Combined functional genome survey of therapeutic targets for clear cell carcinoma of the kidney.

OBJECTIVE: Emerging molecular targeting therapeutics have been incorporated into the management of advanced renal cell carcinoma; however, their efficacy remains limited. The aim of this study was to catalog potential therapeutic target molecules for renal cell carcinoma. METHODS: We first selected genes up-regulated in clear cell renal cell carcinoma relative to surrounding normal kidney tissues in 10 patients (Study Cohort) using high-density exon arrays that detect all potential transcripts predicted in the human genome. The selected genes were subjected to independent validation in another set of 10 patients (Validation Cohort) using real-time reverse transcriptase polymerase chain reaction and functional screening using small interfering RNA in six clear cell renal cell carcinoma cell lines. RESULTS: We identified 164 genes whose expression was significantly elevated in clear cell renal cell carcinoma (P< 0.0001 [Student's t-test] and at least a 3-fold change in transcription signal). We finally extracted 33 genes required for maintaining cell proliferation in at least two clear cell renal cell carcinoma cell lines. The 33 genes included 13 genes known to be associated with the development/progression of renal cell carcinoma, including CAIX and FLT-1, confirming the robustness of the current strategy. CONCLUSIONS: Through a combination of genome-wide expression and functional assays, we identified a set of genes with high potential as targets for drug development. This method is rapid and comprehensive and could be applied to the discovery of diagnostic biomarkers and therapeutic targets for cancers other than clear cell renal cell carcinoma.

Genetic alterations at 13q14 may correlate with differences in the biological behavior of prostate cancer between Japanese and Caucasian men.

The incidence of prostate cancer and the resultant mortality rates in Japanese men are lower compared with the rates for Caucasians; however, the Gleason score at diagnosis is higher in Japanese men compared with Caucasians. Loss of 13q is one of the most common chromosomal alterations in prostate cancer. To elucidate the difference in the rate of loss of 13q between Japanese and Caucasian men, we examined the allelic imbalance (AI) on chromosome 13q in 32 Japanese and 39 German prostate cancer patients with a fluorescent polymerase chain reaction technique using 12 microsatellite markers. Benign and malignant histology was identified by a single pathologist and laser capture microdissection was used to gather cancer cells. Although there were no statistical differences in patient background characteristics, the frequency of AI at 13q14 (D13S1253) and at 13q21 (D13S166) was significantly higher in Japanese patients compared with German patients (p = 0.0128 and p = 0.0078, respectively). The frequency of AI at 13q14 was significantly higher in tumors with high Gleason scores (GS) compared with tumors with low GS (p = 0.0478). The present observations suggest that the frequency of genetic alterations at 13q14 may underlie differences in the biological behavior of prostate cancer between Japanese and Caucasian populations.

Preoperative erythrocyte sedimentation rate is an independent prognostic factor in Japanese patients with localized clear cell renal cell carcinoma.

INTRODUCTION: Few studies have examined the prognostic significance of common laboratory variables in Japanese patients with localized clear cell renal cell carcinoma (CCRCC). We evaluate the prognostic significance of preoperative laboratory variables in Japanese patients with localized CCRCC. PATIENTS AND METHODS: The study included 110 Japanese patients who were pathologically confirmed as nonmetastatic CCRCC (pT1-3 N0M0) after radical nephrectomy at our institution. We assessed the clinical (including laboratory measurements) and pathological findings, with the survival rates after surgery. RESULTS: Tumor stage and erythrocyte sedimentation rate (ESR) were identified as significant independent prognostic factors of progression-free survival in multivariate analysis. As for the prognostic factors for disease-specific survival, tumor stage and ESR had prognostic significance both in univariate and multivariate analyses. When the analysis was limited to pT1, multivariate analysis showed that only ESR was an independent prognostic factor for disease-specific survival. CONCLUSIONS: Preoperative ESR is an independent prognostic factor in Japanese patients with localized CCRCC, especially in patients with pT1.

Discrepancies between cytology, cystoscopy and biopsy in bladder cancer detection after Bacille Calmette-Guerin intravesical therapy.

