Urothelial Carcinoma: Divergent Differentiation and Morphologic Subtypes.

Identifying the histologic subtypes and divergent differentiation in bladder cancer is important to establish the correct diagnosis and classification and may also be clinically relevant and dictates clinical management in certain subtypes. More work is needed to delineate the molecular biology driving these morphologic subtypes and identify predictive and prognostic value biomarkers to devise novel and efficacious therapeutic approaches. Due to the rarity of most of these subtypes, multi-institutional collaborative studies may be needed to produce adequate cohorts and generate meaningful and potentially transformative data.

Urachal carcinomas are extremely rare bladder cancers arising from urachal remnants with an incidence of ranging from 0.01% to 0.7% in Western and European countries. 6 , 104 – 106 Mucosuria is present in up to one fourth of the patients. Urachal carcinomas are usually located in the bladder dome or anterior bladder wall, and often are deeply invasive into perivesical soft tissue. 107 Urachal remnants may be identified adjacent to the neoplasms after adequate sampling, and are usually lined by cuboidal or urothelial lining and may exhibit enteric metaplasia or adenomatous changes. These tumors are generally cystic and attempts are being made to subdivide them, based on the cellularity and atypia, into mucinous cystadenoma, mucinous cystic tumors of low malignant potential and mucinous cystadenocarcinoma. 6 , 108 , 109 Although adenocarcinoma is the most common urachal carcinoma, other tumor types have been also reported including urothelial, squamous, neuroendocrine neoplasms. 107 , 109 Recent genomic profiling of urachal adenocarcinomas identified frequent TP53, KRAS, and SMAD4 mutations; while no mutations were detected in TERT promoter or chromatin-modifying genes. 26 , 110 , 111 Urachal adenocarcinomas have a 5-year survival rate ranging from 40% to 70% despite the locally advanced stage at presentation. In a non-metastatic setting, wide/en bloc resection with negative soft tissue and bladder margins is generally curative. 26 , 112 – 116

MPUC is a rare and aggressive form of UC, reported in 0.6% to 2.2% of cases, and is commonly seen in males with a mean age of 66 years. This subtype frequently presents as a high stage disease and has a natural tendency for lymphatic vascular invasion and nodal metastasis. 6 , 10 Morphologically the reproducible features include presence of multiple small clusters of tumor cells with reversal of polarity, embedded in a back-to-back lacunar/retraction space and lacking a true fibrovascular core ( Figs. 6A and 6B ). Retraction spaces associated with MPUC can often mimic vascular invasion while lacking definitive endothelial lining. It is also not to be confused with the retraction artifacts that are frequently present in bladder tumors as a result of tissue processing. It has been shown that multiple nests within the same lacunar space had the highest association with a diagnosis of MPUC among the morphologic features. 54 Non-invasive urothelial carcinomas with micropapillary architecture should not be regarded as MPUC, although these unrelated features may be associated and not considered as precursors for MPUC. 55 These tumors have consistently been reported to harbor higher rates of ERBB2 gene alterations (including amplification and mutation) than UC-NOS and other UC subtypes ( Figs. 6C and 6D ). By expression profiling, these tumors are typically of luminal subtype and characterized by enrichment of PPARG and suppression of p63 target genes. 56 – 60 The aggressive nature of MPUC has been recognized from the time this entity was coined as this tumor is generally associated with a higher rate of locally advanced disease. However, more studies are showing that when compared with pure UC, MPUC was not associated with increased recurrence or mortality following RC. 61 – 63 PUC is a rare variant (<1%) of UC, histologically exhibiting discohesive single or small clusters of cells that resemble plasma cells or monocytes with minimal stromal response ( Fig. 7 ). 6 , 64 The invasive tumor cells typically infiltrate the bladder wall as single cells, linear cords, solid expansile or loose alveolar nests, or diffuse sheets. 8 These tumors invariably contain signet ring cells with intracytoplasmic vacuoles, but characteristically lack extracellular mucin. Patients with PUC frequently have local recurrences and peritoneal carcinomatosis, with an increased incidence of cancer-specific mortality. Extravesical spread of the disease can be as high as 28% for patients who undergo preoperative chemotherapy, despite the absence of radiographic progression after systemic therapy, rendering them unresectable at the time of surgery. 