Distinct effects of Fgf7 and Fgf10 on the terminal differentiation of murine bladder urothelium revealed using an organoid culture system.

BACKGROUND: Dysregulation of the terminal differentiation of bladder urothelium is associated with the pathogenesis of urinary tract disorders. Fibroblast growth factor (Fgf)7 and Fgf10 stimulate urothelial proliferation; however, their roles in cellular differentiation remain unclear. In this study, we used an organoid system to investigate the roles of these Fgfs in regulating bladder urothelium differentiation and identify their distribution patterns in the mouse bladder. METHODS: Adult bladder epithelia (AdBE) isolated from adult mouse bladder tissues (AdBTs) were used to culture adult bladder organoids (AdBOs) in the presence of Fgf7 and Fgf10. The differentiation status of the cells in AdBTs, AdBEs, AdBOs, and neonatal bladder tissues (NeoBTs) was analyzed via quantitative real-time-PCR for the presence of undifferentiated cell markers (Krt5, Trp63, and Krt14) and differentiated cell markers (Krt20, Upk1a, Upk2, and Upk3a). Organoid cell proliferation was assessed by counting cell numbers using the trypan blue method. The effects of Fgf7 and Fgf10 on organoid differentiation were assessed using different doses of Fgfs, and the involvement of peroxisome proliferator-activated receptor γ (PPARγ) signaling in these processes was tested by introducing a PPARγ agonist (Rosiglitazone) and antagonist (T0070907) to the culture. The expression patterns of Fgf7 and Fgf10 were examined via in situ hybridization of AdBTs. RESULTS: AdBOs showed higher expression of undifferentiated cell markers and lower expression of differentiated cell markers than AdBTs, NeoBTs, and AdBEs, indicating the relatively immature state of AdBOs. Differentiation of AdBOs was enhanced by Rosiglitazone and Fgf7, suggesting an interplay of intracellular signals between Fgf7 and PPARγ. Co-addition of T0070907 suppressed Fgf7-mediated differentiation, demonstrating that PPARγ is activated downstream of Fgf7 to promote cellular differentiation into umbrella cells. Furthermore, we found that Fgf7 is predominantly expressed in the umbrella cells of the urothelium, whereas Fgf10 is predominantly expressed in the urothelium and stroma of AdBTs. CONCLUSIONS: We demonstrated that unlike Fgf10, Fgf7 induces cellular differentiation via PPARγ activity and has a unique tissue distribution pattern in the adult bladder. Further studies on the Fgf7-PPARγ signaling axis would provide insights into the differentiation mechanisms toward functional umbrella cells and the pathogenesis of several urinary tract diseases.

Reduced urothelial expression of uroplakin-IIIa in cystitis areas in bladders of postmenopausal women with recurrent urinary tract infections: pilot study.

PURPOSE: To study human bladder biopsies to investigate urothelial response to UTI, expression of uroplakin, and urothelial response after healing from cystoscopy with electrofulguration (CEF) treatment for antibiotic-recalcitrant RUTI. METHODS: Following IRB approval, cold cup bladder biopsies from "no cystitis" and "cystitis" regions were obtained from women with antibiotic-recalcitrant rUTI undergoing CEF under anesthesia. "No cystitis" and "cystitis" biopsies from 14 patients (5 had prior CEF, 9 naïve) were analyzed by immunofluorescence (IF) confocal microscopy using antibodies against uroplakin-IIIa. For an additional 6 patients (2 prior CEF, 4 naïve), only "cystitis" area biopsies were taken and analyzed. Cytokeratin 5 (marker for squamous metaplasia) staining was performed on 14 patients. RESULTS: In healthy tissue, uroplakin-IIIa staining was observed as a contiguous line on the luminal surface of umbrella cells. In "cystitis" areas for 19/20 patients, there was either no uroplakin-IIIa staining observed or spotty (+) staining. The "cystitis" regions of all patients had less intense uroplakin-IIIa staining compared to the matched "no cystitis" area in the same patient. In patients post-CEF but requiring repeat EF for persistent RUTI lesions, healed areas served as control and in 3 of 7 patients no uroplakin-IIIa staining was observed. Squamous metaplasia was observed in 10 patients. CONCLUSION: In bladders of postmenopausal women with antibiotic-recalcitrant RUTI, areas with visible cystitis expressed less uroplakin-IIIa, supporting the model of urothelial exfoliation in response to UTI.

