Histologic and immunohistochemical characterization and frequency of rare subtypes of canine invasive urothelial carcinoma.

The histologic and molecular heterogeneity of human muscle-invasive bladder cancer (MIBC) is a major contributor to poor treatment outcomes. While most cases of MIBC are diagnosed as conventional urothelial carcinoma (UC), there is growing recognition of histologic subtypes and divergent differentiation within conventional UC. This is clinically significant, as some require modification of therapy and some are associated with more aggressive behavior. Spontaneously occurring UC in dogs has been shown to exhibit histologic and molecular features that closely resemble those of human MIBC. In this study, we evaluated 31 canine UC tumor samples for histologic subtypes and divergent differentiation. Slides were reviewed by a human uropathologist and three board-certified veterinary pathologists and assessed for expression of uroplakin III and E-cadherin. All tumors were classified as high-grade UC. Fifteen cases were identified as conventional UC. Among the remainder, eight displayed glandular differentiation, four were classified as sarcomatoid UC, two showed squamous differentiation, and one case each was classified as large nested and tubular and microcystic subtypes.In summary, this study found a higher frequency of certain histologic subtypes and divergent differentiation in canine UC—particularly sarcomatoid UC and UC with glandular differentiation—compared to previous reports in both canine UC and human MIBC.Conclusion:The relatively high prevalence of the sarcomatoid UC subtype in dogs observed in this study suggests that canine UC may serve as a valuable translational model for evaluating novel therapeutic agents, particularly for this rare and aggressive variant in humans.

The histologic heterogeneity of human Muscle Invasive Bladder Cancer (MIBC) is one of the main contributing factors to poor treatment outcomes. Recent reports on MIBC histological variants have highlighted increased levels of transcription factors (TF) associated with epithelial-mesenchymal transformation (EMT) and epigenetic regulation in the sarcomatoid histology variant of MIBC. Dogs with spontaneously developing MIBC have been shown to display similar histological and molecular features, in addition to closely mimicking the clinical behavior of human MIBC. Here, we add to previously reported data on histological variants in canine MIBC by evaluating 21 tumor biopsies for variant histology and performing bulk RNAseq analyses on 3 organoid cell lines derived from one sarcomatoid subvariant and two conventional urothelial carcinoma (UC). MIBC biopsies from 21 dogs were reviewed for variant histology by a human urologic pathologist. Organoids derived from the urine of canine MIBC patients were cultured, preserved in RNA later, and sequenced for mRNA expression to identify TF associated with sarcomatoid variants versus conventional UC. All canine MIBC biopsies were classified as invasive, high-grade UC. Histologic variants were identified as glandular differentiation in 7/21, sarcomatoid differentiation in 3/21, squamous differentiation in 3/21, micropapillary carcinoma in 1/21 MIBC samples and small nested variants in 1/21 samples; with the rest of the tumors classified as conventional UC. Raw RNAseq analyses revealed no clear clustering of expression profiles between the three organoid cell lines, indicating distinct RNA expression profiles between the sarcomatoid variants and conventional UC of canine MIBC. To evaluate differences in the expression of TF targets between the organoid cell lines, pathway analyses and VIPER algorithms were used. Organoids derived from sarcomatoid variant UC showed increased expression of Snail familiy transcription repressor 1 (SNAI1) along with decreased expression of cadherin 1 (CDH1), indicating a signature of EMT, similar to that previously identified in human MIBC organoids with sarcomatoid variant histology. Canine MIBC organoids derived from conventional UC histology samples showed a TF profile associated with proliferation, metastasis, and progression, such as increased Zinc finger E-box binding homeobox 1 (ZEB1), E2F transcription factor 3 (E2F3), and centromere protein F (CENPF), as previously described in humans with high grade, invasive UC. In conclusion, the frequency of some histologic variants in canine MIBC, such as sarcomatoid UC, appear to be higher than reported in human patients. TF analyses of canine MIBC-derived organoid cell lines showed evidence of EMT, similar to the sarcomatoid variants of MIBC in humans. Organoids derived from canine MIBC could potentially serve as a valuable model for evaluating the efficacy of novel treatment modalities for the rare histologic variants of UC. Citation Format: Karin Allenspach, Mohamed Elbadawy, Hannah Nocholson, Christopher Zdyrski, John Cheville, Eugene Douglass, Elizabeth W. Howerth, Lilian Oliveira, Jonathan P. Mochel. Sarcomatoid Histological Variants of Canine Muscle-Invasive Bladder Cancer: Transcription Factor Activation Highlights Pathways of Epithelial-Mesenchymal Transformation Similar to Humans [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR002.

