumors do not benefit from neoadjuvant chemotherapy, a prospective neoadjuvant trial reported efficacy with aggressive systemic chemotherapy.[25] Although micropapillary UC is associated with a higher rate of advanced disease than conventional UC, a stage-matched trial showed no significant difference in ten year survival after cystectomy.[26] Still, in the absence of data demonstrating comparable survival for bladder preservation compared to cystectomy in patients with T1 micropapillary variant disease, and given the higher likelihood of metastatic spread and advanced stage in many of these cancers, aggressive intervention may be warranted to improve outcomes. HER2 alterations occur at a much higher frequency in micropapillary UC, and ERBB2 gene amplification or other mutations are often seen.[27] Additionally, a recent study demonstrated that ERBB2 amplification is associated with worsened cancer-specific survival in patients with micropapillary UC following radical cystectomy.[28] Although there have been anecdotal reports of response to HER2-targeted therapy in this variant, definitive studies are lacking.

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Lymphoepithelioma-like urothelial carcinoma Lymphoepitheloma-like UC is named for its resemblance to lymphoepithelioma of the nasopharynx. In contrast to the latter, lymphoepithelioma-like UC is unrelated to Epstein-Barr virus infection.[29] This lesion is unusual in that the tumor cells may be masked by intense infiltration of polyclonal B- and T-cells,[30] and awareness of this entity is important in the differential diagnosis. Indeed, at low magnification, many of these lesions may mimic a solitary inflammatory nodule or lymphoma (Fig. 4A). Immunohistochemical stains for epithelial and urothelial markers can be used to highlight the cancer cells, which grow in a syncytial pattern and demonstrate large nuclei and prominent nucleoli (Fig. 4B). In the pure form, this variant appears to have a good prognosis with low metastatic potential, and patients may do well when treated with chemotherapy alone.[30] The good prognosis may be possibly related to the dense inherent inflammatory infiltrate, although this has not been tested experimentally.

Plasmacytoid urothelial carcinoma Plasmacytoid UC is another rare but aggressive variant of UC that is distinguished by the presence of discohesive, individual cells that resemble plasma cells (Fig. 4C,D).[5, 31] One recent change in the diagnosis of this category includes the allowance for cells that contain intracellular mucin, as these had previously been characterized as adenocarcinoma. The urothelial nature of this entity can be confirmed by immunostains for the urothelial markers CK7, p63 and uroplakin III. Patients typically present at an advanced stage. This variant appears to be chemotherapy responsive, but with a high relapse and mortality rate, and with the frequent clinical observance of peritoneal carcinomatosis.[32]

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Loss of E-cadherin, encoded by the gene CDH1, has recently been described in a large cohort of patients with plasmacytoid UC and may account for the marked discohesion of these cancer cells.[31] In this study, loss of E-cadherin protein was demonstrated in 76.2% of plasmacytoid UC in contrast to only 11.1% of conventional UC and was shown to be associated with a worse prognosis.[31] In another recent study, six plasmacytoid tumors were analyzed by whole-exome sequencing, and all six demonstrated CDH1 nonsense mutations, whereas no CDH1 mutations were seen in any of 127 conventional UC.[5] Additional sequencing using targeted exon capture in a larger set of 19 patients showed somatic CDH1 mutations in 14 plasmacytoid UC, which was not seen in 56 non-plasmacytoid variants. Many of the CDH1 alterations were distinct from those seen in lobular breast cancer and gastric cancer, two other lesions in the differential diagnosis of widely disseminated disease. The majority of the remaining cases showed CDH1 methylation as responsible for E-cadherin loss.[5] Further analysis of epithelial-to-mesenchymal transition pathways and the functional role of E-cadherin in the evolution of this variant may be interesting in determining possible alternative targeted therapies in this setting.

