[Three cases of IgG4-related focal retroperitoneal fibrosis].

(Case 1) A 63-year-old man was diagnosed as retroperitoneal fibrosis by the exploratory laparotomy for the pelvic mass with high IgG4 levels. (Case 2) A 64-year-old man had past medical history of autoimmune pancreatitis which was treated by steroid use. Three years later, he was diagnosed as IgG4-related gallbladder tumor by the cholecystectomy. And, then he was diagnosed as right hydronephrosis with high IgG4 levels. (Case 3) A 71-year-old man was diagnosed as left hydronephrosis and pelvic mass by computerized tomography with high IgG4 levels. We reported three cases of IgG4-related focal retroperitoneal fibrosis. All cases had pelvic mass with high IgG4 levels and were also treated effectively with steroid use. Those symptoms of the patients occurred in close association with IgG4 levels. It is necessary to acknowledge that retroperitoneal fibrosis may have aspects of IgG4-related systemic disease and that the measurement of serum IgG4 should be considered for diagnosing and treating the conditions.

Combined inhibition of Wee1 and Hsp90 activates intrinsic apoptosis in cancer cells.

Heat shock protein 90 (Hsp90) is an essential, evolutionarily conserved molecular chaperone. Cancer cells rely on Hsp90 to chaperone mutated and/or activated oncoproteins, and its involvement in numerous signaling pathways makes it an attractive target for drug development. Surprisingly, however, the impact of Hsp90 inhibitors on cancer cells is frequently cytostatic in nature, and efforts to enhance the antitumor activity of Hsp90 inhibitors in the clinic remain a significant challenge. In agreement with previous data obtained using Wee1 siRNA, we show that dual pharmacologic inhibition of Wee1 tyrosine kinase and Hsp90 causes cancer cells to undergo apoptosis in vitro and in vivo. Gene expression profiling revealed that induction of the intrinsic apoptotic pathway by this drug combination coincided with transcriptional downregulation of Survivin and Wee1, an outcome not seen in cells treated separately with either agent. At the translational level, expression of these two proteins, as well as activated Akt, was completely abrogated. These data support the hypothesis that Wee1 inhibition sensitizes cancer cells to Hsp90 inhibitors; they establish combined Wee1/Hsp90 inhibition as a novel therapeutic strategy; and they provide a mechanistic rationale for enhancing the pro-apoptotic activity of Hsp90 inhibitors.

Methoxychalcone inhibitors of androgen receptor translocation and function.

Androgen receptor activity drives incurable castrate-resistant prostate cancer. All approved antiandrogens inhibit androgen receptor-driven transcription, and in addition the second-generation antiandrogen MDV3100 inhibits ligand-activated androgen receptor nuclear translocation, via an unknown mechanism. Here, we report methoxychalcones that lock the heat shock protein 90-androgen receptor complex in the cytoplasm in an androgen-non-responsive state, thus demonstrating a novel chemical scaffold for antiandrogen development and a unique mechanism of antiandrogen activity.

Perioperative complications of radical cystectomy after induction chemoradiotherapy in bladder-sparing protocol against muscle-invasive bladder cancer: a single institutional retrospective comparative study with primary radical cystectomy.

OBJECTIVE: To compare rates of early morbidity after radical cystectomy in patients treated with or without induction chemoradiotherapy (CRT) using a standardized reporting methodology. METHODS: All 193 consecutive patients undergoing radical cystectomy for bladder cancer between 1989 and 2010 were retrospectively reviewed. Induction chemoradiotherapy consists of radiation at 40 Gy to the small pelvis and two cycles of concurrent cisplatin at 20 mg/day for 5 days. Deaths within 90 days after radical cystectomy and complications arising within 30 days were recorded and graded according to the Clavien-Dindo classification. Grades 1-2 were considered minor; Grades 3-5 were considered major. RESULTS: Eighty-seven patients underwent radical cystectomy following chemoradiotherapy (chemoradiotherapy group) while the remaining 106 primarily underwent radical cystectomy (no chemoradiotherapy group). No Grade 4-5 complication was observed. Overall, 118 patients (61%) experienced 36 major and 122 minor complications. There was no significant difference in the incidence of overall complications between the chemoradiotherapy and no chemoradiotherapy groups (67 vs. 57%). Overall urinary anastomosis-related complications and major gastrointestinal complications, most of which were Grade 3 ileus, were more frequent in the chemoradiotherapy group than the no chemoradiotherapy group (11 vs. 2%, P = 0.007; and 14 vs. 4%, P = 0.02; respectively). Multivariate analysis identified induction chemoradiotherapy as an independent risk factor for overall urinary anastomosis-related complications (relative risk 6.0, P = 0.01) but not for major gastrointestinal complications. CONCLUSIONS: Induction chemoradiotherapy at 40 Gy in bladder-sparing protocols against MIBC is unlikely to increase the rate of severe complications of radical cystectomy.