Sarcomatoid urothelial carcinoma (SUC) is a rare histologic subtype with poor prognosis. While there is known intra-tumoral heterogeneity between individual SUC tumors, the relationship between sarcomatoid and conventional urothelial carcinoma (CUC) within the same patient is poorly understood. The objective of this study was to identify differences between the sarcomatoid and CUC tumor microenvironment components that may drive this aggressive phenotype. Using tissue microarrays from eight patient tumors with mixed CUC and SUC, we examined paired CUC, mixed urothelial carcinoma (UC) regions, and SUC using the Nanostring Digital Spatial Profiling platform. We found SUC and mixed UC had higher levels of stromal cells, predominately macrophages and fibroblasts, when compared with CUC within the same tumor. CD14, CD163, and transforming growth factor-beta levels were significantly higher in SUC than in CUC. Immunohistochemical analysis revealed consistently moderate to strong expression of CD163-positive antigen-presenting cells (APCs) in SUC regions, whereas CD68-positive APC expression was generally absent. Thus, in mixed histology SUC, the SUC component preferentially expressed CD163-positive APCs and fibroblasts compared to the CUC component. As CD163-positive APCs and fibroblasts are known to be tumor-promoting and immune-suppressive, this infiltration may contribute to epithelial to mesenchymal transition and other aggressive properties of SUC.

Plasmacytoid urothelial carcinoma (PC-UC) is an aggressive variant of urothelial carcinoma (UC), characterized by loss of E-cadherin (E-Cad)-mediated intercellular adhesion. Loss of E-Cad by immunohistochemistry can help diagnose PC-UC; however, sensitivity is limited. Expression of other cadherin-catenin adhesion complex members, that is, p-120 catenin (p-120) and β-catenin (B-Cat), which are diagnostically useful for lobular breast carcinoma, remains unknown in UC. To determine the utility of p-120 and B-Cat in conventional and variant UC. E-cadherin, B-Cat, and p-120 immunohistochemistry was performed in 25 conventional UCs and 33 variant UCs, including 22 PC-UCs, 6 sarcomatoid UCs (SUCs), and 5 micropapillary UCs. Membranous staining for all biomarkers was considered normal; however, any cytoplasmic staining or an absence of staining was considered diagnostically abnormal. Next-generation sequencing was performed on 8 PC-UC cases. E-cadherin, B-Cat, and p-120 showed membranous staining in all conventional and micropapillary UCs. In contrast, most PC-UCs were negative for E-Cad (17 of 22; 77%) with an additional 2 of 22 cases (9%) showing cytoplasmic with partial membranous staining. p-120 catenin demonstrated cytoplasmic or negative staining in 21 of 22 cases (95%). Most SUCs showed an absence of E-Cad (5 of 6; 83%) and cytoplasmic or negative p-120 in 5 of 6 cases (83%). Staining for B-Cat was also abnormal in a subset of PC-UCs and SUCs. Five PC-UC cases that harbored CDH1 gene variants were p-120 cytoplasmic positive. p-120 catenin is a useful adjunct biomarker to E-Cad in the clinically important distinction of PC-UC and SUC from conventional UC. In particular, the combination of cytoplasmic p-120 and loss of E-Cad is strongly supportive of PC-UC and SUC.

Multiple cardiac complications due to sepsis and cardiac metastasis of a sarcomatoid urothelial cell carcinoma of the urinary bladder: A case report.