Lipid-rich urothelial carcinoma The lipid-rich variant contains lipid-containing cancer cells that are identical in appearance to lipoblasts (immature fat cells; Fig. 4E). Although rare, this variant typically comprises at most 50% of the UC cellular content and may be seen admixed with other variants.[33] One study on a limited number of patients confirmed an identical pattern of loss of heterozygosity between areas of conventional UC and the lipid-rich component, suggesting a common clonal origin.[33] Although a relatively direct diagnosis, the differential diagnosis could potentially include liposarcoma or metastatic carcinoma. However, immunohistochemical stains readily show a staining pattern consistent with

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UC.[34] This variant may be associated with worse outcomes, although the number of published cases is small, and the implications for treatment strategies are unclear.[34]

Clear cell (glycogen-rich) urothelial carcinoma The cytoplasmic clearing seen in this variant is due to an abundance of polysaccharides present within cancer cells (Fig. 4F), which can be confirmed by periodic acid-Schiff special stain that is sensitive to diastase digestion.[35] The immunohistochemical staining pattern is similar to conventional UC, with the exception of CA-125 expression, a finding also seen in tumors of Müllerian origin.[36] In most cases, there is recognizable conventional UC admixed to help in the diagnosis. Given its rarity, treatment strategies and prognosis of this variant are ill-defined.

Sarcomatoid urothelial carcinoma Sarcomatoid UC (formerly known as “carcinosarcoma”) is well-known to be associated with advanced disease and poor outcomes.[37] The major differential diagnosis for this entity is a primary sarcoma of the bladder, especially when epithelial components are not apparent. The morphology of this entity is incredibly variable, with regions mimicking diverse forms of sarcoma by microscopy and immunohistochemical stain (Fig. 4G,H); however, foci positive for at least one epithelial and/or urothelial immunohistochemical marker can be demonstrated in most cases, which supports its derivation from UC.[38] The epithelioid component may be also diverse in appearance and may have unusual patterns, including squamous, rhabdoid, chordoid and many other patterns present. Although the vast majority of patients have a poor outcome, even shorter overall survival may be associated with myxoid or chordoid features within these carcinomas.[38]

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One recent study has shown that markers representative of epithelial-to-mesenchymal transition, including vimentin, FoxC2, SNAIL, and ZEB1, are often over-expressed in sarcomatoid UC. Concurrent loss of E-cadherin and elevated N-cadherin expression is also evident.[38] Although these findings do not indicate any form of specific therapy for this variant, they do highlight an element that may be relevant to its aggressive behavior based on available data.

Giant cell urothelial carcinoma This variant is distinguished by the presence of markedly atypical, often multinucleated cancer cells that are present concurrently with conventional UC in the majority of specimens.[39] However, occasional cases may be composed purely of these pleomorphic giant cells, raising a differential diagnosis of a secondary carcinoma involving the bladder. Immunohistochemical stains support its origin as urothelial in nature,[39] and should be employed in this setting. Likely, this may represent a dedifferentiation of a concurrent or precedent UC.[39] Bizarre, atypical mitotic figures and necrosis are often present, and these carcinomas are often associated with advanced stage and metastasis.

Urothelial carcinoma with osteoclast-like giant cells In contrast to the “giant” cells present as part of divergent trophoblastic differentiation and those associated with poorly differentiated giant cell UC, this entity contains giant cells reminiscent of osteoclast-like giant cells (positive for the macrophage marker CD68) admixed with poorly differentiated UC cells (positive for cytokeratins). Thus, bladder carcinomas with giant cell features can be further substratified through use of immunohistochemical stains. This variant is rare, but cases described in the literature tend to show poor outcomes.[40]

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Toward a Molecular Classification Recent studies have sought to better define the molecular landscape of UC. In contrast to the previously described approaches that reflect variant-specific analysis, analyses of UC using broad-based genomic and transcriptomic approaches have been employed to segregate UC into distinct molecular classifications. However, many of the variant morphologies have not been analyzed in this context. The concept of molecular pathways that define tumor behavior and outcomes in UC has a historical basis, with separation of non-invasive lesions into a papillary Ta pathway that leads to recurrent disease and a CIS pathway that leads to progressive disease, each with unique genomic alterations.[41] This two pathway model in non-invasive lesions associated frequent HRAS and FGFR3 alterations with low-grade papillary Ta lesions,[42] and frequent TP53 and RB alterations with highgrade progressive lesions.[43] Recent more comprehensive studies have re-evaluated this model, identifying significant overlap in molecular alterations between the pathways, with several routes leading to progression, suggesting that the model may somewhat oversimplify the genomic complexity inherent to UC.[44] The first signs of major molecular subtypes in non-muscle invasive bladder carcinoma were observed in early gene expression studies,[45] later shown to have significant overlap with more derived subtypes.[46] Bladder cancer subtypes have also been derived by analysis of all stages and grades.[47] In one of the first efforts to establish such subtypes, whole genome transcriptional analysis was used to identify five categories of tumors that demonstrated differential expression of cytokeratins, FGFR3 mutational status, cell adhesion gene profiles, and cell-cycle regulator gene profiles.[47] The five categories include Urobasal A, Urobasal B, Genomically unstable, Squamous-cell carcinoma-like, and Infiltrated.[47] The three major subtypes, urobasal, genomically unstable, and squamous-cell carcinoma-like, can also be distinguished based on morphology and immunohistochemical profile,

