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Background: Increased expression of programmed cell death 1 ligand 1 (PD-L1) by tumor cells is thought to be a mechanism through which solid cancers promote immune tolerance. However, the association between PD-L1 expression and the prognosis of upper urinary tract urothelial carcinoma (UTUC) remains unknown., Methods: We examined immunohistochemical PD-L1 expression and the tumor-infiltrating lymphocyte density (TILD) in 79 patients with UTUC who underwent nephroureterectomy. We classified the tumors into four types based on the combination of PD-L1 expression and TILD, and studied the clinicopathological characteristics of these four tumor types., Results: Elevated expression of PD-L1 by tumor cells and a higher TILD were associated with a worse histological grade, higher pT stage, and higher peripheral blood neutrophil-to-lymphocyte ratio. Elevated expression of PD-L1 by tumor cells, a higher TILD, and type I, III, or IV tumors with elevated expression of either PD-L1 or TILD showed a positive correlation with poorer differentiation and local invasion. These three variables were associated with shorter progression-free survival and overall survival in univariate analysis, but only the latter was an independent determinant according to multivariate analysis. The patients who had type II tumors with lower PD-L1 expression and a lower TILD showed more favorable survival than the other three groups., Conclusions: These findings suggest that PD-L1 expression and TILs in the tumor microenvironment influence the progression of UTUC. Accordingly, it is important to understand the immunologic characteristics of the tumor microenvironment to develop more effective treatment strategies for this cancer.

Objective: Renal cell carcinoma (RCC) is a hypervascular tumour due to high constitutive production of vascular endothelial growth factor (VEGF), which is activated by hypoxia-inducible factor (HIF). Elevated levels of cardiovascular peptides, including brain natriuretic peptide (BNP), have been reported in patients with cancer, regardless of whether they have overt cardiovascular disease. Furthermore, it has been demonstrated that hypoxia stimulates BNP production by an HIF-dependent manner. However, the clinical implications of such cardiovascular peptides in patients with RCC have not been assessed., Methods: In patients with clear cell RCC who underwent nephrectomy, we investigated the relationship between the serum level of BNP or N-terminal pro-BNP (NT-proBNP) and various clinicopathological characteristics, including serum VEGF and expression of BNP and HIF-2 alpha in the primary tumour., Results: Elevated preoperative serum levels of BNP, NT-proBNP and VEGF, as well as increased tumour expression of HIF-2 alpha, were associated with a worse performance status, local invasion, distant metastasis and shorter overall survival. HIF-2 alpha expression showed a positive correlation with the preoperative serum VEGF level, while there was no relation between the serum levels of BNP/NT-proBNP and VEGF or tumour expression of HIF-2 alpha. BNP expression was very low in both tumour tissues and normal kidney tissues. Serum levels of BNP, NT-proBNP and VEGF all decreased significantly after nephrectomy., Conclusions: Our findings suggested that the preoperative serum levels of BNP and NT-proBNP are markers of tumour progression, as well as indicators of subclinical functional and structural myocardial damage in patients with advanced RCC., Competing Interests: Competing interests: None declared.