65 PUC is associated with higher rates of positive surgical margins and lymph node involvement and can be upstaged in up to 73% cases following RC. 66 Overall survival is still significantly lower following adjuvant or neoadjuvant chemotherapy for PUC compared to UC-NOS, with an overall downstaging rate of ~21%, pathologic complete response of ~12% and response rates ranging from 38–53%. 65 , 66 Genomically, PUC harbors a relatively high mutation rate with a median tumor mutation burden (TMB) of 14.9 mutations/Mb in one study. 65 Notably, these tumors harbor CDH1 truncating mutations, and less frequently CDH1 promoter hypermethylation, which are pathognomonic to this histologic type. Therefore, loss of E-cadherin expression (or less frequently presence of cytoplasmic labeling only; Fig. 7D ), and abnormal expression of p120 (cytoplasmic expression and loss of membranous staining) is a characteristic immunoprofile. 67 , 68 Aside from CDH1 alterations, the overall genomic landscape of PUC is similar to that of UC-NOS, with frequent mutations in chromatin modifiers, cell cycle regulators, and TERT promoter which may be useful to distinguish from other tumors with similar morphology such as lobular mammary carcinoma and diffuse type gastric adenocarcinoma, particularly in metastatic setting. 69 So far, no germline CDH1 mutations have been identified in patients with PUC. 15 , 67 NVUC is a rare variant of UC that despite the characteristic deceptively bland morphologic features, can be associated with aggressive clinical course. 6 , 70 – 72 Histologically, NVUC has a characteristic growth pattern composed of proliferation of small round to ovoid irregular nests of urothelial cells with banal cytology, jagged tumor-stroma interface and minimal desmoplastic response in the superficial compartment making its diagnosis challenging on initial biopsy or transurethral resection ( Figs. 8A – C ). The architectural pattern of the nested component can also be variably admixed with confluent nests, cordlike growth, cystitis cystica-like areas and tubular growth pattern. 73 The large nested subtype of UC, as the name suggests, consists of large irregular and infiltrating nests, associated with stromal reaction and has an overall bland histologic feature ( Fig. 8D ). 71 , 72 Diagnostically, this subtype may be difficult to identify as it invariably shares overlapping morphologic features with a number of benign or non-neoplastic entities such as proliferative cystitis, von Brunn nest hyperplasia, nephrogenic adenoma, or inverted papilloma. 71 , 74 , 75 However, many cases can present with scattered but deeply invasive tumor foci of similar morphologic features, supporting its aggressive nature. By immunohistochemistry, NVUC typically have luminal phenotype with frequent expression of FOXA1, GATA3 and CK20. 76 – 78 Interestingly, despite the overall luminal expression pattern, some NVUC tumors can exhibit CK5/6 expression with distinct localization at the basal layers of tumor nests. 78 Moreover, more than half of NVUC show nuclear PAX8 expression. 79 , 80 Relevant genomic findings in NVUC include the high rate of TERT promoter mutations, which is typically not detected in benign mimickers. 76 , 79 , 81 Additionally, occasional mutations in TP53 , JAK3 , and CTNNB1 have been reported in a study on large nested UC, while FGFR3 mutations were identified in the vast majority of cases. 77 UC exhibiting tubular and microcysic features with overall bland cytologic features have been rarely reported. 82 These two entities are closely related to NVUC due to the bland appearing cells lining the tubules or microcystic structures making their recognition and distinction from NVUC challenging. In fact, microcystic and tubular morphology is present in a significant number of NVUC. The lining cells may exhibit mucinous contents. 6 Similar to nested subtypes, the tumor-stroma interface is jagged and irregular including involvement of muscularis propria, which is an indication of its malignant behavior. 9 The differential diagnosis includes cystitis cystica, cystitis glandularis, and sometimes clear cell adenocarcinomas. 10 , 83 LELC of the bladder is rare and exhibits similar morphologic features to its namesake in the nasopharynx and stomach characterized by a syncytial growth of high-grade epithelial cells with large pleomorphic nuclei, vesicular chromatin, prominent nucleoli, and ill-defined cytoplasmic borders, and are associated dense lymphoplasmacytic infiltrate ( Fig. 8E and 8F ). 84 , 85 It is, however, not associated with Epstein-Barr virus (EBV). 84 , 86 , 87 These tumors are male predominant, commonly seen between 5 th to 7 th decade, and associated with a component of urothelial carcinoma in the majority of cases. 