OBJECTIVES: To evaluate discrepancies in the detection of Bacille Calmette-Guerin (BCG)-resistant bladder cancer by cystoscopy, bladder biopsy and urinary cytology. METHODS: Between January 1992 and August 2006, 127 bladder cancer patients underwent a cycle of eight weekly BCG instillations. Four weeks after the last BCG instillation, urinary cytological analysis and cystoscopy with targeted biopsy in addition to eight-nine selected-site biopsies were performed. RESULTS: Biopsy-proven cancer was found in 11/27 (40.7%), 5/42 (11.9%), and 11/58 (19.0%) of positive, suspicious, and negative cytology cases, respectively. Abnormal and normal cystoscopic findings correlated with a biopsy-proven cancer in 13/53 (24.5%) and 14/74 (18.9%) cases, respectively. The combination of a macroscopic cystoscopic suspicion and a positive cytology missed malignant cases in 15.9% of the cases. In 100 cases without biopsy-proven cancer, the rates of denuded urothelium at biopsy in the cases with positive and non-positive cytology were 7/16 (43.8%) and 16/84 (19.0%), respectively. CONCLUSIONS: According to our study, routine biopsy is recommended in the evaluation of BCG treatment, even if the timing, limitations and disadvantages of the procedure should be taken into account.

Risk of concomitant carcinoma in situ determining biopsy candidates among primary non-muscle-invasive bladder cancer patients: retrospective analysis of 173 Japanese cases.

OBJECTIVES: To determine candidates for bladder biopsies among Japanese primary non-muscle-invasive bladder cancer patients according to the risk of concomitant carcinoma in situ (CIS). METHODS: Between January 1992 and August 2006, 173 primary non-muscle-invasive bladder cancer cases underwent transurethral resection of the bladder tumor with bladder biopsies for the detection of CIS. Correlations between biopsy results and preoperative/pathological features were retrospectively analyzed. RESULTS: Positive cytology was statistically associated with the presence of concomitant CIS in multivariate analysis (P < 0.01). Abnormal cystoscopic appearance outside the tumor almost achieved statistical significance in multivariate analysis among preoperative factors (P = 0.06). In our series, one (12.5%) of eight low-risk, 18 (24.7%) of 73 intermediate-risk and 41 (59.4%) of 69 high-risk cases had CIS in normal-looking sites, respectively. In cases with a single papillary tumor and negative cytology, one of 16 (6.3%) had concomitant CIS in their biopsy specimens at the normal-looking sites. CONCLUSIONS: All non-muscle-invasive bladder cancer patients with positive cytology are candidates for additional random biopsies. Targeted biopsies should be performed for all suspicious areas in the bladder mucosa. Random biopsies should be considered in cases with the macroscopic types of cancer for predicting intermediate- and high-risk cancer.

Effects of hemodialysis on testicular volume and oxidative stress in humans.

PURPOSE: Male infertility is a serious problem in patients on hemodialysis. Our understanding is that end stage renal disease or hemodialysis causes poor semen quality but the mechanism leading to impaired spermatogenesis is largely unknown. MATERIALS AND METHODS: Testicular volume in 120 patients on maintenance hemodialysis was compared with that in age matched healthy controls. Volume was correlated with clinical findings. In 10 testicular biopsy specimens from patients on hemodialysis who visited our infertility clinic Western blotting was performed to examine the generation of 4-HNE modified proteins, which are markers of oxidative stress, and the expression of proliferating cell nuclear antigen. Interstitial fibrosis was determined by Masson's trichrome staining. RESULTS: Mean bilateral testicular volume in patients on hemodialysis was significantly smaller than that in healthy controls (31.7 vs 36.4 ml, p <0.01) in a hemodialysis duration dependent manner (r = -0.32, p <0.01). The increase in serum ferritin correlated inversely with testicular volume (r = -0.25, p <0.01). The generation of 4-HNE modified proteins was significantly increased 3.1-fold in patients on hemodialysis, following the 60% decreased expression of proliferating cell nuclear antigen. Quantitative analysis of Masson's trichrome staining revealed increased interstitial fibrosis in patients on hemodialysis compared with that in controls (41.5% vs 14.8%, p <0.01). Serum ferritin, proliferating cell nuclear antigen expression and interstitial fibrosis correlated with the generation of 4-HNE modified proteins (p <0.05). CONCLUSIONS: Testicular volume, which is a parameter of spermatogenesis, is impaired in patients on hemodialysis and oxidative stress is considered to be involved in the process. Serum ferritin is a useful parameter for predicting oxidative stress in the testis.