Background: Many urologic surgeons refer to biopsy core details for decision making in cases of localized prostate cancer (PCa) to determine whether an extended resection and/or lymph node dissection should be performed. Furthermore, recent reports emphasize the predictive value of prostate-specific antigen density (PSAD) for further risk stratification, not only for low-risk PCa, but also for intermediate- and high-risk PCa. This study focused on these parameters and compared respective predictive impact on oncologic outcomes in Japanese PCa patients., Methods: Two-hundred and fifty patients with intermediate- and high-risk PCa according to the National Comprehensive Cancer Network (NCCN) classification, that underwent robot-assisted radical prostatectomy at a single institution, and with observation periods of longer than 6 months were enrolled. None of the patients received hormonal treatments including antiandrogens, luteinizing hormone-releasing hormone analogues, or 5-alpha reductase inhibitors preoperatively. PSAD and biopsy core details, including the percentage of positive cores and the maximum percentage of cancer extent in each positive core, were analyzed in association with unfavorable pathologic results of prostatectomy specimens, and further with biochemical recurrence. The cut-off values of potential predictive factors were set through receiver-operating characteristic curve analyses., Results: In the entire cohort, a higher PSAD, the percentage of positive cores, and maximum percentage of cancer extent in each positive core were independently associated with advanced tumor stage ≥ pT3 and an increased index tumor volume > 0.718 ml. NCCN classification showed an association with a tumor stage ≥ pT3 and a Gleason score ≥8, and the attribution of biochemical recurrence was also sustained. In each NCCN risk group, these preoperative factors showed various associations with unfavorable pathological results. In the intermediate-risk group, the percentage of positive cores showed an independent predictive value for biochemical recurrence. In the high-risk group, PSAD showed an independent predictive value., Conclusions: PSAD and biopsy core details have different performance characteristics for the prediction of oncologic outcomes in each NCCN risk group. Despite the need for further confirmation of the results with a larger cohort and longer observation, these factors are important as preoperative predictors in addition to the NCCN classification for a urologic surgeon to choose a surgical strategy.

Background: The relationship between the clinicopathological features and molecular changes associated with standardized uptake value (SUV) determined by Positron emission tomography (PET) with [18F] fluorodeoxyglucose (18F-FDG PET) in human renal cell carcinoma (RCC) has not been elucidated. On the other hand, overactivation of the phosphatidylinositol 3'kinase (PI3K), serine/threonine kinase Akt, and mammalian target of rapamycin (mTOR) pathway has been detected in a variety of human cancers, including RCC. So far, little is known about the relationship between the SUV and these proteins in human RCC. Thus, it is important to study the relevance of SUV with clinicopathological features in human RCCs from a molecular point of view., Methods: Seventy-seven consecutive patients with RCC who underwent nephrectomy and pretreatment determination of the maximum SUV (SUVmax) by 18F-FDG PET were analyzed. We investigated the relationship between the SUVmax, phosphorylated-Akt (Ser-473) (pAkt(Ser-473)), phosphorylated-Akt (Thr-308) (pAkt(Thr-308), and phosphorylated-S6 ribosomal protein (Ser-235/236) (pS6) protein levels in the primary tumor and various clinicopathological features., Results: The average SUVmax of the primary tumor was 6.9 (1.5 to 40.3). A higher SUVmax was correlated with higher expression of pAkt(Ser-473), pAkt (Thr-308), and pS6 protein in the primary tumor. A higher SUVmax and increased expression of pAkt (Ser-473), pAkt (Thr-308), and pS6 of the primary tumor was associated with less tumor differentiation, a higher pT stage, regional lymph node involvement, microscopic vascular invasion, and distant metastasis, as well as with early relapse following radical nephrectomy in patients who had localized or locally advanced RCC without distant metastasis (cTanyNanyM0) and with shorter overall survival in all patients., Conclusions: A higher SUVmax on 18F-FDG PET is associated with elevated tumor levels of pAkt and pS6 protein and with aggressive behavior and metastatic potential of RCC, as well as with early relapse following radical nephrectomy and shorter overall survival. These findings suggest that SUVmax may be useful for predicting the biological characteristics of RCC.

Background: Activation of Rho, one of the small GTPases, and its major downstream target Rho-kinase (ROCK) promotes the development and metastasis of cancer. We previously showed that elevation of Rho and ROCK expression was associated with tumor invasion, metastasis, and an unfavorable prognosis in patients with urothelial cancer of the bladder or upper urinary tract., Methods: We investigated the effects of a ROCK inhibitor on the growth, migration, and apoptosis of bladder cancer cells. We also examined phosphorylation of RhoA (RhoA activity) by measuring its GTP-bound active form and assessed the expression of ROCK to explore the underlying molecular mechanisms., Results: Lysophosphatidic acid (LPA) and geranylgeraniol (GGOH) induced an increase of cell proliferation and migration in association with promotion of RhoA activity and upregulation of ROCK expression. The ROCK inhibitor fasudil (HA-1077) suppressed cell proliferation and migration, and also induced apoptosis in a dose-dependent manner. HA-1077 dramatically suppressed the expression of ROCK-I and ROCK-II, but did not affect RhoA activity., Conclusions: These findings suggest that ROCK could be a potential molecular target for the treatment of urothelial cancer.

