Cisplatin resistance driver claspin is a target for immunotherapy in urothelial carcinoma.

Bladder cancer is a major and fatal urological disease. Cisplatin is a key drug for the treatment of bladder cancer, especially in muscle-invasive cases. In most cases of bladder cancer, cisplatin is effective; however, resistance to cisplatin has a significant negative impact on prognosis. Thus, a treatment strategy for cisplatin-resistant bladder cancer is essential to improve the prognosis. In this study, we established a cisplatin-resistant (CR) bladder cancer cell line using an urothelial carcinoma cell lines (UM-UC-3 and J82). We screened for potential targets in CR cells and found that claspin (CLSPN) was overexpressed. CLSPN mRNA knockdown revealed that CLSPN had a role in cisplatin resistance in CR cells. In our previous study, we identified human leukocyte antigen (HLA)-A*02:01-restricted CLSPN peptide by HLA ligandome analysis. Thus, we generated a CLSPN peptide-specific cytotoxic T lymphocyte clone that recognized CR cells at a higher level than wild-type UM-UC-3 cells. These findings indicate that CLSPN is a driver of cisplatin resistance and CLSPN peptide-specific immunotherapy may be effective for cisplatin-resistant cases.

Fluvastatin potentiates anticancer activity of vorinostat in renal cancer cells.

Drug repositioning is an emerging approach to developing novel cancer treatments. Vorinostat is a histone deacetylase inhibitor approved for cancer treatment, but it could attenuate its anticancer activity by activating the mTOR pathway. The HMG-CoA reductase inhibitor fluvastatin reportedly activates the mTOR inhibitor AMP-activated protein kinase (AMPK), and we thought that it would potentiate vorinostat's anticancer activity in renal cancer cells. The combination of vorinostat and fluvastatin induced robust apoptosis and inhibited renal cancer growth effectively both in vitro and in vivo. Vorinostat activated the mTOR pathway, as evidenced by the phosphorylation of ribosomal protein S6, and fluvastatin inhibited this phosphorylation by activating AMPK. Fluvastatin also enhanced vorinostat-induced histone acetylation. Furthermore, the combination induced endoplasmic reticulum (ER) stress that was accompanied by aggresome formation. We also found that there was a positive feedback cycle among AMPK activation, histone acetylation, and ER stress induction. This is the first study to report the beneficial combined effect of vorinostat and fluvastatin in cancer cells.

Ritonavir and ixazomib kill bladder cancer cells by causing ubiquitinated protein accumulation.

There is no curative treatment for advanced bladder cancer. Causing ubiquitinated protein accumulation and endoplasmic reticulum stress is a novel approach to cancer treatment. The HIV protease inhibitor ritonavir has been reported to suppress heat shock protein 90 and increase the amount of unfolded proteins in the cell. If the proteasome functions normally, however, they are rapidly degraded. We postulated that the novel proteasome inhibitor ixazomib combined with ritonavir would kill bladder cancer cells effectively by inhibiting degradation of these unfolded proteins and thereby causing ubiquitinated proteins to accumulate. The combination of ritonavir and ixazomib induced drastic apoptosis and inhibited the growth of bladder cancer cells synergistically. The combination decreased the expression of cyclin D1 and cyclin-dependent kinase 4, and increased the sub-G1 fraction significantly. Mechanistically, the combination caused ubiquitinated protein accumulation and endoplasmic reticulum stress. The combination-induced apoptosis was markedly attenuated by the protein synthesis inhibitor cycloheximide, suggesting that the accumulation of ubiquitinated proteins played an important role in the combination's antineoplastic activity. Furthermore, the combination induced histone acetylation cooperatively and the decreased expression of histone deacetylases was thought to be one mechanism of this histone acetylation. The present study provides a theoretical basis for future development of novel ubiquitinated-protein-accumulation-based therapies effective against bladder cancer.

[CLINICAL ANALYSIS ABOUT PERCUTANEOUS NEEDLE BIOPSY FOR RENAL MASSES].