86 , 87 RNA expression and IHC profiling of LELC revealed that these tumors are enriched for basal-squamous molecular subtype markers (express KRT5 , KRT6 , and KRT14 ), are microsatellite stable and overexpress PD-L1 (~93%). 85 These rare urothelial tumors have large neoplastic cells with optically clear empty multivacuolated cells resembling lipoblasts ( Fig. 8G ). They are typically admixed with UC-NOS, with variable amounts of the lipid-rich areas within the tumor. 6 , 88 The lipid rich contents within the neoplastic cells have been verified by electron microscopy, Sudan Black B and Oil Red stains. 89 , 90 The background urothelial carcinoma is invariably high grade and invasive. The common differential diagnosis includes signet ring component (glandular differentiation) of UC, heterologous liposarcomatous elements of a sarcomatoid carcinoma or a liposarcoma itself. Due to the limited number of reported cases of lipid-rich UC, the associated clinical outcome is difficult to determine. Clear cell (glycogen-rich) urothelial carcinoma is a rare manifestation of UC that is characterized by solid and nested architecture and composed of cells with prominent cytoplasmic membranes with voluminous clear cytoplasm. 6 , 91 Diagnostic considerations may include involvement by clear cell renal cell carcinoma or clear cell adenocarcinoma of Mullerian origin. However, clear cell UC lacks the characteristic vascular pattern typically seen in clear cell RCC and the tubulocystic architecture, hobnailed nuclei and hyalinized stroma that are typically seen in Mullerian type clear cell adenocarcinoma. Periodic acid–Schiff (PAS) and PAS-diastase (PAS-D) stains highlight the presence of glycogen within clear cells. By immunohistochemistry, clear cell UC typically expresses CK7, GATA3, CK5 and CD44, consistent with its urothelial phenotype. Giant cell urothelial carcinoma is a very rare subtype of urothelial carcinoma with only a handful of cases reported in literature and is often accompanied by UC-NOS. 92 – 95 Morphologically, the tumor consists of loosely cohesive expansile and infiltrating nests or single cells of anaplastic giant cells ( Fig. 8H ). Tumor cells have abundant eosinophilic cytoplasm and may contain intracytoplasmic vacuoles. Necrosis and atypical mitotic forms are readily seen. Identifying a classic urothelial carcinoma component or carcinoma in situ may be the key to diagnosis in challenging cases. The giant cells typically express keratins and other markers associated with urothelial phenotype, which helps to distinguish them from osteoclastic or trophoblastic cells, which represent histologic mimickers. 92 , 94 This is a rare form of biphasic bladder cancer, in which a component of the tumor exhibits mesenchymal differentiation, commonly nondescript spindled appearance, which may or may not be associated with heterologous differentiation (osseous, cartilaginous, myogenic, angiogenic, etc.) and myxoid change of the stroma ( Figs. 9A – 9E ). 6 , 96 , 97 Sarcomatoid UC may be associated with prior radiation therapy and/or cyclophosphamide treatment. 97 Morphologically, the epithelial component may contain UC-NOS, squamous, glandular, or high-grade neuroendocrine carcinoma elements, and may imperceptibly merge with sarcomatoid histology. 98 , 99 To establish a diagnosis of sarcomatoid UC, one of the following must be identified: (1) epithelial differentiation on H&E examination; (2) variable keratin expression in the sarcomatous areas, often high molecular keratins CK 34ßE12 or CK5/6; and or (3) GATA3 expression in the sarcomatous areas. 8 The genomic landscape of sarcomatoid UC is overall similar to that of UC-NOS with enrichment of TP53, RB1, and PIK3CA mutations. 100 , 101 By expression profiling, sarcomatoid UC is linked to the basal/squamous subtype with downregulated of luminal markers, dysregulation of epithelial-mesenchymal transition (EMT) network and overexpression of EMT markers, and overexpression of programmed death-ligand 1 (PD-L1). 101 Sarcomatoid UC is associated with higher stage at presentation and independently associated with worse survival compared to UC-NOS. Despite the worse outcome, sarcomatoid UC has similar pathological response rates to neoadjuvant chemotherapy to those of UC-NOS. 102 In the current WHO 2022, this tumor encompasses tumors such as the undifferentiated carcinoma with osteoclastic giant cells and any other tumors in which a substantial component of the neoplasm does not show a definitive line of differentiation ( Fig. 9F ). 9 These tumors often lose lineage specific markers (GATA3, uroplakin 2), however variably express markers of epithelial differentiation (cytokeratins AE1/AE3, Cam 5.2, CK7 and/or EMA). The multinucleated osteoclast-like giant cells in this tumor often express CD68. 103 Presence of conventional UC component or carcinoma in situ is usually diagnostic of poorly differentiated urothelial carcinoma.