Background: Gleason pattern 3 less often has molecular abnormalities and often behaves indolent. It is controversial whether low grade small foci of prostate cancer (PCa) on biopsy could avoid immediate treatment or not, because substantial cases harbor unfavorable pathologic results on prostatectomy specimens. This study was designed to identify clinical predictors for classical and redefined insignificant cancer on prostatectomy specimens in Japanese men with favorable pathologic features on biopsy., Methods: Retrospective review of 1040 PCa Japanese patients underwent radical prostatectomy between 2006 and 2013. Of those, 170 patients (16.3%) met the inclusion criteria of clinical stage ≤ cT2a, Gleason score (GS) ≤ 6, up to two positive biopsies, and no more than 50% of cancer involvement in any core. The associations between preoperative data and unfavorable pathologic results of prostatectomy specimens, and oncological outcome were analyzed. The definition of insignificant cancer consisted of pathologic stage ≤ pT2, GS ≤ 6, and an index tumor volume < 0.5 mL (classical) or 1.3 mL (redefined)., Results: Pathologic stage ≥ pT3, upgraded GS, index tumor volume ≥ 0.5 mL, and ≥ 1.3 mL were detected in 25 (14.7%), 77 (45.3%), 83 (48.8%), and 53 patients (31.2%), respectively. Less than half of cases had classical (41.2%) and redefined (47.6%) insignificant cancer. The 5-year recurrence-free survival was 86.8%, and the insignificant cancers essentially did not relapse regardless of the surgical margin status. MRI-estimated prostate volume, tumor length on biopsy, prostate-specific antigen density (PSAD), and findings of magnetic resonance imaging were associated with the presence of classical and redefined insignificant cancer. Large prostate volume and short tumor length on biopsy remained as independent predictors in multivariate analysis., Conclusions: Favorable features of biopsy often are followed by adverse pathologic findings on prostatectomy specimens despite fulfilling the established criteria. The finding that prostate volume is important does not simply mirror many other studies showing PSAD is important, and the clinical criteria for risk assessment before definitive therapy or active surveillance should incorporate these significant factors other than clinical T-staging or PSAD to minimize under-estimation of cancer in Japanese patients with low-risk PCa.

To clarify the role of serum soluble T cell regulatory molecules in clear cell renal cell carcinoma (CCRCC), we measured the serum levels of soluble interleukin-2 receptor (sIL-2R), soluble B7-H3 (sB7-H3), and soluble cytotoxic T lymphocyte associated antigen-4 (sCTLA-4) in 70 CCRCC patients and 35 healthy controls. We investigated correlations between the serum levels of these soluble T cell regulatory molecules and the pathological grade, clinical stage, and prognosis of CCRCC. We also assessed the relations among each of these soluble molecules. As a result, the serum level of sIL-2R was significantly higher in CCRCC patients than in healthy controls (P < 0.05). In addition, elevation of serum sIL-2R was significantly correlated with the clinical stage (P < 0.001), and the survival of patients with high sIL-2R levels was shorter than that of patients with low sIL-2R levels (P < 0.05). Furthermore, the serum level of sB7-H3 was also significantly correlated with the clinical stage (P < 0.05), while the sIL-2R and sB7-H3 levels showed a positive correlation with each other (R = 0.550, P < 0.0001). These results indicate that the serum level of sIL-2R reflects tumor progression in CCRCC patients. In addition, the possibility was suggested that the IL-2/IL-2R and B7-H3 pathways may be involved in the progression of CCRCC.

