Biomarkers for Monitoring of Changes in Disease Activity in Ulcerative Colitis.

BACKGROUND: In recent years, various biomarkers of ulcerative colitis (UC) have emerged; however, few studies have simultaneously examined the utility of multiple biomarkers for monitoring disease activity. Additionally, serum leucine-rich alpha-2 glycoprotein (LRG), a new biomarker, may show a blunt response to anti-TNF antibody therapy. This prospective study explored effective biomarkers that could monitor disease activity changes in patients with UC. In addition, we examined the effect of anti-TNF antibody therapy on changes in LRG. METHODS: Blood and stool samples were collected twice from patients with UC: at baseline and at least 8 weeks later. Changes in serum LRG, interleukin (IL)-6, prealbumin (pre-Alb), high-sensitivity C-reactive protein (hs-CRP), CRP, and fecal calprotectin (FC) were measured and correlated with changes in disease activity. The relationship between anti-TNF antibody therapy and LRG levels was also examined in patients with the same disease activity. RESULTS: Forty-eight patients with UC (96 samples) were analyzed. ΔLRG and ΔIL-6 correlated strongly with the change in the partial Mayo (pMayo) score between the two time points (ΔpMayo) (r = 0.686, 0.635, respectively). In contrast, FC and IL-6 were particularly accurate predictors of clinical remission, and their area under the curves (AUCs) were significantly higher than that of CRP (AUC: 0.81, 0.76 vs. 0.50; p = 0.001, 0.005). No association was found between the administration of anti-TNF antibody preparations and the LRG values. CONCLUSIONS: Correlations were found between changes in UC disease activity and LRG, IL-6, pre-Alb, hs-CRP, CRP, and FC. LRG reflects disease activity during anti-TNF antibody therapy.

Relationship between Chemotherapy-Induced Diarrhea and Intestinal Microbiome Composition.

BACKGROUND AND AIM: Fluoropyrimidines (FPs) are key drugs in many chemotherapy regimens; however, recipients are often prone to diarrhea due to gastrointestinal toxicity. Disruption of the intestinal epithelial barrier function by FPs leads to dysbiosis, which may exacerbate intestinal epithelial cell damage as a secondary effect and trigger diarrhea. However, despite studies on chemotherapy-induced changes in the intestinal microbiome of humans, the relationship between dysbiosis and diarrhea is unclear. In this study, we aimed to investigate the relationship between chemotherapy-induced diarrhea and the intestinal microbiome. METHODS: We conducted a single-center prospective observational study. Twenty-three patients who received chemotherapy, including FPs as first-line chemotherapy for colorectal cancer, were included. Stool samples were collected before the start of chemotherapy and after one cycle of treatment to analyze intestinal microbiome composition and perform PICRUSt predictive metagenomic analysis. RESULTS: Gastrointestinal toxicity was observed in 7 of 23 patients (30.4%), diarrhea was observed in 4 (17.4%), and nausea and anorexia were observed in 3 (13.0%). In 19 patients treated with oral FPs, the α diversity of the microbial community decreased significantly following chemotherapy only in the diarrheal group. At the phylum level, the diarrheal group showed a significant decrease in the abundance of Firmicutes and a significant increase in the abundance of Bacteroidetes with chemotherapy (p = 0.013 and 0.011, respectively). In the same groups, at the genus level, Bifidobacterium abundance was significantly decreased (p = 0.019). In contrast, in the non-diarrheal group, Actinobacteria abundance increased significantly with chemotherapy at the phylum level (p = 0.011). Further, Bifidobacterium, Fusicatenibacter, and Dorea abundance significantly increased at the genus level (p = 0.006, 0.019, and 0.011, respectively). The PICRUSt predictive metagenomic analysis revealed that chemotherapy caused significant differences in membrane transport in KEGG pathway level 2 and in 8 KEGG pathway level 3, including transporters and oxidative phosphorylation in the diarrhea group. CONCLUSION: Organic-acid-producing bacteria seem to be involved in diarrhea associated with chemotherapy, including FPs.

5­Aminolevurinic acid inhibits the proliferation of bladder cancer cells by activating heme synthesis.