Clinical significance of lymphovascular invasion in upper urinary tract urothelial cancer.

OBJECTIVES: To clarify the significance of lymphovascular invasion (LVI) in patients with pT3N0M0 upper urinary tract (UUT) urothelial carcinoma (UC) relative to prognosis in terms of disease-specific survival, as LVI, which implies both blood vessel and lymph vessel involvement, is reportedly a poor prognostic factor in patients with UUT-UC. PATIENTS AND METHODS: The clinical records of 90 patients who had surgery for UUT-UC were reviewed retrospectively. The median patient age was 71 years and the median follow-up was 42 months. The prognostic significances of LVI (with vs without), T stage (< 1 vs 2-4), grade (1-2 vs 3), N stage (0 vs 1-2), age (< or = 70 vs > 70 years), gender and tumour location (renal pelvis vs ureter) for survival time were evaluated. RESULTS: LVI of UUT-UC was found in 34 patients (37.8%). There were significantly higher frequencies of LVI with advancing stage and lymph node metastasis. Kaplan-Meier analysis showed that LVI was strongly associated with disease-specific survival in all patients (P < 0.001) and in patients with pT3N0M0 disease (P < 0.001). Univariate analyses showed that LVI, T stage, N stage and tumour grade were significantly related to disease-specific survival in all patients (P < 0.001, < 0.001, 0.003 and 0.007, respectively). Multivariate analysis using Cox proportional hazards model showed that LVI was the only prognostic factor with independent significance for disease-specific survival (P < 0.001). CONCLUSIONS: LVI appears to be an important and independent prognostic factor for UUT-UC in patients treated by nephroureterectomy. Our data suggest that the LVI status might be a predictive marker for disease-specific survival in patients with T3N0M0 UTT-UC.

Development of a peritoneal sclerosis rat model using a continuous-infusion pump.

OBJECTIVE: Encapsulating peritoneal sclerosis (EPS) is a serious complication of continuous ambulatory peritoneal dialysis. Previous studies have created peritoneal sclerosis rat models using daily intraperitoneal injection of chlorhexidine gluconate (CG), but this technique is cumbersome and thickening of the peritoneum makes it difficult to evaluate the injection site. We therefore aimed to make a rat model using a continuous-infusion pump. METHODS: Various concentrations of CG (5%, 8%, 10%, 12%, and 14%) in ethanol were dissolved in saline within the infusion pumps, each of which was placed in the lower abdominal cavity of a male Wister rat. After a peritoneal equilibration test was performed, the rats were sacrificed and the lower anterior parietal and visceral peritoneum was removed. Each excised peritoneum was analyzed by macroscopic and microscopic examinations, including immunohistochemistry for the expression of transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and alpha-smooth muscle actin (alphaSMA). The results were compared with those of control rats injected with ethanol dissolved in saline within the infusion pump and with no-pump rats. RESULTS: Two of the 5 rats in the 12% CG group and 3 of the 5 rats in the 14% CG group died of ileus within 14 days. All the rats in the 5%, 8%, and 10% CG groups survived to 28 days. Macroscopic examination in the 10% CG group showed bowel dilatation, bowel adhesion, and bloody ascites, similar to those seen in human EPS patients. All rats in each CG group showed the same extent of thickening of the submesothelial compact zone, proliferation of collagen fibers, and presence of numerous cells and neovascularization. Within same CG groups, an equal degree of thickening was observed at all sites of the peritoneum. TGF-beta1, VEGF, and alphaSMA were highly expressed in the peritoneum of the 10% CG group. CONCLUSION: We developed a novel method of creating a peritoneal sclerosis rat model using a continuous-infusion pump. Our technique is simple and highly reproducible, and will be useful in the study of peritoneal sclerosis mechanisms.

Tumor-infiltrating lymphocytes derived from human renal cell carcinoma: clonal analysis of its characteristics.