Background: The system L amino acid transporter (LAT) has an important role in the transport of various amino acids, and there have been reports about the relation of this system to cancer. Although LATs are highly expressed in the kidneys, little is known about their influence on human renal cancer., Methods: To clarify the role of LATs in human clear cell renal cell carcinoma (RCC), we investigated the expression of mRNAs for LAT1, LAT2, LAT3, LAT4, and 4F2hc in clear cell RCC tissues. The mRNAs of these five genes were analyzed by the real-time reverse transcription polymerase chain reaction in matched sets of tumor and non-tumor tissues obtained at operation from 82 Japanese patients with clear cell RCC. We also measured phosphorylated S6 ribosomal protein (Ser-235/236) proteins levels in 18 paired tumor and non-tumor tissues of the patients by Western blotting., Results: Expression of LAT1 mRNA was significantly increased in tumor tissue compared with non-tumor tissue, while expression of LAT2 and LAT3 mRNAs was reduced. There was no difference in the expression of LAT4 and 4F2hc mRNAs between tumor and non-tumor tissues. Increased expression of LAT1 mRNA was associated with less differentiated tumors, local invasion, microscopic vascular invasion, and metastasis. Kaplan-Meier survival analysis showed that a higher serum LAT1 mRNA level was associated with a shorter overall survival time. Phosphorylated S6 ribosomal protein levels were associated with metastatic potential. LAT1 mRNA levels positively correlated with phosphorylated S6 ribosomal protein proteins levels in primary tumors., Conclusions: These findings suggest that LAT1 mRNA is related to the invasive and progressive potential of clear cell RCC.

We cloned a novel splicing variant for nuclear coactivator p120(alpha), designated as p120beta and studied its function and expression in several human prostate diseases. Transfection assays demonstrated that p120beta functions as a strong coactivator for androgen receptor (AR), but weakly for other nuclear receptors. GST-pull down assay showed that a glutamine-rich region of the p120 bound to the ligand-binding domain of AR. Interestingly, p120beta mRNAs were expressed predominantly in the normal prostate, androgen-responsive prostate cancers and an androgen-sensitive prostate cancer cell line, LNCaP, but weakly in recurrent cancers and the androgen-insensitive prostate cancer cell lines PC3 and DU145. Furthermore, knockdown of p120alpha by siRNA abolished coactivator activity on thyroid hormone receptors (TR) and PPARgamma, but did not affect that of ARs in PC3 cells. In addition, competitive assay with other nuclear receptors demonstrated that TR and PPARgamma did not inhibit p120beta-induced stimulation. These findings suggested that while p120alpha was essential for ligand-dependent stimulation of TRs and PPARgamma, p120beta acted as a coactivating protein predominantly for AR.

Previous studies on prostate blood flow regulation have indicated that androgen regulates prostate blood flow. However, the mechanism responsible for this regulation is unknown. In the present study, we focused on the effects of vascular endothelial growth factor (VEGF), a key factor responsible for angiogenesis and androgenic blood flow regulation. We examined in vivo the effect of VEGF on prostate blood flow and its participation in the androgenic regulation of this blood flow using a castrated rat model following subcapsular intraprostatic injection method. We found that VEGF is involved in blood flow regulation with an activity equal to that of dihydrotestosterone (DHT). The effect of VEGF on prostate blood flow was already seen at 30 min after the administration. The elevating effect of DHT on castrated rat prostate blood flow was abolished by coadministration of DHT with neutralizing anti-VEGF antibody. The change in VEGF-A mRNA expression in response to androgen stimulation was examined by double-fluorescent probe quantitative PCR (Taqman PCR). The results showed that androgenic regulation of VEGF gene expression occurred shortly after androgen stimulation. VEGF gene up-regulation was abolished or down-regulated by coadministration of neutralizing anti-VEGF antibody. This is the first report on the importance of VEGF in the androgenic regulation signaling pathway that affects prostate blood flow. Alternative treatment targeted toward anti-VEGF activity as a substitute for ordinary antiandrogenic therapy may be effective against prostate diseases, especially those with androgen-independent and hyperhemorrhagic status.