Targeting the regulation of androgen receptor signaling by the heat shock protein 90 cochaperone FKBP52 in prostate cancer cells.

Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormone-bound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.

A novel repeat biopsy nomogram based on three-dimensional extended biopsy.

OBJECTIVES: To develop a nomogram based on a cohort examined with 3-dimensional (3D) protocol for diagnosis of prostate cancer on repeat biopsy. METHODS: Of 4074 consecutive men undergoing prostate biopsy at our institutions between 2000 and 2009, 775 men with at least 1 previous negative biopsy underwent repeat biopsy with a 3D protocol. Men with previous atypical glands or atypical small acinar proliferation and/or without available prostate-specific antigen (PSA) kinetics information were excluded. The remaining 515 men constituted the study cohort. We developed a logistic regression-based nomogram with 70% of the cohort selected randomly; we validated it with the remaining 30%. Predictive accuracy and performance characteristics were assessed using the area under the receiver operating characteristic curve (AUC) and calibration plots, respectively. The threshold probability was evaluated with decision curve analysis. RESULTS: We developed a novel repeat biopsy nomogram incorporating age, free to total PSA ratio, prostate volume, history of previous extended biopsy, and PSA doubling time. Validation confirmed predictive accuracy with an AUC value of 0.791. Calibration plots showed good agreement. The decision curve of the nomogram was superior to the decision curve of biopsying all men in a range of threshold probability over 0.15. At the threshold of 0.2, the number of unnecessary biopsies could be reduced by 10 per 100, without missing prostate cancer. CONCLUSIONS: We developed a novel repeat biopsy nomogram based on a cohort examined with 3D protocol. This repeat biopsy nomogram is clinically beneficial, preventing a substantial number of unnecessary biopsies.

Simple prophylactic procedure of inguinal hernia after radical retropubic prostatectomy: isolation of the spermatic cord.

To reduce the incidence of inguinal hernia (IH) after radical retropubic prostatectomy (RRP), a simple prophylactic procedure was carried out during RRP. A consecutive 82 patients who had undergone RRP for clinically localized prostate cancer between July 2002 and October 2006 at Toride Kyodo General Hospital were enrolled. From July 2002 to November 2003, 20 patients underwent conventional RRP. Thereafter, 62 patients underwent conventional RRP with blunt dissection of the peritoneum at the internal inguinal ring and isolation of the spermatic cord from the peritoneum as a prophylactic procedure for IH. There was no significant difference in patient characteristics between the two groups. In the conventional RRP group, IH occurred in 10 patients during a median range follow-up period of 41 (1 to 73) months. In contrast, in the RRP plus prophylactic procedure group, IH occurred in one patient (1.6%) during a median range follow-up period of 41 (25 to 59) months. The incidence of IH after RRP plus the prophylactic procedure was significantly lower than that after conventional RRP, indicating the efficacy of the presented procedure.

[Primary adenocarcinoma of the female urethra treated by multimodal therapy].

A 64-year old female presented with urinary retention. Physical examination revealed a firm mass on the anterior vaginal wall. Magnetic resonance imaging showed a tumor surrounding the urethra, which invaded to the vesical triangle and the anterior vaginal wall. Serum levels of carcinoembryonic antigen and carbohydrate antigen 19-9 were elevated, but squamous cell carcinoma antigen and prostate specific antigen were within normal limits. Pathological examinations of the transurethral and transvaginal needle biopsy specimen suggested mucinous adenocarcinoma. First, the patient received local chemoradiotherapy and systemic chemotherapy using a fluoropyrimidine drug TS-1 and cisplatin. The tumor markers declined to within normal limits after this preoperative therapy. Then she underwent total cysto-urethrectomy with anterior vaginal wall resection, pelvic lymphadenectomy, and urinary diversion with ureterocutaneous fistula. Histopathological examination of the surgical specimen showed mutinous adenocarcinoma invading to the vesical triangle and the anterior vaginal wall. No metastasis was found in the lymph nodes. The final diagnosis was urethral adenocarcinoma, pT4N0, Stage IV. Five months after surgery, local recurrence and distant metastases appeared, and she died 14 months after surgery.