SqD is one of the most frequent forms of divergent differentiation observed in up to ~40% of high-grade and/or high-stage disease. 4 , 6 , 10 , 11 It is important to note that conventional urothelial carcinoma (UC-NOS) not uncommonly exhibits many morphologic features also seen with squamous cell carcinoma, including polygonal cell shape, abundant light eosinophilic cytoplasm, distinct cell border, and peritumoral lymphocytic aggregates. Therefore, it is recommended that the term of squamous differentiation in UC is used only when more specific features of squamous morphology are seen, such as intercellular bridges or keratinization ( Figs. 1A and 1B ). Tumors with SqD characteristically cluster with the basal/squamous subtypes on gene expression profiling, characterized by overexpression of basal and stemlike markers (CD44, CK5, CK6, and CK14), epidermal growth factor receptor (EGFR) and desmocollins (DSC1–3) and desmogleins (DSG1–4), TGM1 (transglutaminase 1), and PI3 (elafin); and low expression of markers associated with urothelial phenotype and histogenesis (uroplakins, GATA3, FOXA1, PPARG, and thrombomodulin). 12 – 15 In the series of radical cystectomy (RC), UC with SqD is associated higher stage at presentation; however, the recurrent free survival (RFS) and cancer specific survival (CSS) in cases with SqD was similar to UC-NOS in stage-matched analysis. 16 , 17 In addition, the clinical outcome appeared more favorable compared to the other subtypes of UC. 17 Primary SCC of the urinary bladder ( Figs. 1C and 1D ) is extremely rare in the Western world with an overall worldwide incidence of 3.4% females and 1.3% in males. SCC is the most frequent type of bladder cancer following UC, comprising 2.1–6.7% of all bladder malignancies. 18 In addition to the usual risk factors such as smoking, arsenic and aromatic amines exposure, important predisposing risk factors include chronic irritation associated with chronic urinary tract infection, calculi, prolonged catheterization (such as patients with spinal cord injury or neurogenic bladder) and Schistosomiasis. 18 Rarely, human papillomavirus (HPV) has been associated with bladder SCC, with similar morphology to those seen in cervical or oropharyngeal counterparts with basaloid, poorly differentiated appearance and little to no keratin formation ( Figs. 1E and 1F ). 19 Given the rarity of these cases, diagnostic consideration should also include HPV-related carcinoma arising from cervical, anal, urethral, or penile origin. The diagnosis of pure SCC is generally reserved for keratin-forming squamous neoplasm, lacking any identifiable conventional UC component (invasive or non-invasive). Areas of keratinization and glassy pink cytoplasm are recognizable within the carcinoma in many cases. Grading pure SCC of the bladder is generally dependent on the amount of keratinization. 6 The histological spectrum can be variable and may include large nest formation, clear cell change, pseudocystic changes, bizarre atypia and spindle cell morphology and sarcomatoid transformation. The precursor surface lesions, i.e. keratinizing squamous metaplasia/dysplasia or extensive condyloma acuminata may be observed in the vicinity with characteristic cystoscopy findings. Oncologic outcomes remain unclear when compared to UC with SqD, as some studies found no survival differences between pure SCC and UC with SqD if treated with RC and pelvic lymph node dissection, 16 whereas other studies reported bladder SCC to be more aggressive than UC-NOS after adjusting for stage and other prognostic parameters. 18 , 20 Verrucous carcinoma of the urinary bladder is an extremely rare and clinically indolent tumor in the urinary tract with an exophytic filiform growth pattern. It is commonly observed in males in their 5 th decade with higher incidences in areas endemic to Schistosomiasis. 18 , 21 , 22 The relationship with HPV is uncertain although progression from condyloma acuminatum has been documented in anecdotal case reports. 