Voiding defects in acute radiation cystitis driven by urothelial barrier defect through loss of E-cadherin, ZO-1 and Uroplakin III.

Long term-side effects from cancer therapies are a growing health care concern as life expectancy among cancer survivors increases. Damage to the bladder is common in patients treated with radiation therapy for pelvic cancers and can result in radiation (hemorrhagic) cystitis (RC). The disease progression of RC consists of an acute and chronic phase, separated by a symptom-free period. Gaining insight in tissue changes associated with these phases is necessary to develop appropriate interventions. Using a mouse preclinical model, we have previously shown that fibrosis and vascular damage are the predominant pathological features of chronic RC. The goal of this study was to determine the pathological changes during acute RC. We identified that radiation treatment results in a temporary increase in micturition frequency and decrease in void volume 4-8 weeks after irradiation. Histologically, the micturition defect is associated with thinning of the urothelium, loss of urothelial cell-cell adhesion and tight junction proteins and decrease in uroplakin III expression. By 12 weeks, the urothelium had regenerated and micturition patterns were similar to littermate controls. No inflammation or fibrosis were detected in bladder tissues after irradiation. We conclude that functional bladder defects during acute RC are driven primarily by a urothelial defect.

Histone deacetylase inhibitor givinostat alleviates liver fibrosis by regulating hepatic stellate cell activation.

Hepatic fibrosis, a common pathological manifestation of chronic liver injury, is generally considered to be the end result of an increase in extracellular matrix produced by activated hepatic stellate cells (HSCs). The aim of the present study was to target the mechanisms underlying HSC activation in order to provide a powerful therapeutic strategy for the prevention and treatment of liver fibrosis. In the present study, a high­throughput screening assay was established, and the histone deacetylase inhibitor givinostat was identified as a potent inhibitor of HSC activation in vitro. Givinostat significantly inhibited HSC activation in vivo, ameliorated carbon tetrachloride­induced mouse liver fibrosis and lowered plasma aminotransferases. Transcriptomic analysis revealed the most significantly regulated genes in the givinostat treatment group in comparison with those in the solvent group, among which, dermokine (Dmkn), mesothelin (Msln) and uroplakin­3b (Upk3b) were identified as potential regulators of HSC activation. Givinostat significantly reduced the mRNA expression of Dmkn, Msln and Upk3b in both a mouse liver fibrosis model and in HSC­LX2 cells. Knockdown of any of the aforementioned genes inhibited the TGF­ß1­induced expression of α­smooth muscle actin and collagen type I, indicating that they are crucial for HSC activation. In summary, using a novel strategy targeting HSC activation, the present study identified a potential epigenetic drug for the treatment of hepatic fibrosis and revealed novel regulators of HSC activation.

Nicotine-induced hypoxia in rat urothelium deteriorates bladder storage functions.