Glucocorticoids suppress tumor lymphangiogenesis of prostate cancer cells.

PURPOSE: Glucocorticoids such as prednisone, hydrocortisone, and dexamethasone are known to provide some clinical benefit for patients with hormone-refractory prostate cancer. However, the underlying mechanisms by which glucocorticoids affect hormone-refractory prostate cancer progression are not well established as yet. Our previous study has shown that glucocorticoids inhibit tumor angiogenesis possibly by down-regulation of vascular endothelial growth factor (VEGF) and interleukin 8. Here, we hypothesized that the therapeutic effect of dexamethasone on hormone-refractory prostate cancer can be partly attributed to a direct inhibition of lymphangiogenesis through the glucocorticoid receptor by down-regulating a major lymphangiogenic factor, VEGF-C. EXPERIMENTAL DESIGN: The effects of dexamethasone on the expression of VEGF-C and its receptor, VEGF receptor-3 (VEGFR-3), were examined using an androgen-independent human prostate cancer cell line, DU145, which expresses glucocorticoid receptor. The effects of dexamethasone on tumor-associated lymphangiogenesis in DU145 xenografts were determined by analyzing VEGF-C gene expression, lymphatic vessel density, and relative lymphatic vessel area. RESULTS: Dexamethasone significantly down-regulated VEGF-C gene expression and protein production by 48% (P = 0.003) and 44% (P = 0.002), respectively, under normoxic condition. Similarly, hydrocortisone down-regulated VEGF-C gene expression. The effects of dexamethasone were completely reversed by the glucocorticoid receptor antagonist RU486. Even under hypoxia-like conditions, dexamethasone inhibited VEGF-C gene expression. In DU145 xenografts, dexamethasone significantly down-regulated VEGF-C gene expression and decreased lymphangiogenesis. Dexamethasone did not affect VEGFR-3 gene expression in vitro and in vivo. CONCLUSION: Glucocorticoids suppressed tumor-associated lymphangiogenesis by down-regulating VEGF-C through glucocorticoid receptor in androgen-independent prostate cancer cells in vivo.

Glucocorticoids suppress tumor angiogenesis and in vivo growth of prostate cancer cells.

PURPOSE: Glucocorticoids, such as prednisone, hydrocortisone, and dexamethasone, are known to produce some clinical benefit for patients with hormone-refractory prostate cancer (HRPC). However, the underlying mechanisms by which glucocorticoids affect HRPC growth are not well established as yet. Here, we hypothesize that the therapeutic effect of glucocorticoids on HRPC can be attributed to a direct inhibition of angiogenesis through the glucocorticoid receptor by down-regulating two major angiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). EXPERIMENTAL DESIGN: The effects of dexamethasone on VEGF and IL-8 expression and cell proliferation were examined using DU145, which expresses glucocorticoid receptor. The effects of dexamethasone on DU145 xenografts were determined by analyzing VEGF and IL-8 gene expression, microvessel density, and tumor volume. RESULTS: Dexamethasone significantly down-regulated VEGF and IL-8 gene expression by 50% (P < 0.001) and 89% (P < 0.001), respectively, and decreased VEGF and IL-8 protein production by 55% (P < 0.001) and 74% (P < 0.001), respectively, under normoxic condition. Similarly, hydrocortisone down-regulated VEGF and IL-8 gene expression. The effects of dexamethasone were completely reversed by the glucocorticoid receptor antagonist RU486. Even under hypoxia-like conditions, dexamethasone inhibited VEGF and IL-8 expression. In DU145 xenografts, dexamethasone significantly decreased tumor volume and microvessel density and down-regulated VEGF and IL-8 gene expression, whereas dexamethasone did not affect the in vitro proliferation of the cells. CONCLUSION: Glucocorticoids suppressed androgen-independent prostate cancer growth possibly due to the inhibition of tumor-associated angiogenesis by decreasing VEGF and IL-8 production directly through glucocorticoid receptor in vivo.