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enabling identification in standard pathological examination.[48] Furthermore, in a recent study the authors showed significant differences in outcome in stage pT1 patients when stratifying for molecular subtype.[49] A recent study of 460 non-muscle invasive UC identified three molecular subtypes (Classes 1-3), where tumors from clinical high risk patients (Class 2 – highly similar to the “Genomically unstable” tumors) harbored frequent TP53 and ERBB2 alterations and APOBEC-related mutations, while tumors from low risk patients (Class 1 - highly similar to the “Urobasal A” tumors) were enriched for FGFR3 mutations.[46] Class 3 tumors showed basal-like characteristics and pronounced expression of long non-coding RNAs. Mutations from APOBEC enzymes are frequent in multiple cancers including UC, and the observation that they are enriched in high risk patients indicates that they may drive disease progression. Molecular subtypes of muscle invasive UC have now also been thoroughly investigated, an especially challenging task given that UC possesses among the highest frequency of genetic and epigenetic alterations when compared to other cancer types.[4] The high rate of chromatin remodeling gene alterations may additionally contribute to the molecular complexity of these lesions.[4] Over the past several years, a number of groups have independently developed molecular classification systems for invasive UC, and these classifiers show significant overlap suggesting inherent commonalities in subsets of UC.[4, 7, 47, 50, 51] A simple classification system was recently published that divides UC into “luminal” and “basallike” categories in a system analogous to breast cancer. Whereas luminal carcinomas expressed genes frequently associated with superficial umbrella cells and appeared similar to superficial papillary tumors, basal-like carcinomas expressed genes more characteristic of urothelial basal cells and also had a significantly worse prognosis but may be more responsive to neoadjuvant chemotherapy.[51, 52] In

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addition to the luminal and basal-like tumors, some classification systems have identified a third group of tumors, “p53-like,” that are associated with resistance to neoadjuvant chemotherapy.[50, 52] The concept of molecular classifiers was supported by data from the Cancer Genome Atlas Project that undertook complete molecular characterization of 131 muscle-invasive carcinomas,[4] although inclusion of variant morphologies in this study was limited. Integration of mRNA, microRNA, and protein expression platforms were used to delineate four discrete clusters of UC. Cluster I often showed “papillary” features and frequent mutations or amplifications in FGFR3. Cluster II is similar to cluster I, but lacks the papillary features and FGFR3 alterations. Both clusters I and II were compared to the luminal subtype of breast cancer, based on comparable expression patterns of GATA3 and FOXA1, as well as increased Her2/neu and estrogen receptor beta signaling,[4] although such pathway activation is not unique to these two organ systems. Cluster III was described as “basal/squamous-like”, based on expression of EGFR and basal-type cytokeratins characteristic of squamous differentiation at multiple anatomic sites.[4] Cluster IV was similar to cluster III, but did not show the same characteristic histologic squamous features as cluster III.[4] Although the molecular classification categories vary in number from two to five, this variation can be primarily attributed to the decision to either group tumors into broad categories or further substratify them into more discrete subtypes. The molecular alterations appear to remain consistent between studies and are not the basis for separate classification schema or nomenclature. One commonality between these classification systems is that UC can be broadly divided into luminal and basal subtypes, with basal tumors having a worse prognosis, yet often showing EGFR activation and sensitivity to anti-EGFR therapy.[53] As our knowledge about the biological properties and clinical implications continues to emerge, so too will a consensus molecular classification system.