AIMS: To measure the effects of nicotine on lower urinary tract symptoms (LUTS), bladder blood flow, and the urothelial markers hypoxia-inducible factor 1α (HIF1α), uroplakin III (UPIII), and aquaporin 3 (AQP3). METHODS: Ten-week-old female Sprague Dawley rats were subcutaneously injected with 2 mg/kg nicotine (n = 17) or vehicle (control, n = 18) twice daily for 13 days. Some nicotine-treated rats (n = 10) were injected daily with 1 mg/kg tadalafil for the last 6 days of nicotine treatment. One day before cystometry, the bladders of some nicotine-treated and control rats were instilled with 0.08% acetic acid. Urinary frequency and volume were measured 1 day after treatment. Blood flow in the bladder neck was measured, and the urothelia were immunochemically assayed for HIF1α, UPIII, and AQP3. RESULTS: Following acetic acid treatment, both voiding interval and micturition volume of the nicotine-treated rats were significantly lower than controls. Nicotine-treated rats had lower blood flow than controls, and the urothelial expression of HIF1α was higher than controls. Simultaneously, the expressions of UPIII and AQP3 were decreased. Tadalafil treatment increased bladder blood flow, and nicotine-treated rats had increased voiding interval and micturition volume. Further, the expression of HIF1α decreased, and both UPIII and AQP3 levels increased. CONCLUSIONS: Nicotine-treated rats stimulated by intravesicular acetic acid instillation exhibited deterioration of bladder storage functions. Changes in tissue markers in the nicotine-treated rats were consistent with hypoxia and loss of urothelial function. Restoration of blood flow reversed the nicotine effects. Nicotine may induce LUTS through reduced bladder blood flow and urothelial hypoxia.

Membrane Microdomains as Platform to Study Membrane-Associated Events During Oogenesis, Meiotic Maturation, and Fertilization in Xenopus laevis.

Studies on the egg plasma membrane-associated tyrosine kinase Src have shed light on the identity of the molecular machinery that is responsible for gamete interaction and possibly fusion in African clawed frog Xenopus laevis. Here we describe our protocol for identifying and analyzing molecular and cellular machinery that contributes to a variety of biological processes in the course of oogenesis, oocyte maturation, egg fertilization, and early embryogenesis in Xenopus. Our current special interest is to evaluate the hypothesis that the oocyte/egg membrane microdomain (MD)-associated uroplakin III-Src system is responsible for mediating sperm-egg membrane interaction/fusion signal to the oocyte/egg cytoplasm to initiate embryonic and zygotic development in this species. Therefore, this chapter contains a brief introduction to biology of oocytes and eggs in Xenopus and addresses the following questions: (1) What is oocyte/egg MD? (2) Why do we study oocyte/egg MD? (3) How to manipulate oocyte/egg MD? (4) What has been achieved by oocyte/egg MD studies? (5) What are the next steps in oocyte/egg MD studies?

Immunohistochemical Expression of Cyclin D1, Cytokeratin 20, and Uroplakin III in Proliferative Urinary Bladder Lesions Induced by o-Nitroanisole in Fischer 344/N Rats.

o-Nitroanisole is an intermediate in the manufacture of azo dyes. In a National Toxicology Program stop-exposure study,o-nitroanisole induced hyperplasia, papillomas, and papillary carcinomas in the urinary bladder of Fischer 344/N rats.o-Nitroanisole was investigated since occupational or environmental exposure to aniline and azo dyes is a risk factor for urinary bladder cancer in humans. The current study describes the morphology of urinary bladder neoplasms seen in rats with respect to those observed in humans. This study also evaluated immunohistochemical expression of the cell cycle-related proteins cyclin D1 and p53 and the differentiation markers cytokeratin 20 and uroplakin III in hyperplastic (n= 11) and neoplastic (n= 6 papillomas,n= 11 carcinomas) lesions of the urinary bladder epithelium from rats treated with o-nitroanisole and in normal (n= 6) urinary bladders from untreated rats. The tumors observed were more similar to the papillary type rather than the muscle-invasive type of urinary bladder cancer in humans. The preneoplastic and neoplastic lesions observed suggest progression from hyperplasia to papilloma to papillary carcinoma. With neoplastic progression (hyperplasia to papilloma to carcinoma), cyclin D1 immunoreactivity progressively increased in intensity, percentage of cells staining, and distribution. Overexpression of p53 was not found. Cytokeratin 20 staining decreased in superficial cells, while uroplakin III staining increased in intermediate and basal cells with progression from hyperplasia to carcinoma. The results are consistent with increased cell cycle dysregulation or proliferation (cyclin D1), decreased differentiation (cytokeratin 20), and abnormal differentiation (uroplakin III) as lesions progress toward malignancy.

[Clinicopathologic features observation of ovarian transitional cell tumors].