Suppression of VEGF transcription in renal cell carcinoma cells by pyrrole-imidazole hairpin polyamides targeting the hypoxia responsive element.

Hypoxia inducible factor (HIF), a master regulator of critical genes for cell survival under hypoxic conditions, is known to be related to tumorigenesis and progression of renal cell carcinoma. N-methylpyrrole (Py)-N-methylimidazole (Im) hairpin polyamides are synthetic organic compounds that recognize and bind to the minor grooves of specific DNA sequences. We synthesized three Py-Im hairpin polyamides targeting the flanking sequences of hypoxia responsive element (HRE; a binding site of HIF) in the promoter region of the vascular endothelial growth factor (VEGF) gene. The effects of the polyamides on HIF-induced transcription were evaluated by a luciferase assay using a reporter plasmid containing a VEGF promoter. Real time reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay were performed to examine the effects of the polyamides on the transcription and secretion of VEGF in A498 renal cell carcinoma cells, which have a frame-shift mutation in the von Hippel-Lindau gene. A combination of three Py-Im hairpin polyamides suppressed HIF-induced transcription in reporter assays using 293 cells and successfully suppressed transcription and translation of the VEGF gene in A498 cells. Inhibition of the HIF-HRE interaction was confirmed by an electrophoresis mobility shift assay. An approach using Py-Im hairpin polyamides may be a new strategy for the treatment of renal cell carcinoma.

[A case of idiopathic retroperitoneal fibrosis accompanied by asynchronous bilateral urinoma].

The patient was a 68-year-old female with the complaint of left flank pain. On examination, hydronephrosis of the left kidney and the urinoma of left retroperitoneal space were disclosed by abdominal computed tomographic (CT) scan. The urinoma disappeared spontaneously, but 4 months later right hydronephrosis and the urinoma of ipsilateral retroperitoneum emerged. Enhanced CT scan revealed aneurysm of bilateral common iliac arteries and perianeurysmal fibrotic mass. After steroid therapy under the diagnosis of idiopathic retroperitoneal fibrosis (IRPF), the fibrotic mass reduced in size and the urinoma disappeared. We should be aware that urinomas, which are thought to be caused by sudden obstruction of the upper urinary tract, could be brought about by IRPF.

Hypotaurocyamine kinase evolved from a gene for arginine kinase.

Hypotaurocyamine kinase (HTK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase (AK). HTK is found only in sipunculid worms, and it shows activities for both the substrates hypotaurocyamine and taurocyamine. Determining how HTK evolved in sipunculids is particularly insightful because all sipunculid-allied animals have AK and only some sipunculids have HTK. We determined the cDNA sequence of HTK from the sipunculid worm Siphonosoma cumanense for the first time, cloned it in pMAL plasmid and expressed it in E. coli as a fusion protein with maltose-binding protein. The cDNAderived amino acid sequence of Siphonosoma HTK showed high amino acid identity with molluscan AKs. Nevertheless, the recombinant enzyme of Siphonosoma HTK showed no activity for the substrate arginine, but showed activity for taurocyamine. Comparison of the amino acid sequences of HTK and AK indicated that the amino acid residues necessary for the binding of the substrate arginine in AK have been completely lost in Siphonosoma HTK sequence. The phylogenetic analysis indicated that the HTK amino acid sequence was placed just outside the molluscan AK cluster, which formed a sister group with the arthropod and nematode AKs. These results suggest that Siphonosoma HTK evolved from a gene for molluscan AK. Moreover, to confirm this assertion, we determined by PCR that the gene for Siphonosoma HTK has a 5-exon/4-intron structure, which is homologous with that of the molluscan AK genes. Further, the positions of splice junctions were conserved exactly between the two genes. Thus, we conclude that Siphonosoma HTK has evolved from a primordial gene for molluscan AK.

Down-regulation of vascular endothelial growth factor in renal cell carcinoma cells by glucocorticoids.