(Objective) We investigated the efficacy and safety of percutaneous renal mass biopsy retrospectively. (Methods) A retrospective review was performed of 44 patients (46 renal masses) who received ultrasound and/or computed tomography guided percutaneous biopsy of a solid renal mass between April 2004 and December 2012 in National Defense Medical College Hospital. (Results) The median renal mass size was 45 (range 15-140) mm with a median of 2 (1-5) cores taken. Thirteen masses were biopsied for differential diagnosis between RCC and other malignancies (or benign renal tumors), 11 were biopsied for differential diagnosis between RCC and renal pelvic urothelial carcinoma, 10 unresectable masses were biopsied to confirm the diagnosis pathologically before starting medication, and 12 small masses were biopsied before radio-frequency ablation. Of the initial 46 biopsies, 38 (82.6%) were diagnostic. The median lesion sizes in the diagnostic and nondiagnostic biopsy specimens were 45 (15-140) mm and 43 (17-128) mm. The median numbers of diagnostic and nondiagnostic cores were 2 (1-5) and 1.5 (1-4). These size and core number differences between the diagnostic and nondiagnostic biopsy specimens are not statistically significant. Of initial nondiagnostic 8 masses, 3 masses that were performed repeat biopsy resulted in determined diagnosis finally. There were mild postprocedural hematomas not requiring blood transfusion. There was no tumor dissemination after renal mass biopsy. (Conclusions) Percutaneous biopsy of renal masses is a safe procedure that provides diagnostic information.

[Metastatic Renal Cell Carcinoma in Paranasal Sinus for which Periodic Drug Withdrawal Schedule of Axitinib was Effective : A Case Report].

A 73-year-old male patient underwent a right nephrectomy for renal cell carcinoma in 2008, and interferon-alpha was initiated as adjuvant treatment. Computed tomography (CT) scans showed lymphadenopathy above the left diaphragm, and treatment with interferon-2 was subsequently initiated in 2009. Nasal bleeding manifested in February 2010, and CT scans showed a soft-tissue density mass mainly located in the ethmoid sinus. A biopsy of the lesion was performed, and metastatic renal cell carcinoma was diagnosed. Treatment with sorafenib was consequently initiated and the paranasal metastasis showed a temporary partial response (PR). However, the metastatic lesion increased in size and caused repeated nasal bleeding that required blood transfusion. Although treatment with everolims was initiated, adverse events, such as rush, hypertensionnemia, and anemia due to nasal bleeding, developed. Treatment with axitinib was subsequently initiated. However, because adverse events, such as severe diarrhea, renal dysfunction and proteinuria manifested, the dose of axitinib was gradually decreased, and a periodic drug withdrawal schedule (11 days on, 3 days off) was finally initiated, which controlled these adverse events. The metastatic lesions showed a PR for 31months following axitinib administration.

Clinical significance of p21-activated kinase 1 expression level in patients with upper urinary tract urothelial carcinoma.

OBJECTIVE: The p21-activated kinase serine/threonine kinases have been outlined as the main cytoskeletal remolding regulators. The same holds true for cell proliferation and motility. They additionally have a part in cellular invasion and carcinogenesis, but the effect of p21-activated kinase 1 expression on the progression of upper urinary tract urothelial carcinoma remains unclear. Therefore, we assessed the relation of p21-activated kinase 1 positivity level to clinicopathological features in patients with upper urinary tract urothelial carcinoma. METHODS: Immunohistochemical staining was performed using formalin-fixed and paraffin-embedded specimens, which were all from 124 patients with upper urinary tract urothelial carcinoma. The determination of staining level was based on the intensity of the staining along with portion of cells stained. Correlation of p21-activated kinase 1 positivity with clinicopathological parameters, including disease-specific or extravesical-recurrence-free survival, was evaluated. RESULTS: Statistically significant association was observed between moderate or more than moderate p21-activated kinase 1 positivity and higher tumor grade, pathological T stage, lymphovascular invasion, history of adjuvant chemotherapy and extravesical recurrence. Positivity for p21-activated kinase 1 had a significant association with shortened disease-specific survival in a multivariate analysis among clinicopathological parameters. Strongly positive p21-activated kinase 1 expression was also one of the independent factors for shortened extravesical-recurrence-free survival time in N0M0 upper urinary tract urothelial carcinoma patients in another multivariate analysis as well as histology and lymphovascular invasion (P = 0.0304, hazard ratio = 4.425). CONCLUSIONS: We conclude that our findings can help us continue a careful follow-up for upper urinary tract urothelial carcinoma patients with high p21-activated kinase 1 expression in surgical specimens.