23 , 24 Grossly, these tumors are solitary, exophytic, fungating and filiform. Microscopically, the tumor is frequently characterized by papillomatosis, acanthosis and hyperkeratosis with pushing borders. The cytological atypia is minimal. High-grade areas or frank stromal infiltration makes the diagnosis of verrucous carcinoma unsuitable. Overall prognosis is highly favorable although the data is very limited. 21 Glandular differentiation is common in UC and can be seen in up to 18% cases. 4 True glandular differentiation is defined by the presence of glandular spaces resembling tubular or enteric glands ( Figs. 2A and 2B ) with occasional tumors exhibiting abundant extracellular mucin and mimicking colloid carcinoma. Intracytoplasmic mucin can be observed in 14–63% cases. 6 The expression of MUC5AC and CDX2 by immunohistochemistry might be useful in highlighting glandular differentiation. Recent studies suggested that UC with glandular differentiation is genomically related to UC-NOS with similar frequency of alterations in TERT promoter (~76%), chromatin-modifying genes (e.g., KDM6A, KMT2D, and ARID1A ), and DNA damage response (DDR) genes. These findings can help to distinguish UC with glandular differentiation from pure adenocarcinomas of the urinary bladder, which typically have a molecular profiles similar to colorectal adenocarcinomas (see additional discussion of adenocarcinoma below). 12 , 25 , 26 Similar to SqD, UC with glandular differentiation had a higher stage at presentation; however it is not associated with adverse survival outcome compared with conventional UC after adjusting for stage. 11 , 17 , 26 Primary adenocarcinoma of the bladder is rare accounting for 0.5 – 2% of all bladder cancers in the US. Implicated predisposing factors include long-standing intestinal metaplasia, bladder exstrophy, chronic irritation and obstruction due to non-functioning bladder or endemic schistosomiasis. Grossly, the tumors are frequently solid or sessile. Microscopically, primary bladder adenocarcinoma can be of enteric, mucinous, mixed and not otherwise specified (NOS) types. The enteric type is identical to its gastrointestinal counterpart, exhibiting acinar, cribriform, villous or solid architectural patterns, lined by pseudostratified epithelium and luminal necrosis ( Figs. 2C and 2D ). The mucinous type is composed of abundant extracellular mucin with floating tumor cells that may appear signet-ring, gland-forming or sheet-like. The mixed type includes an admixture of enteric and mucinous types. Adenocarcinoma NOS is applicable to tumors not fulfilling any of the above features. There is not a uniformly accepted grading system for bladder adenocarcinoma. Surface glandular changes may coexist and include villous adenoma and high-grade dysplasia. By immunohistochemistry, bladder adenocarcinoma is usually positive for CK20 and CDX2 which cannot distinguish between primary adenocarcinoma from metastasis or direct extension from a colorectal primary. 27 , 28 Knowledge of precedent clinical history of a primary gastrointestinal malignancy and radiologic and/or endoscopic evaluation is generally helpful in establishing a site of origin of bladder adenocarcinomas. Recent genomic studies on adenocarcinomas of the bladder demonstrated a high incidence of oncogenic alterations in TP53 , KRAS , and PIK3CA , similar to frequencies observed in colorectal adenocarcinoma, and significantly lower incidence of APC alterations compared to colorectal adenocarcinoma. They typically don’t harbor alterations commonly seen in urothelial carcinoma such as mutations in TERT promoter, FGFR3 or chromatin modifying genes. 12 , 25 , 26 , 29 When matched for stage, primary bladder adenocarcinoma has a similar outcome to urothelial carcinoma. 30 Primary treatment modalities include surgery followed by adjuvant chemotherapy to control distant metastasis and or adjuvant radiotherapy for better local control. Müllerian-type adenocarcinoma may rarely develop in the bladder and is thought to either arise from a pre-existing Müllerian precursor within deeper layers of the bladder wall such endometriosis and Mullerianosis, or be of urothelial derivation. 