Metastatic renal cell carcinomas (RCC) remain highly resistant to systemic therapy. RCCs are highly vascular tumors, which overproduce angiogenic peptides such as vascular endothelial growth factor (VEGF) even under normoxic conditions. A potential suggested role of antiangiogenic therapeutic strategies is the treatment of RCC by inhibiting VEGF production. The down-regulation of VEGF expression by glucocorticoids has recently been demonstrated in several cells. In this study, the direct effects of glucocorticoids on VEGF production by RCC cells were evaluated. Four RCC cell lines A498, RCC270, Caki1, and ACHN were treated with dexamethasone (DEX), hydrocortisone (HC), 5-alpha-dihydrotestosterone (DHT), or estradiol (E2). RU486 was used as a glucocorticoid receptor (GR) antagonist. Cell growth was studied with MTS assays. VEGF mRNA and protein were evaluated with quantitative real-time RT-PCR and ELISA, respectively, and GR expression was examined using RT-PCR and immunocytochemistry. All four RCC cell lines expressed GR. DEX at 100 nM down-regulated VEGF secretions by more than 50% in three lines (A498, RCC270, and Caki1) and had a weak inhibitory effect on ACHN cells. The effect of DEX on reducing VEGF mRNA levels in A498 cells was concentration-dependent and maximal at 100 nM (80% inhibition). HC had similar but weaker effects on VEGF production in the RCC cells, but E2 and DHT had no effect. RU486 reversed the effects of DEX. DEX at 1-1000 nM did not affect cell growth in any of the four RCC cell lines. This is the first study showing that glucocorticoids, at concentrations achievable in vivo by oral administration of low doses of DEX, have an inhibitory effect on VEGF mRNA expression and protein secretion of RCC cells possibly through the GR pathway. Furthermore, DEX might have a potential role in antiangiogenic therapeutic strategies by inhibiting VEGF production during metastatic RCC treatment.

p51/p63 Controls subunit alpha3 of the major epidermis integrin anchoring the stem cells to the niche.

p51/p63, a member of the tumor suppressor p53 gene family, is crucial for skin development. We describe here identification of ITGA3 encoding integrin alpha(3) as a target of its trans-activating function, proposing that p51/p63 allows epidermal stem cells to express laminin receptor alpha(3)beta(1) for anchorage to the basement membrane. When activated by genotoxic stress or overexpressed ectopically in non-adherent cells, p51/p63 transduced a phenotype to attach to extracellular matrices, which was accompanied by expression of ITGA3. Motifs matching the p53-binding consensus sequence were located in a scattered form in intron 1 of human ITGA3, and served as p51/p63-responsive elements in reporter assays. In addition to the trans-activating ability of the TA isoform, we detected a positive effect of the DeltaN isoform on ITGA3. The high level alpha(3) production in human keratinocyte stem cells diminished upon elimination of p51/p63 by small interfering RNA or by Ca(2+)-induced differentiation. Furthermore, a chromatin immunoprecipitation experiment indicated a physical interaction of p51/p63 with intron 1 of ITGA3. This study provides a molecular basis for the standing hypothesis that p51/p63 is essential for epidermal-mesenchymal interactions.

[A case bilharziasis in a Japanese male].

Bilharziasis is an endemic disease distributed mostly in African countries and the Middle East, and causes severe disturbances of urinary tract secondarily. Although it used to be a very rare disease in Japan, modern human mobility and jet travel have brought this tropical disease into our country far from endemic areas. A 25-year-old Japanese male presented to our hospital with macroscopic hematuria. He had an experience of traveling to Malawi two years before. Cystourethroscopy demonstrated so-called 'bilharzial tubercles', many yellowish specks of mucosa at the posterior wall and dome of the bladder. The diagnosis of bilharziasis was made by detection of Schistosoma haematobium eggs in urine and histological specimen obtained by transurethral biopsy. In this case, radiographic and pathological examinations revealed neither obstructive uropathy nor urothelial malignancy. He was treated with praziquantel, and the disease is under good control.

C-H to N substitution dramatically alters the sequence-specific DNA alkylation, cytotoxicity, and expression of human cancer cell lines.