[Vesicoappendiceal Fistula: A Case Report].

A 65-year-old man had been aware of pieces of food occasionally in the urine since February 2008. In November 2010, he came to an outpatient clinic with high fever and cloudy urine and was diagnosed with urinary tract infection. Because he had fecaluria, interconnection between urinary bladder and digestive tract was suspected. Although excretory urography showed no remarkable findings, an outflow of contrast media into the appendix was demonstrated in the cystography. By cystoscopy, the fistula hole was confirmed on the posterior wall of the bladder and inflow of feces from the hole was noticed. Operation was performed under the diagnosis of vesicoappendiceal fistula. The appendix was adhesive to the ileum, the right side of the bladder and the upper side of the rectum, and an en bloc resection was performed. Because the fecalith existed near the fistula, appendicitis appeared to induce inflammatory change and abscess formation around the appendix, and the abscess might have perforate into the bladder.

[RENAL CELL CARCINOMA PRESENTING WITH HIGH-OUTPUT HEART FAILURE DUE TO ARTERIOVENOUS FISTULA].

A 64-year-old woman who has a history of congestive heart failure and atrial fibrillation was admitted to our hospital with the exacerbation of exertional dyspnea and urinary retention due to severe gross hematuria. Contrast-enhanced computed tomography showed a tumor involving the inferior and middle poles of the right kidney with no nodal involvement, or distant metastases, but that was accompanied by markedly proliferated blood vessels around the inferior vena cava and right renal vein, seemingly a result of an arteriovenous fistula. After embolization of the right renal artery, right radical nephrectomy was performed via a thoracoabdominal incision. The histological diagnosis of the tumor was clear cell renal cell carcinoma, G2 > G3, Fuhrman nuclear grade3, pT2a. Although the presence of an arteriovenous fistula was not confirmed histologically, the severely condensed proliferation of the blood vessels in the renal hilum is consistent with the diagnosis of an arteriovenous fistula accompanying renal cell carcinoma. Immediately after the operation, her symptoms of congestive heart failure, including dyspnea, subsided and her serum BNP levels and CTR value returned to normal levels. Two years after the operation, she shows no signs of recurrence or metastasis. To the best of our knowledge, there have been 25 cases of arteriovenous fistulas accompanied by renal cell carcinoma but only a few in which the symptoms were those of severe congestive heart failure. Clinicians should be aware that renal cell carcinoima could be a cause of heart failure.

Panobinostat synergizes with bortezomib to induce endoplasmic reticulum stress and ubiquitinated protein accumulation in renal cancer cells.

BACKGROUND: Inducing endoplasmic reticulum (ER) stress is a novel strategy used to treat malignancies. Inhibition of histone deacetylase (HDAC) 6 by the HDAC inhibitor panobinostat hinders the refolding of unfolded proteins by increasing the acetylation of heat shock protein 90. We investigated whether combining panobinostat with the proteasome inhibitor bortezomib would kill cancer cells effectively by inhibiting the degradation of these unfolded proteins, thereby causing ubiquitinated proteins to accumulate and induce ER stress. METHODS: Caki-1, ACHN, and 769-P cells were treated with panobinostat and/or bortezomib. Cell viability, clonogenicity, and induction of apoptosis were evaluated. The in vivo efficacy of the combination was evaluated using a murine subcutaneous xenograft model. The combination-induced ER stress and ubiquitinated protein accumulation were assessed. RESULTS: The combination of panobinostat and bortezomib induced apoptosis and inhibited renal cancer growth synergistically (combination indexes <1). It also suppressed colony formation significantly (p <0.05). In a murine subcutaneous tumor model, a 10-day treatment was well tolerated and inhibited tumor growth significantly (p <0.05). Enhanced acetylation of the HDAC6 substrate alpha-tubulin was consistent with the suppression of HDAC6 activity by panobinostat, and the combination was shown to induce ER stress and ubiquitinated protein accumulation synergistically. CONCLUSIONS: Panobinostat inhibits renal cancer growth by synergizing with bortezomib to induce ER stress and ubiquitinated protein accumulation. The current study provides a basis for testing the combination in patients with advanced renal cancer.

[Case of primary scrotal sclerosing lipogranuloma : review of 227 cases reported in Japan].