OBJECTIVE: To assess clinical and pathological features of ovarian transitional cell tumors. METHODS: Fourteen cases of ovarian transitional cell carcinoma (TCC) were selected and investigated for their clinical and pathological features. Their immunohistochemical profiles were compared with 12 cases of serous adenocarcinoma (SC) admixed with TCC and 4 cases of EC admixed with TCC 20 cases of pure high-grade serous adenocarcinoma (HG-SC), 15 cases of endometrioid adenocarcinoma (EC), 6 cases of Brenner tumor (BT, 2 cases of malignant BT and 4 cases of benign BT). RESULTS: The patients' age ranged from 36-63 years (mean, 56 years). All cases underwent surgery and postoperative chemotherapy with TP or CAP program. Clinical follow-up was available in 9 cases, of which 2 patients died. Histologically, all cases showed features of transitional cell carcinoma without BT component. Immunohistochemically, 13 of 14 TCCs were positive for WT-1 and all were positive for CK7, ER, PR and CA125, but negative for Uroplakin III and CK20.Similar immunohistochemical staining patterns were seen in SC admixed with TCC and pure HG-SC. Percentage of the 14 TCC cases were also diffusely positive for BRCA1. All SCs admixed with TCC and pure HG-SCs were diffusely or heterogeneously positive for WT-1, with a sharp contrast and mottled distribution pattern in the heterogeneous cases. All TCCs were diffusely and strongly positive for p53, while 16 of 20 cases of pure HG-SC were positive. The positive ratio of p53 in SCs admixed with TCC cases was 11/12.WT-1 expression in TCCs was significantly higher than BTs, ECs and ECs admixed with TCC (P < 0.01), while no obvious difference was seen when compared with SCs admixed with TCC and pure HG-SCs.SCs admixed with TCC, TCCs and EC were positive for BRCA1 except pure ECs and BTs. The positive rate of Ki-67 of BTs was low, while it was higher in TCCs, SCs admixed with TCC and pure HG-SCs. Only BTs expressed Uroplakin III. CONCLUSIONS: Ovarian TCC has characteristic morphological and immunohistochemical features, similar to SC but different from BT. Therefore, TCC should be considered as a morphological variant of HG-SC.

Differences in expression of uroplakin III, cytokeratin 7, and cyclooxygenase-2 in canine proliferative urothelial lesions of the urinary bladder.

The expression of immunohistochemical markers that have been used in diagnosis and/or prognostication of urothelial tumors in humans (uroplakin III [UPIII], cytokeratin 7 [CK7], cyclooxygenase-2 [COX-2], and activated caspase 3) was evaluated in a series of 99 canine proliferative urothelial lesions of the urinary bladder and compared to the lesion classification and grade as defined by the World Health Organization / International Society of Urologic Pathology consensus system. There were significant associations between tumor classification and overall UPIII pattern (P = 1.49 × 10(-18)), loss of UPIII (P = 1.27 × 10(-4)), overall CK7 pattern (P = 4.34 × 10(-18)), and COX-2 pattern (P = 8.12 × 10(-25)). In addition, there were significant associations between depth of neoplastic cell infiltration into the urinary bladder wall and overall UPIII pattern (P = 1.54 × 10(-14)), loss of UPIII (P = 2.07 × 10(-4)), overall CK7 pattern (P = 1.17 × 10(-13)), loss of CK7 expression (P = .0485), and COX-2 pattern (P = 8.23 × 10(-21)). There were no significant associations between tumor classification or infiltration and caspase 3 expression pattern.

Upk3b is dispensable for development and integrity of urothelium and mesothelium.