AIM: To assess the characteristics of activated tumor-infiltrating lymphocytes (TIL), we report the isolation, growth response, and functional analysis of a CD4(-) CD8(+) TIL-clone derived from human renal cell carcinoma (RCC). METHODS: Bulk TILs were expanded from a human RCC and the lymphocytes were separated into a CD8(+) enriched population. Subsequently, using the limiting dilution technique, a TIL clone was established and its growth response, phenotype and cytotoxic activity were analyzed. RESULTS: A clone, T16-13, by day 94 numbering 1 x 10(7) cells, was harvested and characterized as a CD4(-) CD8(+) clone. On day 144, the cytotoxic activity of this clone against the autologous tumor was relatively high (2.3 +/- 0.7 LU(30)/10(6) cells). Meanwhile, against allogeneic renal tumors, there was no cytotoxic activity (-0.1 LU(30)/10(6) cells). CONCLUSIONS: A TIL clone possessing modest autologous tumor-specific cytotoxicity can be isolated from human RCC. The characteristics analysis of various TIL clones may provide a better understanding of an RCC tumor microenvironment and may help to establish new modalities for the treatment of patients with metastatic kidney cancer.

Gain of 5p15.33 is associated with progression of bladder cancer.

OBJECTIVE: To search for a biological marker to distinguish low-risk from high-risk bladder cancer indicating disease progression. METHODS: The whole genome-wide copy numbers were screened in 18 patients with bladder cancer using array comparative genomic hybridization (CGH) consisting of 4,030 bacterial artificial chromosome clones. RESULTS: Gain of 5p15.33, including TPPP (tubulin polymerization-promoting protein)and ZDHHC11 (zinc finger DHHC domain-containing protein 11) genes, was detected in 5 of 9 (55.6%) high-grade bladder cancers and no (0%; n = 9) low-grade bladder cancer. To confirm the preliminary data, 5p15.33 gain was studied by fluorescence in situhybridization (FISH) in 100 patients, and the results were compared with biological characteristics. In FISH analysis, gain of 5p15.33 was significantly correlated with higher histological grade (p < 0.0001) and advanced pathological stage (p = 0.0284). Tumors with a gain of 5p15.33 had a significantly higher progression-free survival rate than those without (p = 0.0006, log-rank test). Multivariate analysis revealed that gain of 5p15.33 was a predictor for disease progression in bladder cancer (hazard ratio: 1.887, 95% confidence interval: 1.215-2.968, p = 0.0050). CONCLUSION: These data suggest that gain of 5p15.33 (TPPP and ZDHHC11) may become a potential biomarker identifying high-risk patients with disease progression in bladder cancer.

Haploinsufficiency of 8p22 may influence cancer-specific survival in prostate cancer.

Although Knudson's two-hit hypothesis with functional loss of a tumor suppressor gene has been widely accepted, accumulating evidence suggests that several genes are regulated by the quantity of their product in a dose-dependent manner (gene dosage effect). The study was designed to identify the influence of gene dosage effect of 8p22 on patient prognosis. With a median age of 71 years, 40 patients with prostate cancer (11 organ-confined, 13 capsular penetrating, and 16 nodal and/or distant metastatic) were followed for a median of 68.5 months. A fluorescence in situ hybridization (FISH) technique was applied using a region-specific cosmid probe combined with centromeric probe. Allelic losses of 8p22, 8p21.3, 8p21.1 approximately 2, and 8p12 were found in 23, 22, 14, and 9 patients, respectively. A Cox proportional hazard model revealed that decreased fraction (i.e., the fraction of nuclei with a lesser number of cosmid signals than of centromeric probe signals) of 8p22 proved to be the sole independent prognostic factor predicting cancer-specific death, as well as disease progression--but allelic loss of 8p22 was not predictive. Cytogenetic estimation of 8p22 by FISH can yield quantitative evaluation of relevant gene dosage, which may become a useful biomolecular marker predicting poor patient prognosis.

Overexpression of BUBR1 is associated with chromosomal instability in bladder cancer.

BUBR1, a mitotic checkpoint protein, is a key component of the mitotic spindle checkpoint machinery. Defective BUBR1 has been proposed to contribute to chromosomal instability (CIN). To elucidate the relationship of BUBR1 expression with CIN, expression of BUBR1, numbers of centrosomes, numerical aberrations of chromosomes, and DNA ploidy were examined, and BUBR1 expression status was compared with clinicopathological parameters in 104 human urothelial bladder carcinomas. Expression of BUBR1 and numbers of centrosomes were assessed by immunohistochemistry. Numerical aberrations of chromosomes 7, 9, and 17 were evaluated by fluorescence in situ hybridization. Cancers with a large intercellular variation in centromere copy number were designated as CIN cancers. Tumors with BUBR1 overexpression were associated with CIN, DNA aneuploidy, and centrosome amplification. Array CGH revealed that BUB1B amplification and loss rarely occurred, indicating that the overexpression of BUBR1 in these bladder cancers was independent of BUB1B copy number. Overexpression of BUBR1 significantly correlated with higher histological grade, advanced pathological stage, and high cell proliferation. Overexpression of BUBR1 predicted tumor recurrence and disease progression. These data suggest that overexpression of BUBR1 is potentially a new tumor marker for estimating biological characteristics of bladder cancer.