We report a case of primary scrotal sclerosing lipogranuloma. A 39-year-old man who had complained of a painless intrascrotal mass was introduced to our hospital for detailed examinations. He denied having received any injection of exogenous substances or having suffered from any trauma. Physical examination revealed a U-shaped elastic hard mass surrounding the penile shaft in the scrotum. T2-weighted magnetic resonance imaging (MRI) demonstrated an ill-defined U-shaped lesion which exhibited relatively low signal intensity. Primary scrotal sclerosing lipogranuloma was the most suspected. The mass gradually disappeared after 47 days from tumor open biopsy for definitive diagnosis. We found 227 cases reported in Japan, and we discuss the diagnosis, treatment and clinical features with reference to previous reports.

[Evaluation of clinical results in patients undergoing laparoscopic nephroureterectomy].

We evaluated the efficacy of laparoscopic radical nephroureterectomy (LNUx) by comparing the clinical results in 55 patients undergoing LNUx at the National Defense Medical College since 2001 with those in patients undergoing open radical nephroureterectomy (ONUx) there over the same period. LNUx was performed successfully in 54 of the patients, but one patient required conversion to ONUx. The mean operating time for LNUx was not significantly different from that for ONUx, and the mean estimated blood loss during LNUx was significantly lower than that during ONUx. Major complications occurring early in our laparoscopic series of cases were injury of the duodenum and bleeding from the inferior vena cava. Oncological outcomes were compared between LNUx (n＝50) and ONUx (n＝50) in patients with pathologically-confirmed urothelial carcinoma. The pT stage, histological grade, percentage of lymph node dissection and percentage of high grade hydronephrosis did not differ significantly between the two groups. Because the intravesical recurrence-free survival rates, extravesical recurrence-free survival rates, and disease specific survival rates were also similar in the two groups, we concluded that LNUx is an appropriate treatment for upper urinary tract urothelial carcinoma.

Impact of pulse duration alterable laser ureterorenoscopic lithotripsy for upper urinary tract calculi.

To assess the effectiveness of a pulse duration alterable Holmium-YAG (Ho:YAG) laser on the stone-free rate (SFR) compared to a conventional pulse duration fixed laser after ureterorenoscopic lithotripsy (URSL). The medical records from patients with upper urinary tract calculi of ≥ 9 mm and < 30 mm were retrospectively investigated. URSL using a conventional Ho:YAG Laser (group C) or a pulse duration alterable Ho:YAG system (group A) was included. In total, 228 and 188 patients were enrolled in groups C and A, respectively. A 272 µm optical core bare-ended, reusable laser fiber was used, and the laser system was set to a standard 0.8 J and 10 Hz (8 W of average power) in both groups. URSL adopts active fragmentation using an extraction approach. SF was defined as the complete absence of stone fragments on computed tomography (CT) 1-2 months after URSL. Sex, BMI, stone length, stone volume, stone density, and the number of patients with positive preoperative urine cultures were not significantly different between the groups. However, age, rate of preoperative febrile urinary tract infection (fUTI), and pre-stenting were significantly higher in group A, and the operative times and incidence of postoperative fUTI were comparable. The SFRs were 71.5% and 80.3% in groups C and A, respectively (P = 0.035). Multivariate logistic regression revealed that the use of conventional laser was associated with non-SF (odds ratio [OR] 1.090, 95% confidence interval [CI] 1.01-1.18, P = 0.040). The present study revealed the superior performance of a pulse duration alterable Ho:YAG laser on the SFR after URSL compared to a conventional pulse duration fixed laser delivery system.

[The efficacy of direct vision internal urethrotomy for male urethral stricture].