The mesothelium, the lining of the coelomic cavities, and the urothelium, the inner lining of the urinary drainage system, are highly specialized epithelia that protect the underlying tissues from mechanical stress and seal them from the overlying fluid space. The development of these epithelia from simple precursors and the molecular characteristics of the mature tissues are poorly analyzed. Here, we show that uroplakin 3B (Upk3b), which encodes an integral membrane protein of the tetraspanin superfamily, is specifically expressed both in development as well as under homeostatic conditions in adult mice in the mesothelia of the body cavities, i.e., the epicardium and pericardium, the pleura and the peritoneum, and in the urothelium of the urinary tract. To analyze Upk3b function, we generated a creERT2 knock-in allele by homologous recombination in embryonic stem cells. We show that Upk3bcreERT2 represents a null allele despite the lack of creERT2 expression from the mutated locus. Morphological, histological and molecular analyses of Upk3b-deficient mice did not detect changes in differentiation or integrity of the urothelium and the mesothelia that cover internal organs. Upk3b is coexpressed with the closely related Upk3a gene in the urothelium but not in the mesothelium, leaving the possibility of a functional redundancy between the two genes in the urothelium only.

Value of uroplakin III in distinguishing variants of primary bladder urothelial carcinoma from malignancy metastatic to the urinary bladder.

BACKGROUND: Urothelial carcinoma (UC) variants can be difficult to differentiate from carcinoma metastatic to the bladder. MATERIALS AND METHODS: We examined immunostaining for uroplakin III in 43 cases of primary bladder UC variants including micropapillary UC (n=19), nested variant of UC (n=2), pleomorphic giant-cell carcinoma (n=8), plasmacytoid UC (n=4), lymphoepithelioma-like carcinoma (n=2), large cell undifferentiated carcinoma (n=2), UC with abundant myxoid stroma (n=3) and lipid cell variant (n=3) and in 11 tumors from other organs metastatic to the bladder. These tumors included invasive ductal carcinoma of the breast (n=2), colorectal adenocarcinoma (n=4), endometrioid adenocarcinoma (n=1) and serous papillary carcinoma of the uterus (n=1) melanoma (n=1), embryonal carcinoma of the testis (n=1), and renal clear cell carcinoma (n=1). RESULTS: Out of the 43 UC variants, 35 (81%) were positive for uroplakin III, including micropapillary, lipid cell variant and UC with abundant myxoid stroma. Pleomorphic giant cell carcinoma, plasmacytoid UC and nested variant of UC were less commonly positive. Of the 11 metastatic tumors, six were found to be positive for uropIakin III: metastatic colorectal adenocarcinoma, clear cell carcinoma of the kidney and embryonal carcinoma of testis. CONCLUSION: UP III Positivity for uroplakin III is not found only in primary bladder UC variants, but in some tumors that have metastatized to the bladder. Staining for uroplakin III alone should not be taken as evidence of UC.

A newly developed uroplakin II antibody with increased sensitivity in urothelial carcinoma of the bladder.

CONTEXT: Uroplakin II is a 15-kDa protein component of the urothelial plaques that enhance the permeability barrier and strength of the urothelium. Studies have shown uroplakin II messenger RNA to be expressed in bladder cancer tissues and peripheral blood of patients with urothelial carcinoma. Little is known about the protein expression of uroplakin II in urothelial carcinoma, possibly because of the absence of a commercially available uroplakin II antibody. Pathologists have used the uroplakin III antibody (AU1) to identify tumors of urothelial origin; however, the use of AU1 is limited because of its poor sensitivity. OBJECTIVES: To evaluate a newly developed mouse monoclonal uroplakin II antibody (BC21) in urothelial carcinoma and to compare it with previously developed mouse monoclonal uroplakin III (BC17 and AU1). DESIGN: Uroplakin II and III antibodies were optimized for staining using a horseradish peroxidase-polymer detection system and were visualized with 3,3'-diaminobenzidine. RESULTS: BC21, BC17, and AU1 demonstrated sensitivities in urothelial carcinoma of the bladder of 79% (44 of 56), 55% (31 of 56) (P = .002), and 34% (19 of 56) (P < .001), respectively. Subsequently, the increased staining sensitivity and intensity of BC21, compared with BC17, was validated in a larger study (134 of 174; 77% and 94 of 174; 54%, respectively) (P < .001). BC21 was found to be highly specific when evaluated in various normal and neoplastic tissues, including prostatic and renal carcinomas. CONCLUSIONS: The mouse monoclonal uroplakin II antibody (BC21) demonstrated superior sensitivity and specificity in urothelial carcinoma, compared with uroplakin III (BC17 and AU1), suggesting its advantages in the differential diagnosis of urothelial carcinoma and in the detection of tumors of unknown origin.