To clarify the effect of androgen on the microcirculation in seminal vesicles of adult Wistar rats, we investigated the organ blood flow and morphological features in the capillaries after castration and subsequent testosterone supplementation. Testosterone (T) was subcutaneously injected every 12 hours after castration and its doses were set to 10(-2), 10(-1), 10(0) and 10(1) mg/kg-body weight (T10(-2), T10(-1), T10(0) and T10(1) groups, respectively). Organ blood flow was measured using laser Doppler flowmetry, and the subepithelial capillaries were analyzed using transmission electron microscopy. The capillaries were morphologically classified into 3 types; oval opened (type 1), intermediate (type 2) and collapsed (type 3), and their luminal areas were measured using a computed image analyzer. The organ blood flow was significantly reduced from 36.3+/-5.1 to 21.9+/-2.7 ml x min(-1)/100 g tissue, and the luminal area of the capillaries was significantly reduced from 9.02+/-1.28 to 4.85+/-0.82 microm2 with the shift of the type 1 and type 2 to type 3 after castration. The reduction of the luminal area and the blood flow reduction, and shift of the capillary type were significantly protected by gradated testosterone supplementation. These results indicate that the maintenance of the blood flow and morphological profiles in capillaries depend on androgen-supplementation levels in seminal vesicles.

The purpose of this study was to clarify the androgen-ablation-induced morphological changes in the capillaries and stroma near the epithelial cells in prostate and seminal vesicles (SV) using transmission electron microscopy (TEM). In ventral prostate (VP) and SV of immature male rats, the luminal areas of subepithelial capillaries and the width of the stromal layers between endothelia and epithelia were measured quantitatively after castration using TEM and a computed image analyzer. The luminal areas of the capillaries were significantly reduced in VP and SV in the short-term after castration. In the stromal layer, the width of the collagen layer surrounding the capillary significantly increased in VP and SV in the long-term after castration. These data suggest that the reduction of the capillaries and the thickening of collagen surrounding them are related to the involution of glandular epithelial cells in VP and SV after castration.

Interleukin (IL)-15 is a pleiotropic cytokine that is important for innate and adaptive immune cell homeostasis. The expression of IL-15 protein is controlled by posttranscriptional mechanisms. Here, we constructed a human IL-15 expression vector consisting of the human IL-2 signal peptide, the human IL-15 mature peptide-coding sequences, and an out-of-frame human growth hormone gene. Human prostate cancer cells, PC-3, transfected with this highly secretable form of the IL-15 gene, successfully secreted abundant bioactive IL-15 protein. In nude mice, the growth of PC-3 cells producing IL-15 was remarkably retarded. NK cell-depletion using anti-asialo GM1 antibody restored tumorigenicity. Histologically, tumors derived from IL-15-producing PC-3 cells contained necrotic areas with high apoptotic index. Splenocytes incubated with supernatant of transfectants killed target PC-3 cells and expressed a significantly high level of mIFN-gamma mRNA. These observations suggest that NK cell-mediated, anti-tumor effects of IL-15 could provide a potential rationale for gene therapy of prostate cancer.

It has been suggested that prostate homeostasis is regulated indirectly by androgens through stromal-epithelial interactions in part by factors from the stromal cells acting on receptors in epithelial cells. In this report, the role of fibroblast growth factor (FGF)-10 in prostatic epithelial proliferation was investigated. The expression of FGF-10 mRNA was apparent in primary-cultured stromal cells, but not in epithelial cells derived from human tissue from patients with benign prostatic hyperplasia (BPH). The mitogenic activity of human recombinant FGF-10 assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation was demonstrated in isolated epithelial cells, but not in cultured stromal cells. No mitogenic activity of dihydrotestosterone (DHT) for either epithelial or stromal cells could be demonstrated, but quantitative PCR (real-time PCR) with a double-labeled fluorogenic probe demonstrated that expression of FGF-10 in stromal cells was enhanced 5.3-fold at a DHT concentration of 100 pM. Androgen receptor mRNA levels showed no significant change with DHT at concentrations less than 100 pM, but were reduced to 50% of control levels at a DHT concentration of 10 nM. These results suggest that stromal-derived FGF-10 stimulates human prostatic epithelial growth and its mRNA expression is induced by androgens, without an increase in the androgen receptor mRNA. Moreover, FGF-10 may be involved in the development or support of human BPH.