OBJECTIVE: Direct vision internal urethrotomy (DVIU) has been considered to be a low invasive and widely used therapeutic modality for male urethral stricture. However, its efficacy is still controversial. We herein evaluated the efficacy of DVIU for male urethral stricture. PATIENTS AND METHODS: Nineteen patients 27 to 78 years old (median age = 59) underwent DVIU for urethral strictures at our hospital were included in this study. Strictures were at bulbar urethra in 17 patients, membranous urethra in 1 patient, and pendulous urethra in 1 patient. The stricture lengths estimated on retrograde urethrography were less than 1 cm in 13 patients, 1-2 cm in 2 patients, and more than 2 cm in 4 patients. The etiology of stricture was straddle injury in 7 patients, post transurethral surgery in 7 patients, pelvic fracture in 1 patient, and unknown in 4 patients. The operation was done by cold knife incision using guidewire. The duration of postoperative urethral catheterization was 5 to 35 days (mean 12.8 days). Follow up duration ranged from 1 month to 139 months (mean 48.2 months). The definition of postoperative re-stricture was the confirmation of re-stricture on retrograde urethrography or deterioration of symptom. RESULTS: While no severe complication was observed, postoperative re-stricture was seen in 13 patients. Stricture-free rates at 3 months, 6 months, and 5 years after the first DVIU were 44.4%, 38.1%, 20.3% respectively. Although second DVIU was done for 7 patients with re-stricture, six patients resulted in failure. Stricture-free rates at 3 months, 6 months, and 5 years after the second DVIU were 42.2%, 28.6%, 14.3% respectively. Though the third DVIU was done for two of them, they were unable to void just immediately after the removal of urethral catheters. Stricture-free rate in stricture less than 1 cm was higher than that in 1 cm or longer, though it did not reach significant difference (p = 0.1813). CONCLUSION: The efficacy of DVIU is lesser than we expected. DVIU seems to be excessively applied to male urethral strictures and should not be performed for long and recurrent urethral stricture.

Ataxia telangiectasia and Rad3-related inhibition by AZD6738 enhances gemcitabine-induced cytotoxic effects in bladder cancer cells.

The ataxia telangiectasia and rad3-related-checkpoint kinase 1 (ATR-CHK1) pathway is involved in DNA damage responses in many cancer cells. ATR inhibitors have been used in clinical trials in combination with radiation or chemotherapeutics; however, their effects against bladder cancer remain unclear. Here, the efficacy of combining gemcitabine with the novel ATR inhibitor AZD6738 was investigated in vitro in three bladder cancer cell lines (J82, T24, and UM-UC-3 cells). The effects of gemcitabine and AZD6738 on cell viability, clonogenicity, cell cycle, and apoptosis were examined. The combined use of gemcitabine and AZD6738 inhibited the viability and colony formation of bladder cancer cells compared to either treatment alone. Gemcitabine (5 nM) and AZD6738 (1 µM) inhibited cell cycle progression, causing cell accumulation in the S phase. Moreover, combined treatment enhanced cleaved poly[ADP-ribose]-polymerase expression alongside the number of annexin V-positive cells, indicating apoptosis induction. Mechanistic investigations showed that AZD6738 treatment inhibited the repair of gemcitabine-induced double-strand breaks by interfering with CHK1. Combining AZD6738 with gemcitabine could therefore be useful for bladder cancer therapy.

[Hemodialysis case in which RCC was identified after disappearance of perirenal hematoma following spontaneous renal rupture treated by transcatheter arterial embolization].

A 55-year-old man who had been undergoing hemodialysis for 9 years visited our institution after the sudden onset of severe left flank pain. He presented with hypotension and was admitted immediately because computed tomography (CT) revealed a massive perirenal hematoma. Renal arteriography showed contrast media leakage at the lower branch of the left renal artery, and spontaneous renal rupture was diagnosed. Five months after the bleeding was stopped by selective transcatheter embolization of the branch of renal artery, CT showed an enhanced mass at the upper pole of left kidney and renal cell carcinoma (RCC) was suspected. Radical nephrectomy was performed, the pathological diagnosis was clear cell carcinoma, and the man has not experienced recurrence within 36 months after the surgery. RCC did not appear to be the cause of the original hemorrhage because there was a small residual hematoma in the middle of the renal parenchyma that was separated from the RCC. In cases of spontaneous renal rupture, re-evaluation by imaging studies is mandatory after disappearance of perirenal hematoma because imaging studies at the time of the rupture sometimes do not reveal the cause of the hemorrhage.

Inhibition of checkpoint kinase 1 potentiates anticancer activity of gemcitabine in bladder cancer cells.