31 , 32 These carcinomas resemble their Mullerian-type counterparts in the female genital tract, such as clear cell adenocarcinoma (CCA) and endometrioid adenocarcinoma (EdCA). They are most commonly pure but may rarely be mixed with a urothelial component. CCA is the more common of these two entities and, in contrast to other bladder carcinomas, these tumors have a female predominance. 33 – 35 Macroscopically, CCA occurs most commonly in the bladder neck and appears as a papillary or sessile, large solitary mass. 33 Microscopically, it resembles the female ovarian counterpart exhibiting tubulocystic pattern with basophilic secretions, hyalinized papillary cores or solid sheet-like growth patterns. Hobnail nuclei with appreciable cytological atypia mitotic activity are frequently seen with accompanying hemorrhage and necrosis ( Fig. 3 ). The histopathological appearance of EdCA is similar to its counterparts in the ovary and endometrium, often containing back-to-back endometrioid-type glands with squamous metaplasia or other metaplastic changes. 18 , 36 Useful immunohistochemical (IHC) markers for CCA include PAX8, keratin 7, EMA, HNF1ß, and CA125. 6 , 34 IHC for (loss of) ARID1A expression has been proposed as multiple recent genomic studies have found CCA to have frequent ARID1A mutations, although the clinical utility in bladder tumors remains to be evaluated. 37 Other genomic characteristics of CCA include PIK3CA alterations, single nucleotide alterations (SNVs), copy number alterations (CNVs) with absence of TERT alterations (when exclusively pure CCA), hence supporting a non-urothelial differentiation. 34 , 38 , 39 The main differential diagnosis of CCA includes nephrogenic adenoma. Due to the shared PAX8 immunoreactivity, the distinction is often entirely morphological based on significant cytological atypia, high mitotic rate, necrosis and deeply invasive glands with stromal changes in CCA. The immunoprofile for EdCA is similar to the female genital counterparts, including ER, PR expression. Prognosis and management of these tumors is currently uncertain due to scarcity of cases. Small cell carcinoma (SmCC) of urinary bladder is rare and associated with an aggressive course. 40 , 41 Morphologically, it is indistinguishable from its pulmonary lung counterpart and exhibits small cells with high nuclear to cytoplasmic ratio, nuclear molding, abundant mitotic figures and necrosis ( Figs 4A – C ). Although the majority of SmCC is associated with UC-NOS suggesting its urothelial origin, pure SmCC in the urinary bladder also exists. By IHC, the tumor often expresses neuroendocrine markers CD56, synaptophysin, chromogranin and INSM1. 8 , 42 , 43 Various keratins are also expressed, mostly as focal and perinuclear dot-like patterns, reflective of the limited amount of cytoplasm in tumor cells. Recently, novel neuroendocrine markers NEUROD1, ASCL1, POU2F3, YAP1, DLL3 identified subgroups of bladder SmCC similar to those recently reported in lung SmCC. 44 , 45 ASCL1 and NEUROD1 expression was generally associated with higher expression of traditional neuroendocrine markers synaptophysin, chromogranin, and INSM1; and mutually exclusive with POU2F3 expression. In contrast, POU2F3+ tumors were associated with lower expression of the same traditional neuroendocrine markers. The clinical significance of this classification is yet to be determined. The preferred treatment modality for SmCC consists of a combination of neoadjuvant chemotherapy and further consolidation by RC. 41 , 46 By expression profiling, SmCC is characterized by a urothelial-to-neural phenotypic switch associated with downregulation of both luminal and basal markers and dysregulation of the epithelial-to-mesenchymal transition network. 47 , 48 The genomic profile of SmCC is generally similar to that of urothelial carcinoma with high levels of APOBEC mutation signature, high level of chromosomal instability and genomic doubling, and higher rates of TP53 , RB1 , and TERT promoter mutations. 40 , 47 – 49 The preferred treatment modality for SmCC consists of a combination of neoadjuvant chemotherapy and further consolidation by radical cystectomy. 41 , 46 Large cell neuroendocrine carcinoma (LCNEC) is a rare and still poorly characterized tumor of the bladder that is traditionally generally included with small cell/high grade neuroendocrine carcinoma category. 