Epithelial cells of the rat seminal vesicle stained positively for nuclear estrogen receptor alpha (ER alpha). We studied these cells using immunohistochemical means. We demonstrated in a previous study that some glandular epithelial cells of the seminal vesicles of immature castrated rats treated with estrogen for 1-2 weeks had multilayer features. The present study shows that these glandular epithelial cells are nuclear ER and basal cell-specific cytokeratin (34betaE12) positive. These findings suggested characteristics of basal cells. Moreover, we demonstrated that these cells express transforming growth factor beta1 (TGFbeta1) as a result of castration and estrogen treatment. Our findings indicate that glandular epithelial cells with multilayer features, which stained positively for nuclear ER alpha have basal cell features and may play an important role in the expression of TGFbeta1 through an epithelial-stromal interaction.

To elucidate the autoregulation of androgen receptor (AR) by androgen and antiandrogen, Western blot analysis and immunohistochemical study were performed. Castration reduced the immunodetected AR content, and nuclear staining was lost without cytoplasmic staining. Testosterone (T) supplement restored AR content. Quick response of AR content restoring following single administration of T was observed 48 hours after castration. The recovery of AR content detected by Western blot under each condition was accompanied by recovery of the reduced unclear staining intensities in the epithelia. Neither steroidal nor non-steroidal antiandrogens, chlormadinone acetate and flutamide, altered the AR content in normal rat ventral prostate 5, 12, 24 or 48 hours after single administration. Furthermore, neither of the drugs at various doses altered AR levels 12 hours after single administration. In summary, the rat AR is upregulated by androgen. Single administration of antiandrogens have no effect on immunodetected AR content.

We studied the changes in the tissue localization of nuclear estrogen receptor (ER) positively stained cells in the seminal vesicle of immature castrated rats under various environmental conditions of sex hormones by immunohistochemical methods. In castrated rats of 6 weeks of age, the percentage of nuclear ER positively stained cells showed remarkable increase in the periglandular stroma region, but not in the epithelium and the peripheral stroma region. Estrogen administration to castrated rats dramatically increased the percentage of nuclear ER positively stained cells in both the epithelium and the peripheral stroma region, whereas cessation of estrogen treatment caused a significant percentile decrease. These results suggest that the nuclear ER expression in both the epithelial cells and the peripheral stromal cells seems to respond to estrogen. The concomitant treatment of estradiol-17 beta (E2) with 5 alpha-dihydrotestosterone (DHT) completely inhibited these E2 mediated ER expression in the epithelium and the stroma. This result suggests that ER works only when E2 is given in the absence of DHT in the seminal vesicle of immature castrated rats.

Stroma and the heparin-binding fibroblast growth factor (FGF) family influence normal epithelial cell growth and differentiation in embryonic and adult tissues. The role of stromal cells and the expression of isoforms of the FGF ligand and receptor family were examined during malignant progression of epithelial cells from a differentiated, slowly growing, nonmalignant model rat prostate tumor. In syngeneic hosts, a mixture of stromal and epithelial cells resulted in nonmalignant tumors which were differentiated and slowly growing. In the absence of the stromal cells, epithelial cells progressed to malignant tumors which were independent of the stroma and undifferentiated. The independence of the malignant epithelial cells from stromal cells was accompanied by a switch from exclusive expression of exon IIIb to exclusive expression of exon IIIc in the FGF receptor 2 (FGF-R2) gene. The FGF-R2(IIIb) isoform displays high affinity for stromal cell-derived FGF-7, whereas the FGF-R2(IIIc) isoform does not recognize FGF-7 but has high affinity for the FGF-2 member of the FGF ligand family. The switch from expression of exclusively exon IIIb to exclusively exon IIIc in the resident FGF-R2 gene was followed by activation of the FGF-2 ligand gene, the normally stromal cell FGF-R1 gene, and embryonic FGF-3 and FGF-5 ligand genes in malignant epithelial cells. Multiple autocrine and potentially intracrine ligand-receptor loops resulting from these alterations within the FGF-FGF-R family may underlie the autonomy of malignant tumor cells.