The egg membrane microdomain-associated uroplakin III-Src system becomes functional during oocyte maturation and is required for bidirectional gamete signaling at fertilization in Xenopus laevis.

In Xenopus laevis, sperm-egg interaction promotes partial proteolysis and/or tyrosine phosphorylation of uroplakin III (UPIII) and the tyrosine kinase Src, which both localize to the cholesterol-enriched egg membrane microdomains (MDs). Here we show that sperm promote proteolysis and/or tyrosine phosphorylation of UPIII and Src in MDs isolated from ovulated and unfertilized eggs (UF-MDs). An antibody against the extracellular domain of UPIII interferes with these events. Inhibition of fertilization by anti-UPIII antibody is rescued by co-incubation with UF-MDs. This suggests that, like MDs in intact eggs, the isolated UF-MDs are capable of interacting with sperm, an interaction that does not interfere with normal fertilization but rather augments the ability of sperm to fertilize eggs pretreated with anti-UPIII antibody. This unexpected effect of UF-MDs on sperm requires UPIII function in UF-MDs and protein kinase activity in sperm. MDs isolated from progesterone-treated mature oocytes, but not ovarian immature oocytes, are similarly functional as UF-MDs. The anti-UPIII extracellular domain antibody binds more effectively to the surface of mature than immature ovarian oocytes. We propose that the structural and functional competency of the UPIII-Src signaling system in MDs is strictly regulated during oocyte maturation and subsequently in sperm-mediated egg activation and fertilization. The fertilization-related signaling properties seen in UF-MDs can be partially reconstituted in MDs of human embryonic kidney 293 cells (293-MDs) expressing UPIII, Src and uroplakin Ib. However, 293-MDs expressing a proteolysis-resistant mutant of UPIII are less functional, suggesting that the availability of UPIII to protease action is important for MD function.

Significance of uroplakin III expression in recurrence of solitary muscle non-invasive bladder cancer.

Numerous immunohistochemical biomarkers for patients with urothelial bladder cancer have been identified in order to predict their biological behavior. The aim of this present study was to examine the uroplakin III (UPIII) expression in homogenous group of non-muscle invasive bladder cancer and to correlate its value with clinico-pathological characteristics of patients and moreover with COX-2 expression and tumor infiltrating lymphocytes (TILs). Tumor specimens from 127 patients with non-muscle invasive bladder cancer, divided into two groups: patients who developed recurrent disease during the first five post-operative years (N=78) and patients without recurrent disease during a follow-up of minimum 5 years (N=49), were retrieved for tissue microarrays construction. On paraffin sections, the immunohistochemical analysis of UPIII expression was performed and staining was semiquantitatively evaluated. Expression of UPIII, including luminal, membranous and cytoplasmic one, was found in more than half of the tumors (57%). Specific staining pattern for UPIII was not associated with age and gender of patients, pathological grade, tumor size, disease stage or recurrence of disease. There was no association between UPIII, COX-2 and TILs, except for a negative moderate association between UP and COX-2 in the group of patients without recurrent tumor, and a strong association between UPIII and in the group with tumor recurrence. The present work gives an insight into the very complex mechanisms involved in tumor biology and progression. Moreover, it highlights the importance of further studies that should include multiple molecular markers in models designed to predict the outcome of non-muscle invasive bladder cancer.

Epithelial urinary bladder tumors from cows with enzootic hematuria: structural and cell cycle-related protein expression.