6 These tumors are high-grade, exhibiting neuroendocrine features and high mitotic activity by light microscopy, and showing evidence of neuroendocrine differentiation by immunohistochemistry. In contrast to SmCC, LCNEC consist of large high grade polygonal tumor cells with variable amounts of cytoplasm and prominent nucleoli. They have variable architectural patterns such as nests, trabeculae, organoid and palisaded ( Figs. 4D – F ). They may be pure or, more often, admixed with components of urothelial, glandular, squamous or SmCC. Many of the tumors may be misclassified as poorly differentiated or glandular when associated with UC-NOS due to their growth patterns. By immunostains, LCNEC tumors express pancytokeratins and neuroendocrine markers. There is limited data on the molecular features of LCNEC, but early evidence suggests a similar genomic profile to SmCC, characterized by high tumor mutational burden and consistently harboring TP53 , RB1 and TERT promoter mutations. 50 The current preferred treatment modality is similar to SmCC whenever possible (neoadjuvant chemotherapy followed by cystectomy) with immunotherapy as an alternative option in the metastatic setting. 41 , 50 Trophoblastic differentiation can occur in up to 5.5% of UC in the urinary bladder and 10.2% in the upper urinary tract. 51 UC with trophoblastic differentiation can be subdivided into (a) UC with scattered syncytiotrophoblasts ( Fig. 5A ), (b) UC with choriocarcinomatous differentiation and (c) urothelial carcinoma with expression of β-HCG but no recognizable trophoblasts. 10 IHC stain for β-HCG can be positive in majority of the cases ( Fig. 5B ), although the expression can frequently be seen in adjacent UC-NOS. 51 , 52 Some studies have suggested that urine or serum β-HCG levels may serve as a biomarker in assessing the response to therapy. 53 Positivity of other germ cell markers such as hydroxyl-δ−5-steroid dehydrogenase (HSD3B1) or Sal-like protein 4 (SALL4) have also been demonstrated, but the clinical utility remains to be further investigated. 52 Due to the limited case number in the literature, the molecular pathogenesis of trophoblastic differentiation in UC has not been well-defined. Trophoblastic differentiation appears to be associated with higher stage at presentation, but the presence of trophoblastic differentiation did not seem to predict adverse outcomes in multivariate analysis. 51 , 52

Bladder cancer is the sixth most common cancer in the US, with estimated 83,730 (4.4% of all cancer cases) new cases and 17,200 deaths in 2021 (2.8% of all cancers). 1 – 3 Cancers arising in the bladder cancers demonstrate a wide spectrum of histopathologic features which can be focal or extensive. It is estimated that approximately 15% to 25% of invasive urothelial carcinoma (UC) exhibit morphologic variations, 4 , 5 which can occur in the form of “divergent differentiation” along other epithelial lineages such as squamous, glandular, trophoblastic, or small cell/high-grade neuroendocrine differentiation, singly or in combination. 6 – 8 Additionally, several “subtypes” (formerly known as “variants”) of urothelial carcinoma have been described with distinctive histologic, and to some extent, immunohistochemical features, with evidence suggesting derivation from a urothelial origin. The histologic complexity is supported by distinct molecular alterations in at least a subset of these subtypes, which further substantiates the biology of intratumoral and intertumoral heterogeneity in UC, and its potential clinical applications. Similar to the previous editions, the 5th edition of the WHO classification recognizes the morphological diversity of UC with 10 subtypes ( Table 1 ), and the guidelines recommend reporting the percentage of divergent differentiation and or subtype(s)/histologic variant(s) when possible. 9 This review will highlight the various morphological criteria for the diagnosis of subtypes of UC and their clinical relevance. Updates on the molecular characteristics of these subtypes will also be discussed.