Chromosome 20q13.2 gain may predict intravesical recurrence after nephroureterectomy in upper urinary tract urothelial tumors.

PURPOSE: Amplification or gain of copy number of chromosome 20q13.2 has been implicated as a causal factor for chromosome instability. We investigated the impact of chromosomal instability and its causative molecular markers, 20q13.2 gain and centrosome amplification, on patient outcome in upper urinary tract transitional cell carcinoma (UUT-TCC). EXPERIMENTAL DESIGN: The number of centrosomes was assessed by immunohistochemistry. Numerical aberrations of chromosomes 7, 9, and 17 that allowed the estimation of chromosomal instability and 20q13.2 gain were evaluated by fluorescence in situ hybridization in 96 frozen specimens from UUT-TCC and compared with clinicopathologic background and patient outcome. RESULTS: Chromosomal instability, 20q13.2 gain, and centrosome amplification were detected in 62 of 96 (64.6%), 61 of 96 (63.5%), and 45 of 90 (50.0%) tumors, respectively. 20q13.2 Gain was significantly associated with tumor stage (P = 0.042), chromosomal instability (P < 0.0001), and centrosome amplification (P < 0.0001). Kaplan-Meier analysis showed that 20q13.2 gain was strongly associated with intravesical recurrence-free survival in all patients (P = 0.0050), as well as in patients with grade 2 tumors (P = 0.0011, log-rank test). On multivariate analysis, 20q13.2 gain was found to be the sole independent prognostic factor predicting subsequent intravesical recurrence (hazard ratio, 1.65; 95% confidence interval, 1.03-2.90; P = 0.036). CONCLUSIONS: 20q13.2 gain was strongly associated with a reduced time to intravesical recurrence in all patients. Our data suggest that 20q13.2 gain may be a predictive marker of intravesical recurrence in patients with UUT-TCC.

Biological characteristics in bladder cancer depend on the type of genetic instability.

PURPOSE: Malignant tumors show an inherent genetic instability that can be classified as microsatellite instability (MSI) or chromosomal instability (CIN). To elucidate the differences in biological characteristics of bladder cancer between the two types of genetic instability, the expression of the mismatch repair (MMR) proteins, Aurora-A and p53 proteins, the number of centrosomes, numerical aberrations of chromosomes and 20q13, and DNA ploidy were examined in 100 human urothelial carcinomas of the bladder. EXPERIMENTAL DESIGN: Expressions of the MLH1, MSH2, Aurora-A, and p53 proteins and the numbers of centrosomes were immunohistochemically assessed. Numerical aberrations of chromosomes 7, 9, 17, and 20q13 spots were evaluated by fluorescence in situ hybridization, and DNA ploidy was assessed by laser scanning cytometry. RESULTS: The expression levels of the MMR related-proteins decreased in 9 of 100 tumors. Tumors with low MLH1 or MSH2 expression (designated as MSI cancers) were not linked with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, 20q13 gain, DNA aneuploidy, and disease progression. MSI cancers showed a favorable prognosis. CIN cancers (49 cases), defined as tumors with a large intercellular variation in centromere copy numbers, were associated more frequently with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, and 20q13 gain than the others (51 cases). Tumors with disease progression were included in the CIN cancer group. CONCLUSIONS: The present observations suggest that there are differences in the biological characteristics of the two types of genetic instability.

Evaluation of lower urinary tract symptoms and how bothersome it was with or without urinary incontinence in apparently healthy persons of both sexes.