Thirty-nine epithelial bladder tumor samples from 37 animals affected with bovine enzootic hematuria (BEH) were selected for immunohistochemical studies. The expression of structural proteins such as uroplakin III (UPIII) and cytokeratin 7 (CK7) and the cell cycle-related proteins cyclin D1 and p53 were evaluated in urothelial papillomas and carcinomas. Loss of UPIII and CK7 expression was seen in both high-grade and high-stage urothelial carcinomas (P < .001 and P < .02). Cyclin D1 expression showed no statistically significant association with grade or stage. In contrast, p53 immunoreactivity was positive in high-grade and high-stage carcinomas (P < .05 and P < .01), confirming its association with the highest malignant behavior of the bladder tumors in BEH.

[Plasmacytoid urothelial carcinoma of the urinary bladder: a clinicopathologic study of 16 cases].

OBJECTIVE: To study the clinicopathologic features and prognosis of plasmacytoid urothelial carcinoma (PUC) of the urinary bladder. METHODS: The clinical and pathologic findings of 16 cases of PUC were retrospectively reviewed. Immunohistochemical study (MaxVision method) was carried out. The follow-up data were analyzed. RESULTS: There were altogether 15 males and 1 female. The age of patients ranged from 40 years to 85 years (median = 64 years). Most patients (15/16) presented with hematuria. The tumor cells were small to medium in size and contained eccentric nuclei and moderate to abundant eosinophilic cytoplasm, assuming a plasmacytoid appearance. The architectural pattern varied from loosely cohesive sheets to cords, papillae, small nests or gland-like structures. Most tumors invaded into the lamina propria or muscularis propria. Twelve of the 16 cases had concurrent conventional urothelial carcinoma component. Immunohistochemical study showed that the tumor cells in all cases were strongly positive for AE1/AE3, epithelial membrane antigen, CK7 and CK18. CK20 and uroplakin III were also expressed in 9 cases. CEA, p53, CD138, p63 and E-cadherin were positive in 12, 13, 15, 11 and 10 cases, respectively. Ki-67 index ranged from 5% to 70% (mean = 30%). All tumors were negative for vimentin, LCA, kappa/lambda light chains, S-100 protein, HMB 45,Melan A, smooth muscle actin and desmin. Follow-up information was available in 13 patients. The duration of follow up ranged from 3 months to 10 years. Three patients died of distant metastasis at 3, 27 and 60 months after the operation, respectively. One patient was alive with disease at 25 months. One was alive at 43 months with a prior recurrence. Another 8 patients were alive and disease free at 7 to 120 months. CONCLUSIONS: PUC of the urinary bladder is a rare variant of high-grade urothelial carcinoma. Immunohistochemical study with positivity for CK7, CK20, p63 and uroplakin III and negative staining for vimentin and LCA may be helpful in the differential diagnosis. PUC is a malignant tumor with high invasiveness, high recurrence rate and poor prognosis. Radical cystectomy is considered as the first line treatment for PUC.

Cytokeratin 15 marks basal epithelia in developing ureters and is upregulated in a subset of urothelial cell carcinomas.

The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules have been defined which regulate ureteric bud initiation and drive the differentiation of ureteric mesenchyme into peristaltic smooth muscle. Less is known about mechanisms underlying the developmental patterning of the multilayered epithelium characterising the mature ureter. In skin, which also contains a multilayered epithelium, cytokeratin 15 (CK15), an acidic intermediate filament protein, marks cells whose progeny contribute to epidermal regeneration following wounding. Moreover, CK15+ precursor cells in skin can give rise to basal cell carcinomas. In the current study, using transcriptome microarrays of embryonic wild type mouse ureters, Krt15, coding for CK15, was detected. Quantitative polymerase chain reaction analyses confirmed the initial finding and demonstrated that Krt15 levels increased during the fetal period when the ureteric epithelium becomes multilayered. CK15 protein was undetectable in the ureteric bud, the rudiment from which the ureter grows. Nevertheless, later in fetal development, CK15 was immunodetected in a subset of basal urothelial cells in the ureteric stalk. Superficial epithelial cells, including those positive for the differentiation marker uroplakin III, were CK15-. Transformation-related protein 63 (P63) has been implicated in epithelial differentiation in murine fetal urinary bladders. In wild type fetal ureters, CK15+ cells were positive for P63, and p63 homozygous null mutant ureters lacked CK15+ cells. In these mutant ureters, sections of the urothelium were monolayered versus the uniform multilayering found in wild type littermates. Human urothelial cell carcinomas account for considerable morbidity and mortality. CK15 was upregulated in a subset of invasive ureteric and urinary bladder cancers. Thus, in ureter development, the absence of CK15 is associated with a structurally simplified urothelium whereas, postnatally, increased CK15 levels feature in malignant urothelial overgrowth. CK15 may be a novel marker for urinary tract epithelial precursor cells.