465 Background: SUC is a rare histology with aggressive behavior. We evaluated outcomes and recurrence patterns of patients (pts) with SUC, in comparison with conventional urothelial carcinoma (CUC). Methods: We retrospectively assessed our radical cystectomy (RC) database to identify pts with cT2-4 SUC (any %) or CUC, at RC or transurethral resection specimens. Clinicopathologic/treatment data were captured and compared with t and χ2 tests, as appropriate. Overall survival (OS; diagnosis to death) and recurrence-free survival (RFS; RC to recurrence or death) were estimated (KM method). Significant factors in univariable (UVA) Cox regression for OS were included in multivariable analysis (MVA). Results: We identified 38 consecutive pts with cT2-4 SUC and 287 with CUC (2003-2018); 17 (45%) and 162 (56%) received neoadjuvant chemotherapy (NAC). The primary non-mesenchymal component was urothelial in all SUC cases. SUC had higher rates of pT3/4 (66% vs. 35%, p < .001) but comparable rates of pN+ disease (26% vs. 20%, p = .38). Complete response (ypT0N0) after NAC was lower for SUC (6% vs. 35%, p = .02). Median follow-up was 73.6 months (95%CI 62.6 – 84.7). Median RFS and OS was inferior among pts with SUC (9.4 vs 109.8 months, p < .001, 19.7 vs. 130.4 months, p < .001 respectively). On MVA, SUC was independently associated with worse OS ( Table). Of 17 (45%) pts with SUC who recurred post-RC, 5 presented with abdomino-pelvic cystic masses, with an average time to recurrence < 5 months. Conclusions: SUC was associated with high rates of extravesical spread at RC and worse NAC response, RFS and OS, vs. CUC. Development of abdomino-pelvic fluid collections should raise suspicion of recurrence among pts with this histology. [Table: see text]

BackgroundIn humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC.ResultsWhole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside.ConclusionCanine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-023-03026-4.

Bladder cancers are the ninth most frequently diagnosed cancer worldwide. More than 90% of bladder cancers are of transitional cell origin and are classified as urothelial carcinomas (UC). UC remains amongst the most genetically diverse tumours and presents with numerous unique histological variants. The most common variants are squamous differentiated UC and glandular differentiated UC. Both of these variants tend to present at a later disease stage than conventional UC and are associated with worse patient outcomes. Rarer UC variants include trophoblastic differentiated UC, nested UC, micropapillary UC, plasmacytoid UC, and sarcomatoid UC. They also present at more advanced disease states than conventional UC, resulting in worse patient outcomes. Limited data is available for the pleomorphic giant cell UC and lipid-rich UC variants, but it suggests morbid outcomes with high patient mortality. The only UC variant with better prognosis than conventional UC is lymphoepithelioid-like UC. Proper identification of the histological variant of UC is important, as it aids the physician in clinical decision-making and can lead to better patient outcomes.

The association between variant histology and prognostic, histomorphological and clinical aspects of bladder urothelial carcinoma