We evaluated the effect of urinary incontinence on the degree of being bothersome in apparently healthy males and females by a questionnaire survery. From March to May, 2003 apparently healthy subjects underwent multiphasic health screening after informed of the nature of this study and were asked to fill out the questionnaires of International Prostate Symptom Score (IPSS) with IPSS QOL index (IPSS-QI) and the short form version of the Urogenital Distress Inventory (UDI-6). The data were subjected to analytical studies. Of the 388 participants who responded completely to both questionnaires, 172 (44.3%) had urinary incontinence; 143 were women (36.9%) and 29 men (7.5%). The mean age of the women was 46.0 years (range 18.0 to 76.0) and that of men was 47.5 years (range 22.0 to 76.0). Compared with continent participants, women and men with mixed urinary incontinence had a significantly higher IPSS severity (P = 0.0002 and P = 0.0014, respectively). In terms of contribution on QOL impairment, the women and men with mixed urinary incontinence considered it significantly more bothersome compared with continent participants (P = 0.0004 and P = 0.0003, respectively). These data showed that urinary incontinence was relatively common among apparently healthy women, but not men, and type of incontinence had a different impact on the degree of being bothersome in both sexes.

Evaluation of hematuria and proteinuria positivity in relation to ageing in 6,651 apparently healthy men and women.

We examined the positivity of hematuria and proteinuria in relation to ageing in 6,651 apparently healthy persons (2,556 women and 4,095 men) who underwent multiphasic health screening in our Medical Checkup Center. Commercially available dipsticks were used. The time from urine collection to dipstick analysis was within 60 minutes. The mean age of women was 48.2 years (range 10 to 82) and that of men was 49.9 years (range 7 to 89). Approximately 30.1, 1.5, and 0.7% of the women had hematuria, proteinuria, and hematoproteinuria, respectively; and 11.4, 4.0, 1.5% of the men had the corresponding urine abnormalities, respectively. Hematuria was 2.6 times more common in women than in men, and proteinuria was 2.7 times more common in men than in women. The positivity of hematuria increased linearly with age in women (Rs = 0.943, P = 0.0350). On the other hand, the positivity of proteinuria or hematoproteinuria was not correlated with age (P = 0.8386 and P = 0.0639, respectively). In men, the positivity of hematuria or hematoproteinuria was not correlated with age (P = 0.0845 and P = 0.0845, respectively). However, the positivity of proteinuria in those more than 30-year age group increased linearly with age (Rs = 1.000, P = 0.0455). The true meaning of such gender- and/or age-related differences in urinary abnormalities remains to be determined.

[Does alpha1-blocker provide additional improvement of quality-of-life in patients with overactive bladder?--a multi-center prospective randomized trial between anti-cholinergic (propiverine chloride) with and without alpha1-blocker (urapidil)].

AIM: Storage/filling symptoms caused by overactive bladder (OAB) are bothersome to patients. The aim of this study is to clarify if alpha1-blocker provides additional benefit in combination with anticholinergic treatment in patients with OAB. METHODS: In total, 100 patients (men/women: 43/57, mean age: 71.3 years) who had frequency (more than eight times a day) and urgency (more than three times a week) were prospectively randomized, and allocated to two groups (monotherapy group [n = 52]: propiverine alone or combination group [n = 48]: propiverine plus urapidil). The primary end point was to compare the improvement of storage symptoms (numbers of frequency, urgency, disappearance of urge incontinence) as well as patients' quality of life (QOL) assessed by King's Health Questionnaires (KHQ) at baseline, 2 weeks, and 6 weeks after the start of treatment in both groups. The second end point was to evaluate the safety of these agents. RESULTS: Statistically significant improvements in terms of urgency and frequency were observed in both groups at two-weeks after the start of treatment as compared with baseline (p < 0.01 and < 0.05, respectively), while no inter-group difference was observed between the two groups. Significant improvement of QOL was observed after six weeks treatment in overall mean score, general health perception, incontinence impact, sleep/energy domains in both groups as compared with baseline. No significant difference was observed in terms of toxic events between the two groups. CONCLUSIONS: Although both groups showed identical improvement of storage symptoms and tolerability, no additional benefit of alpha1-blocker was observed.

Evidence that FTY720 induces rat thymocyte apoptosis.