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Despite this emerging molecular classification landscape, the issue of how variant morphologies fit into the paradigm remains. There is perhaps enough information on some variants to propose possible correlations. For example, tumors with squamous differentiation exhibit cytokeratin 5 and p63 expression and absence of cytokeratin 20 expression, a finding often associated with basal-type cancers. Basal-type cancers also demonstrate markers of epithelial-to-mesenchymal transition, which has been reported in sarcomatoid UC,[38] although these two variants demonstrate remarkably different outcomes. By contrast, micropapillary UC frequently shows amplification and/or increased expression of ERBB2, a finding more consistent with the luminal subtype, despite its more aggressive biological behavior. Other variants such plasmacytoid UC may be completely distinct, as loss of E-cadherin appears to be common in this variant, although may not be per se associated with epithelial-tomesenchymal transition or the basal subtype. Many of the other variants have not been characterized to even allow speculation as to their molecular classification. These new characterization schemas built around the emerging molecular data for bladder cancer hold much clinical promise. The identification of molecularly-defined subgroups may provide clinically relevant prognostic data to better inform patient care and treatment planning. In many other tumor types, the detection of driver mutations is directly linked to the selection of the specific targeted therapy agents. Even without the identification of specific pathway dysfunction, the molecularly-based classifications may themselves be associated with general response to classes of therapy, such that one group may respond better to immunotherapy and another to cytotoxic chemotherapy. All of these advances in molecular classification have the potential to improve and standardize the clinical care of bladder cancer patients, especially those with these rare, non-conventional variants.

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Conclusion Despite a relatively high-degree of interobserver variability, as well as the challenges inherent in defining optimal treatments for a rare disease, diagnosis of variant morphologies in UC substantially informs patient care. Although molecular knowledge of some variants has increased, the application of this knowledge to diagnosis is still in its infancy, and the relationship of these variants to the overarching molecular classifiers of UC remains unclear. Future studies will likely link variants more closely to these molecular classifiers and may perhaps even supersede the need for microscopic distinction for some of these entities.

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Figure Legends Fig. 1. Divergent differentiation is the most common variant of urothelial carcinoma. Variants of urothelial carcinoma are often found in association with conventional urothelial carcinoma (A), with occasional exception. The presence of a conventional in situ or invasive component is helpful to exclude the possibility of a metastasis or secondary spread from a non-bladder carcinoma to the bladder wall. (B) Squamous differentiation is the most common form of divergent differentiation and does not require the presence of keratin. Carcinomas that show extensive squamous differentiation cannot be always reliably distinguished from pure squamous cell carcinoma. (C) Glandular differentiation occurs in many forms and this case shows concurrent urothelial carcinoma features in the right of the panel. (D) Trophoblastic cells (arrows) may be present scattered throughout an urothelial carcinoma.

Fig. 2. Problematic distinction between bland carcinomas and benign processes if the sampling is superficial. (A) Nested urothelial carcinoma has morphological features similar to that of normal urothelium, but is distinguished by its ability to deeply invade. In this example, the cancer nests are seen diffusely spreading deep into the bladder wall. (B) In contrast, a benign proliferation of von Brunn nests may look very similar at the superficial level, although this benign process is restricted to immediately below the urothelium. (C) A similar situation is seen with microcystic urothelial carcinoma, in which bland urothelial nests are deeply infiltrating the bladder wall. (D) A benign nodule of cystitis cystica et glandularis appears mass-like at cystoscopy, but shows a superficial defined lesion on microscopy. It may take multiple rounds of biopsy to solidify the diagnosis in such cases.

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Fig. 3. Significant inter-observer variability may be seen in micropapillary urothelial carcinoma. (A) Classic features of this variant include multiple clusters of carcinoma cells present within a single retraction space, polarization of the nuclei to the outer rim of the cancer cell clusters, and absence of a fibrovascular core. (B) This example shows significant crowding of the cell clusters, but does have “ring” features that may be a feature of micropapillary urothelial carcinoma. (C, D) Are these cases micropapillary? Two examples that do not fit the classic profile, but may be considered within the range of micropapillary features, depending on which pathologist reviews the case. Unfortunately, we do not have a more objective metric to classify these currently as ERBB2 amplification has not been validated as part of the standard work-up on such cases and may not capture all lesions.