Sir: Telomerase reverse transcriptase (TERT) is a ribonucleoprotein involved in maintaining the length of telomeres. In the absence of TERT expression, differentiated cells can only divide a finite number of times before undergoing cellular senescence – often referred to as the Hayflick limit. Mutations within the promoter region of TERT that create consensus binding sequences for ETS family transcription factors are a common mechanism by which neoplastic cells increase TERT expression and overcome this limit.1 TERT promoter mutations are common in many cancer types, including 60–80% of urothelial carcinomas (UC).2, 3 Given the high frequency of these mutations in UC and the absence of these mutations in non-neoplastic/benign mimics of UC,4 TERT promoter mutations may serve as potential biomarkers for monitoring patients with a history of malignancy. Multiple studies have reported detecting TERT mutations in specimens used commonly for monitoring UC patients, such as urine.2, 3, 5 However, in order to be a reliable marker of residual/recurrent disease, TERT mutation status must be a stable and uniform attribute shared among all neoplastic cells and preserved over time. To our knowledge, no previous study has compared TERT promoter genotypes within spatial, temporal and morphologically distinct components of UC. In order to evaluate the stability and uniformity of TERT mutations within a given UC, we developed an allele-specific polymerase chain reaction (PCR) assay targeting the most common TERT promoter mutations: c.-146C>T (Chr.5:1295250C>T), c.-124C>T (Chr.5:1295228C>T), c.-138_139CC>TT (Chr.5:1295242_1295243CC>TT) and c.-124_125CC>TT (Chr.5:1295228_1295229CC>TT). Using this assay, we evaluated 102 DNA samples extracted from formalin-fixed paraffin-embedded tissues from 50 patients with invasive, high-grade UC. The age range of the patients in this cohort was 50–88 years, with the majority of the patients demonstrating pathological stage pT2b–pT4 at the time of cystectomy.6 In order to determine if TERT mutation status varies among spatially distinct regions of UC, microdissection was performed to isolate distinct regions within the same block for 19 cases and within separate blocks for 20 cases, including three metastatic foci (Figure 1, Supporting information, Table S1). For 26 UC patients, microdissection was performed in order to evaluate conventional UC and components with divergent differentiation separately, including sarcomatoid (five), nested and tubular (eight), micropapillary (seven), squamous (nine), glandular (two), single cell/diffuse/plasmacytoid (two) and neuroendocrine (one). The variant morphologies were assigned as instructed by the WHO classification of tumours of the urinary system and male genital organs, 4th edition.7 To evaluate the temporal stability of TERT mutations, specimens from multiple time-points were evaluated for 11 patients (mean: 2.9 years apart; range: 0.2–8.8 years). Fourteen single-sample cases (conventional UC and divergent differentiation) were also included to establish the frequency of TERT promotor mutations in comparison with previous studies. Overall, TERT mutations were found in in 76.0% (38 of 50) of UC cases, similar to previous studies;2, 3 -124C>T was the most common (34 patients), followed by -146C>T (seven patients) and a single instance of -138_-139CC>TT. TERT status was conserved temporally in all cases evaluated. For morphologically and spatially disparate components, we found TERT mutation status to be conserved perfectly in all but one case. This case harboured a -138_-139CC>TT mutation within conventional UC and a -124C>T mutation within a separate block showing squamous differentiation. These results were confirmed after re-extraction and repeated TERT testing. Further evaluation of these two specimens using the Ion AmpliSeq Cancer Hotspot Panel showed that both components shared a PIK3CA E542K mutation. However, a PTEN R130Q mutation was present with the squamous component, but not in the conventional UC. These results suggest that while these two components are related clonally to one another, each component represents a morphologically and molecularly distinct subclonal population. In conclusion, TERT promoter mutations are conserved in the majority of morphologically, spatially and temporally distinct components of a given urothelial carcinoma. These findings corroborate the notion that components of UC with divergent differentiation remain related clonally to the conventional UC. In rare cases, spatially and morphologically distinct components of UC also show differing TERT genotypes. In this study, we showed that these genotypical differences reflect subclonal populations (intratumoral heterogeneity) within some cases of UC. TERT promoter mutations represent secondary alterations in the pathogenesis of UC and other neoplasms.1, 8 That TERT status is conserved spatially and temporally in most cases reflects the fact that these secondary mutations generally occur early in the pathogenesis of UC.3 Overall, TERT promoter mutation status is a stable biomarker in most cases and may therefore be useful in disease monitoring. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

To determine whether a limited immunohistochemical panel can help differentiate metastatic colonic adenocarcinoma from primary enteric-type adenocarcinoma of the urinary tract and urothelial (transitional cell) carcinoma with glandular differentiation, which appear morphologically similar but most often necessitate different treatment protocols. We examined lower urinary tract tumors (5 urinary bladder, 2 urethral) from 7 patients with a history of colonic adenocarcinoma. The differential diagnoses in these cases included metastatic colonic adenocarcinoma, primary enteric-type adenocarcinoma of the urinary tract, and urothelial carcinoma with glandular differentiation. An immunohistochemical panel consisting of cytokeratin 7 (CK-7), cytokeratin 20 (CK-20), and villin was evaluated in all cases. Four primary enteric-type adenocarcinomas of the urinary tract and 5 conventional urothelial carcinomas were also studied to compare morphologic features and immunohistochemical staining patterns. Of the 7 cases, 6 were determined to be metastatic colonic adenocarcinoma and 1 was diagnosed as a primary urothelial carcinoma with glandular differentiation. All 6 metastatic colonic adenocarcinomas, 6 of the 7 primary colonic adenocarcinomas, and all 4 primary enteric-type adenocarcinomas of the urinary tract were CK-20 positive (1 was CK-20 negative), villin positive, and CK-7 negative. The single urothelial carcinoma with glandular differentiation and all 5 control cases of urothelial carcinoma were CK-7 and CK-20 positive, and villin negative. We conclude that (1) villin is expressed in primary enteric-type adenocarcinoma of the urinary tract; (2) in difficult cases, urothelial carcinoma with glandular differentiation can be distinguished from colonic adenocarcinoma because the former is CK-7 positive, CK-20 positive, and villin negative, whereas the latter is CK-20 positive, villin positive, and CK-7 negative; (3) clinical information is essential when evaluating lower urinary tract tumors that are clinically and morphologically similar to enteric-type adenocarcinoma of the urinary tract; and (4) the similar immunohistochemical profiles of metastatic colonic adenocarcinoma and primary enteric-type adenocarcinoma of the urinary tract may be in keeping with the hypothesis that the latter arise from intestinal metaplasia.