We designed and synthesized sequence-specific alkylating conjugates 1 and 2, which selectively alkylate matched sequences at nanomolar concentrations. Conjugates 1 and 2 differ only in that the C-H is substituted by an N in the second ring, which precisely recognizes and effectively alkylates DNA according to the recognition rule of Py-Im polyamides. We investigated sequence-specific DNA alkylation, cytotoxicity in 39 human cancer cell lines, and the effect on expression levels in cancer cell lines by Py-Im conjugates 1 and 2. The COMPARE analysis of the mean graphs showed that conjugates 1 and 2 did not correlate well with each other (r = 0.65) despite having a common DNA alkylating mechanism (purine N3 alkylation). Array-based gene expression analysis demonstrated that there are several oppositely regulated genes. The results suggest the intriguing possibility that DNA alkylating agents recognizing longer base-pair sequences may provide a promising approach for developing new types of antigene agents.

Prognostic significance of endothelial Per-Arnt-sim domain protein 1/hypoxia-inducible factor-2alpha expression in a subset of tumor associated macrophages in invasive bladder cancer.

PURPOSE: Endothelial Per-Arnt-Sim domain protein 1 (EPAS1) is induced under hypoxia and it transactivates a series of genes involved in angiogenesis and energy metabolism. Recent studies showed that EPAS1 is expressed in tumor associated macrophages (TAMs), which have multifaceted roles in tumor progression. We hypothesized that EPAS1 expressed in TAMs may contribute to bladder cancer progression. MATERIALS AND METHODS: Clinicopathological and followup data on 69 patients undergoing radical cystectomy for T1-4N0-2M0 high grade bladder urothelial carcinoma were reviewed. Quantitative immunohistochemical analysis of TAMs and EPAS1 was performed separately in invasive front and in other superficial parts of carcinoma tissues. TAM counts and EPAS1 positive cell counts were compared with pathological variables and cancer specific survival (CSS). RESULTS: The 5-year CSS rate in the 69 patients was 69% at a median followup of 58 months (range 2 to 196). EPAS1 expression was restricted to a small subset of TAMs. Although TAM counts were not associated with T stage or lymph node metastasis, EPAS1 expressing TAM counts were significantly associated with higher T stage. On univariate and multivariate analyses higher EPAS1 expressing TAM counts in invasive front along with higher T stage and positive lymph node metastasis were significantly associated with shorter CSS, while total TAM counts or EPAS1 expressing TAM counts in other superficial parts did not. CONCLUSIONS: Despite limited prognostic effects of total TAMs EPAS1 expressing TAMs were associated with a poor prognosis of invasive bladder cancer, suggesting that EPAS1 expressed in a subset of TAMs mediates bladder cancer progression.

Impaired p63 expression associates with poor prognosis and uroplakin III expression in invasive urothelial carcinoma of the bladder.

PURPOSE: p63 is proposed to play roles in normal development and differentiation of stratified epithelia including urothelium. We recently reported that impaired p63 expression is a common feature of high-grade invasive urothelial carcinomas and associates with reduced beta-catenin. On the basis of these facts, we proposed that impaired p63 expression contributes to biological aggressiveness of urothelial neoplasms. Uroplakin (UP) III expression was also evaluated to investigate a possible association between loss of p63 expression and terminal urothelial differentiation. EXPERIMENTAL DESIGN: Expression of p63, beta-catenin, and UP III was immunohistochemically analyzed in 75 cystectomy specimens of high-grade invasive bladder carcinoma. p63 expression was semiquantified and compared with pathological parameters, expression of beta-catenin and UP III, and cancer-specific survival. RESULTS: Lower p63 expression was significantly associated with higher Tumor-Node-Metastasis (TNM) stage (P = 0.0004), lymph-node metastasis (P = 0.013), and reduced beta-catenin expression (P = 0.003). By univariate analysis, lower p63 expression, along with TNM stage and lymph-node status, were significantly associated with a poor prognosis (P = 0.0005), whereas reduced beta-catenin was not. By multivariate analysis, the prognostic effect of p63 expression was independent of TNM stage and lymph-node status with marginal statistical significance (P = 0.074). UP III expression was restricted to a subset of p63-negative carcinoma cells, including even anaplastic carcinoma cells. CONCLUSIONS: Impaired p63 expression characterizes biological aggressiveness of high-grade invasive urothelial carcinomas. Moreover, loss of p63 expression is a prerequisite for UP III expression. Our data suggest that p63 plays critical roles in tumor progression and biochemical terminal differentiation of urothelial neoplasms.