Fig. 4. A-B. Lymphoepithelioma-like carcinoma can mimic a lymphoproliferative process or infection. (A) Whereas low magnification may be problematic to the diagnosis, (B) high magnification can more readily identify the presence of syncytial sheets of cancer cells. C-D. Plasmacytoid urothelial carcinoma may be challenging to detect in small specimens. (C) Low magnification view taken from a cystectomy specimen appears to harbor only inflammation in the lamina propria. However, carcinoma cells are present deep in the wall (arrows). (D) High magnification shows how these individual cells may be difficult to detect by microscopy. E-F. Urothelial carcinoma variants with cytoplasmic clearing. (E) Lipoid differentiation is seen focally in this case and is demonstrated by cells with pale, loculated cytoplasm that is reminiscent of lipoblasts. (F) By contrast, the cytoplasmic clearing in clear cell urothelial carcinoma shows an “emptiness” of cytoplasmic contents, although special stains will confirm the presence of glycogen. It is unclear the mechanism by which these two products are synthesized in these subsets of urothelial carcinoma. G-H. Sarcomatoid urothelial carcinoma can easily mimic a broad range of non-epithelial neoplasms. (G) One example shows how this carcinoma

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can mimic a high-grade sarcoma, an entity that could occur in the bladder wall. (H) A second example shows how these carcinomas can form osteoid and even potentially an osteosarcoma.

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Variant

Table 1. Urothelial Carcinoma Variants, Diagnostic Challenges, and Molecular Associations Key Challenge(s)That May Impact Clinical Management or Prognosis Molecular Association(s) Divergent differentiation

Squamous

Glandular Trophoblastic cells/ -HCG immunoreactive

 Wide frequency range due to subjectivity in diagnosis of squamous differentiation  Distinction between pure squamous cell ca and urothelial ca with extensive squamous differentiation difficult and relies on clinical history and prior pathology  Infrequently, glandular differentiation may be extensive raising the differential of a pure adenocarcinoma primary to or secondarily involving the bladder  Identification of trophoblastic cells is rare by microscopy, although -HCG reactivity occurs in up to 35% of urothelial carcinomas  Increased serum/tissue -HCG associated with increased pathological stage

 Generally associated with the basal molecular classifier  HPV does not appear causal  Unknown  Unknown

Variants that can mimic benign urothelial nests Nested Microcystic

   

Often diagnosed late (into detrusor muscle) due to bland appearance May be misdiagnosed as von Brunn nest proliferation Often diagnosed late (into detrusor muscle) due to bland appearance May be misdiagnosed as cystitis cystica et glandularis

 Appears similar to conventional urothelial ca  Appears similar to conventional urothelial ca

Marked immune cell infiltrate Lymphoepitheliomalike

 Carcinoma cells may be missed due to marked polyclonal immune cell infiltrate

Micropapillary

 Very high degree of inter-observer variability may result in over-treatment  Micropapillary features in in situ disease is not the same as micropapillary carcinoma  If no obvious carcinoma component is evident and there is no history of bladder cancer, there is potential to misdiagnose as sarcoma  Carcinoma cells may be mistaken for lymphoma for signet ring carcinoma

 Unrelated to EBV infection  Cause of inflammation unknown

Aggressive variants with more recently described molecular alterations

Sarcomatoid Plasmacytoid

 Often has ERBB2 gene amplification or mutations  Shows EMT protein expression, but unknown genomic driver  CDH1 loss in most cases due to mutation or methylation

Variants with pale cytoplasm Lipid rich Clear cell (glycogen rich)

 Rarely a diagnostic dilemma, but differential could potentially include liposarcoma  Rarely a diagnostic dilemma, but differential could potentially include clear cell carcinoma of the gynecologic tract

 Appears similar to conventional urothelial ca  Unknown

Non-trophoblastic giant cells present 23

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Giant cell Osteoclast-like giant cells

 Rarely a diagnostic dilemma since giant cells positive for cytokeratin  Rarely a diagnostic dilemma, but may be confused with other giant cellcontaining tumors if CD68 immunostain not performed

 Unknown  Unknown

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