Objective To determine whether a limited immunohistochemical panel can help differentiate metastatic colonic adenocarcinoma from primary enteric-type adenocarcinoma of the urinary tract and urothelial (transitional cell) carcinoma with glandular differentiation, which appear morphologically similar but most often necessitate different treatment protocols. Design We examined lower urinary tract tumors (5 urinary bladder, 2 urethral) from 7 patients with a history of colonic adenocarcinoma. The differential diagnoses in these cases included metastatic colonic adenocarcinoma, primary enteric-type adenocarcinoma of the urinary tract, and urothelial carcinoma with glandular differentiation. An immunohistochemical panel consisting of cytokeratin 7 (CK-7), cytokeratin 20 (CK-20), and villin was evaluated in all cases. Four primary enteric-type adenocarcinomas of the urinary tract and 5 conventional urothelial carcinomas were also studied to compare morphologic features and immunohistochemical staining patterns. Results Of the 7 cases, 6 were determined to be metastatic colonic adenocarcinoma and 1 was diagnosed as a primary urothelial carcinoma with glandular differentiation. All 6 metastatic colonic adenocarcinomas, 6 of the 7 primary colonic adenocarcinomas, and all 4 primary enteric-type adenocarcinomas of the urinary tract were CK-20 positive (1 was CK-20 negative), villin positive, and CK-7 negative. The single urothelial carcinoma with glandular differentiation and all 5 control cases of urothelial carcinoma were CK-7 and CK-20 positive, and villin negative. Conclusions We conclude that (1) villin is expressed in primary enteric-type adenocarcinoma of the urinary tract; (2) in difficult cases, urothelial carcinoma with glandular differentiation can be distinguished from colonic adenocarcinoma because the former is CK-7 positive, CK-20 positive, and villin negative, whereas the latter is CK-20 positive, villin positive, and CK-7 negative; (3) clinical information is essential when evaluating lower urinary tract tumors that are clinically and morphologically similar to enteric-type adenocarcinoma of the urinary tract; and (4) the similar immunohistochemical profiles of metastatic colonic adenocarcinoma and primary enteric-type adenocarcinoma of the urinary tract may be in keeping with the hypothesis that the latter arise from intestinal metaplasia.

GATA3 expression in sarcomatoid urothelial carcinoma of the bladder.

PD55-12 NEOADJUVANT CHEMOTHERAPY FOR VARIANT HISTOLOGY MUSCLE INVASIVE BLADDER CANCER: PATHOLOGIC DOWNSTAGING AND SURVIVAL OUTCOMES

It is controversial if urothelial carcinoma of the bladder with squamous and/or glandular differentiation is a more aggressive neoplasm than conventional urothelial carcinoma. A total of 165 transurethral resections of the bladder were reviewed. A group with squamous and/or glandular differentiation was compared to a group without this finding. The chi-square test was used to assess the association of the groups with stage (TNM, 1997). Of the total of 165 transurethral resections of the bladder, 153 (92.72%) were conventional urothelial carcinomas and 12 (7.27%) showed squamous and/or glandular differentiation. The distribution according to stage was 84 (54.9%), 35 (22.9%) and 34 (22.2%) for the group without differentiation and 0 (0%), 3 (25%) and 9 (75%) for the group with squamous and/or glandular differentiation, respectively for stages pTa, pT1 and pT2. Tumors with squamous and/or glandular differentiation showed a significant statistical correlation to higher stage at clinical presentation (p < 0.0001). There was no significant statistical relation according to age (p = 0.8433), sex (p = 0.5672) or race (p = 0.3137). The results suggest that urothelial bladder carcinomas with squamous and/or glandular differentiation are more aggressive neoplasms. There was a significant statistical correlation between tumors with this differentiation and higher stage at clinical presentation.