Checkpoint kinases (CHKs) are involved in the DNA damage response in many cancer cells. CHK inhibitors have been used in clinical trials in combination with chemotherapeutics; however, their effect against bladder cancer remains unclear. Here, we investigated the efficacy of combining gemcitabine with MK-8776, a novel CHK1 inhibitor, in four bladder cancer cell lines. The effects of gemcitabine and MK-8776 on cell viability, clonogenicity, cell cycle, and apoptosis were examined alongside in vivo efficacy using murine xenograft tumor models. Combined treatment inhibited the viability and colony formation of bladder cancer cells compared to either single treatment. Although gemcitabine (10 nM) alone increased the cell number in S-phase, it increased the cell number in sub-G1 phase when combined with MK-8776 (0.5 µM). Combined treatment enhanced cleaved poly[ADP-ribose]-polymerase expression alongside the number of annexin-V-positive cells, indicating the induction of apoptosis. In vivo, administration of gemcitabine and MK-8776 was well tolerated and suppressed tumor growth. Mechanistically, the combined treatment elevated γH2A.X and suppressed Rad51 expression. Our study demonstrates that MK-8776 and gemcitabine combined induces apoptosis and suppresses proliferation in bladder cancer cells by inhibiting CHKs and DNA repair. Therefore, CHK1 inhibition combined with gemcitabine may be a potential treatment for bladder cancer.

Ubiquitin-proteasome System Is a Promising Target for Killing Cisplatin-resistant Bladder Cancer Cells.

BACKGROUND/AIM: Activation of the ubiquitin-proteasome system (UPS) has been shown to be associated with drug resistance in cancer. Using bladder cancer cells, we investigated the association between UPS activation and cisplatin resistance and also the efficacy of UPS-targeting drugs. MATERIALS AND METHODS: We established cisplatin-resistant bladder cancer cells (J82-cisR, T24-cisR) and examined the activation status of the UPS and the efficacy of MLN7243, oprozomib, ixazomib, and RTS-V5. RESULTS: The UPS in cisplatin-resistant bladder cancer cells was activated compared to that in their parental controls. All the UPS-targeting drugs induced apoptosis and inhibited growth more effectively in the cisplatin-resistant bladder cancer cells than they did in the parental controls. Furthermore, these UPS-targeting drugs induced endoplasmic reticulum stress by causing unfolded protein accumulation at lower concentrations in the cisplatin-resistant bladder cancer cells. CONCLUSION: Targeting the UPS could be an effective strategy for treating cisplatin-resistant bladder cancer.

[Urothelial carcinoma clear cell variant of the urinary bladder : a case report].

A 69-year-old woman who had been treated with hemodialysis for 16 months for chronic renal failure presented with a chief complaint of gross hematuria in March 2007. Cystoscopic examination revealed a non-papillary, pedunculated tumor located in the right wall of the urinary bladder. A transurethral resection of the bladder tumor (TURBT) was performed in September 2007. The pathological diagnosis was urothelial carcinoma clear cell variant. The patient is now apparently free of disease 20 months after TURBT. Urothelial carcinoma clear cell variant of the urinary bladder is a rare primary tumor, and this is the fourth case of urothelial carcinoma clear cell variant of the bladder to be reported.

Prostate cancer recurrence mimicking invasive urothelial cancer of the bladder.

A 74-year-old male patient with stage D1 prostate cancer with the initial prostate-specific antigen (PSA) level of 5570 ng/mL had received androgen deprivation therapy and the serum PSA level had decreased to 0.23 ng/mL when he developed macroscopic hematuria. MRI and cystoscopy suggested invasive urothelial cancer of the bladder, and transurethral resection was performed. The tumors were pathologically diagnosed as a Gleason score 9 prostate cancer with no PSA expression. Prostate cancer patients who develop novel symptoms should be screened for prostate cancer recurrence even if they have very low PSA levels.

Rapid progression of mucinous tubular and spindle cell carcinoma of the kidney without sarcomatoid changes: A case report.

We report a case of unusually aggressive behavior of a mucinous tubular and spindle cell carcinoma (MTSCC) of the kidney with no sarcomatoid changes. A 43-year-old man was referred to our hospital for a mass on his left kidney. Computed tomography revealed a tumor at the upper pole of the kidney and swollen lymph nodes. Left radical nephrectomy with lymph node dissection was performed and the tumor was diagnosed as MTSCC. Peritoneal dissemination was detected 4 months after the surgery. The patient received systemic treatments, which were not effective. He finally died of the disease 12 months after the surgery.