FTY720, a novel immunomodulator with the potential to improve immunosuppressive therapy after organ transplantation, is currently under clinical investigation. FTY720 drastically decreases blood lymphocytes, especially T cells, accelerating lymphocyte homing to secondary lymphoid organs. However, its immunosuppressive effects remain unknown. We investigated these effects in rat thymocytes. Rats were intramuscularly injected with 10mg/kg/day FTY720 or saline for 7days. Thymuses were removed on days 0, 1, 3, 5, 7 and 14 after treatment. Three-color analysis was performed with a flow cytofluorometer. Apoptotic nuclei in the tissue sections were identified by TUNEL. Genomic DNA was then extracted and samples were electrophoresed on 2.0% agarose gel. FTY720 reduced the total number of thymocytes and, with time, significantly reduced the percentage of CD4+8+ TCRalphabeta(negative/low) thymocytes. Light microscopy of thymuses of FTY720-treated rats revealed obvious reductions in the size of the cortical region. TUNEL analysis showed that FTY720 induced thymocyte apoptosis in the cortical region. Furthermore, DNA fragmentation was observed in thymocytes treated with FTY720, indicating thymocyte apoptosis. FTY720 reduced the number of CD4+8+ thymocytes before TCRalphabeta expression resulting in impaired thymocyte differentiation and maturation. This might be an immunosuppressive effect of FTY720.

Salvage chemotherapy with paclitaxel, ifosfamide, and nedaplatin in patients with urothelial cancer who had received prior cisplatin-based therapy.

BACKGROUND AND AIMS: The aim of the present phase II study was to evaluate the efficacy of combination chemotherapy of paclitaxel, ifosfamide, and nedaplatin (PIN regimen) in patients with recurrent urothelial cancer who had been treated with cisplatin-based chemotherapy. PATIENTS/METHODS: Eligible patients were those with histologically confirmed urothelial cancer who had progressed or relapsed after cisplatin-based chemotherapy. The PIN regimen consisted of paclitaxel 175 mg/m(2) on day 1; ifosfamide 4.5 g/m2 divided over days 1, 2, and 3; and nedaplatin 70 mg/m(2) on day 1; PIN was given every 28 days. RESULTS: Among the 32 patients enrolled in the study (median age, 66 years), complete and partial responses were obtained in 5 patients and 19 patients, respectively, with an overall response rate of 75% (95% confidence interval [CI], 59-91%). The median time to progression was 8 months (range, 0-50+ months) and the median survival was 22 months (range, 4-52+ months). The 1- and 2-year overall survival rates were 53.7 and 42.9%, respectively. All patients experienced Grade 3 or 4 neutropenia, while Grade 3 or 4 thrombocytopenia was seen in 8 patients; Grade 3 or 4 anemia was seen in 6 patients; Grade 3 neuropathy was observed in 1 patient, for whom the PIN therapy was discontinued. There were no treatment-related deaths. CONCLUSION: The PIN combination was highly active and tolerable in previously treated patients with urothelial cancer as a second-line treatment.

Identification of genes linked to gefitinib treatment in prostate cancer cell lines with or without resistance to androgen: a clue to application of gefitinib to hormone-resistant prostate cancer.

Understanding the molecular action of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, might allow us to perform more effective therapies for hormone-independent advanced prostate cancer. A DNA microarray study was undertaken to comprehensively analyze the alteration of levels of 1,081 genes after gefitinib treatment in androgen-independent PC3 and DU145 cells and androgen-dependent LNCaP cells. The proliferation of PC3, DU145 and LNCaP cells was significantly inhibited by 50.2%, 83.8% and 55.2%, respectively, 6 days after 10 microM gefitinib administration. Of the above 1,081 genes, we identified 23, 13 and 33 genes with significantly different expression in PC3, DU145 and LNCaP cells, respectively, 24 h after 10 microM-gefitinib exposure. Among the identified genes, only Quiescin Q6, a negative cell cycle regulator, was increased after gefitinib treatment in all three cell lines regardless of gefitinib sensitivity. Except for Quiescin Q6, there were no overlapping genes between PC3 and DU145 cells. However, levels of several oncogenes or proliferation-related genes were changed after gefitinib treatment in the 2 androgen-independent cell lines. We also identified 7 unique genes [glycyl-tRNA synthetase, interferon, alpha-inducible protein, stratifin, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, dual specificity phosphatase 9, guanine nucleotide binding protein (G protein) beta polypeptide 2, neural retina leucine zipper] whose levels were altered exclusively after gefitinib administration in gefitinib-resistant PC3 and LNCaP cells, but not in DU145 cells, suggesting that these 7 genes could be targets for overcoming gefitinib resistance. Collectively, our molecular profiling data will serve as a framework for understanding the molecular action of gefitinib for prostate cancer.