Multiple joint metastasis of a transitional cell carcinoma in a dog.

An 8-year-old castrated male hound mix was referred to the Purdue University Veterinary Teaching Hospital for severe lameness, pollakiuria, and dyschezia. On presentation, the dog was nonweight bearing on the right rear limb and the right carpus was diffusely swollen. Synovial fluid analysis from the right carpus revealed a population of epithelial cells displaying marked anisocytosis, anisokaryosis, multinucleation, and prominent, variably sized nucleoli. A metastatic carcinoma with presumed prostatic or urothelial origin was diagnosed based on cytomorphology. Subsequent cytologic evaluation of peripheral lymph nodes revealed the presence of a similar neoplastic population. The dog was euthanized and synovial fluid from both stifle joints, as well as impression smears of the prostate gland, were collected. Carcinoma cells were identified in each stifle joint and in the prostate gland. Immunocytochemistry was performed on synovial fluid smears from 2 of the joints (right stifle and right carpus) and on impression smears of the prostate gland. The neoplastic population in the joints and prostate gland showed strong immunoreactivity to uroplakin III, a urothelial marker, indicating metastasis of a transitional cell carcinoma to multiple joints. In addition, evidence for epithelial to mesenchymal transition was identified using cytokeratin, an epithelial marker, and vimentin, a mesenchymal marker. A necropsy was performed and histopathology confirmed the presence of metastatic transitional cell carcinoma in various tissues. This case illustrates the importance of considering metastatic disease when a patient is presented with severe lameness and joint pain, and the clinical utility of synovial fluid cytology for diagnosis of metastasis in these cases.

Immunohistochemical profile to distinguish urothelial from squamous differentiation in carcinomas of urothelial tract.

Urothelial neoplasms with squamous morphology raise the differential diagnosis between pure primary squamous cell carcinoma, urothelial carcinoma with squamous differentiation and secondary involvement by squamous cell carcinoma, for example, from uterine cervix. Accurate identification between these entities is critical due to differing prognosis and therapeutic strategies. We evaluated the utility of an immunohistochemical panel of 3 urothelial-associated antibodies (uroplakin III, S100P, and GATA3) and two squamous-associated antibodies (CK14 and desmoglein-3) in 50 primary urothelial neoplasms: 15 pure urothelial carcinomas, 12 pure squamous cell carcinomas and 23 urothelial carcinomas with squamous differentiation. Squamous differentiation was defined by intercellular bridges or evidence of keratinization. Pure squamous cell carcinomas were positive for CK14 (100%) and desmoglein-3 (75%), negative for GATA3 and uroplakin III; one case was S100P positive (9%). Pure urothelial carcinomas had an opposite pattern and were positive for S100P (93%), GATA3 (93%), and uroplakin III (67%) and were negative for desmoglein-3; CK 14 was positive in 27% of cases; 74% of urothelial carcinomas with squamous differentiation had expression of urothelial and squamous associated markers (S100P, 83%; GATA3, 35%; uroplakin III, 13%; CK14, 87%; and desmoglein-3, 70%), although reactivity for individual markers within some tumors did not always correspond with morphologic differentiation. Of the remaining 26%, 4 showed an overall "squamous" immunoprofile, whereas 2 cases showed a "urothelial" immunoprofile. Our study showed that a panel of five antibodies identifies squamous and urothelial differentiation in most instances suggesting potential diagnostic utility.