Iron is an essential nutrient that facilitates cell proliferation and growth, and it can contribute to tumor growth. Although iron chelators have shown great potential in preclinical cancer models, they can cause adverse side­effects. The aim of the present study was to determine whether treatment with 5­aminolevurinic acid (5­ALA) has antitumor effects in bladder cancer, by reduction of mitochondrial iron without using an iron chelator, through activation of heme synthesis. T24 and MGH­U3 cells were treated with 5­ALA. Ferrochelatase uses iron to convert protoporphyrin IX into heme, thus additional groups of T24 and MGH­U3 cells were transfected with synthesized ferrochelatase small interfering RNA (siRNA) either to silence ferrochelatase or to provide a negative siRNA control group, and then cell viability, apoptosis, mitochondrial Fe2+, the cell cycle, and ferritin expression were analyzed in all groups and compared. As an in vivo assessment, mice with orthotopic bladder cancer induced using N­butyl­N­(4­hydro­oxybutyl) were treated with 5­ALA. Bladder weight and pathological findings were evaluated, and immunohistochemical analysis was performed for ferritin and proliferating cell nuclear antigen (PCNA). In the cells treated with 5­ALA, proliferation was decreased compared with the controls, and apoptosis was not detected. In addition, the expression of Fe2+ in mitochondria was decreased by 5­ALA, expression of ferritin was also reduced by 5­ALA, and the percentage of cells in the S phase of the cell cycle was significantly increased by 5­ALA. In T24 and MGH­U3 cells with silenced ferrochelatase, the inhibition of cell proliferation, decreased expression of Fe2+ in mitochondria, reduced expression of ferritin, and increased percentage of cells in the S phase by treatment with 5­ALA were weakened. In vivo, no mouse treated with 5­ALA developed muscle­invasive bladder cancer. The expression of ferritin was weaker in mice treated with 5­ALA and that of PCNA was higher than that in mice treated without 5­ALA. It was concluded that 5­ALA inhibited proliferation of bladder cancer cells by activating heme synthesis.

[Management of Internal Ureteral Stent (Double J-Stent) Occlusion].

Occlusion of internal ureteral stents commonly called double-J (DJ) stent leads to renal dysfunction, urinary tract infection, and difficulty in replacing the stent. We investigated the cause of stent occlusion and whether DJ stent occlusion persisted with change in the type of stent. The internal ureteral stent, Bird® Inlay™ Optima or Boston Scientific® Tria™, was inserted in 43 ureters of 33 patients who underwent replacement more than three times between September 2017 and June 2020. We defined stent occlusion as follows: a guide wire could not be passed through a stent during the replacement. In the first occlusion, the type of stent was changed. In the second occlusion, the stent placement interval was shortened from 12-13 weeks to 6-8 weeks. The presence of urinary stone and insertion of a urethral catheter had a high risk of DJ stent occlusion. Stent occlusion was observed in 20 of the 43 ureters. After the type of stent in 20 ureters with stent occlusion was changed, there were no DJ stent occlusions in 16 of the 20 ureters. Nevertheless, in 4 of the 20 ureters, even if we changed the type, DJ stent occlusion was still present; hence, the replacement interval was shortened. Therefore, changing the type of stent may be a recommended intervention for DJ stent occlusion.

Risk Factors for Nephrotoxicity due to Tacrolimus Therapy for Ulcerative Colitis.

BACKGROUND AND AIMS: The calcineurin inhibitor tacrolimus is reportedly effective for moderate/severe ulcerative colitis (UC); however, it is also reportedly associated with nephrotoxicity. We investigated the risk factors for tacrolimus-induced nephrotoxicity and whether renal impairment adversely affected the outcomes of tacrolimus treatment in patients with UC. METHODS: We conducted a retrospective study of 93 patients with UC who were administered tacrolimus leading to high trough levels (10-15 ng/mL) for 2 weeks and low trough levels (5-10 ng/mL) for 3 months. RESULTS: Acute kidney injury (AKI) occurred in 44 patients (47.3%) during tacrolimus treatment. Of these patients, 34 (36.6%) developed AKI during the high trough phase and 17 (18.3%) developed AKI when the trough value exceeded the original target value of 15 ng/mL. Multivariate logistic regression analysis revealed that the male sex was significantly associated with AKI (p = 0.002, AOR = 4.38, 95% CI [1.69-11.3]). Clinical remission rate after 4, 8, 12, and 24 weeks of tacrolimus treatment in patients with AKI was lower than that in patients without AKI. Six patients (6.5%) had chronic kidney disease (CKD) after tacrolimus treatment completion, and all patients with CKD developed AKI during treatment. The median duration of treatment with no improvement in AKI was significantly longer in patients with CKD than in those without CKD (p = 0.016). CONCLUSION: We revealed the risk factors for tacrolimus-induced nephrotoxicity. Renal impairment occurrence adversely affected the tacrolimus treatment outcome; therefore, it is important to carefully administer tacrolimus to prevent renal impairment.

Therapeutic Drug Monitoring of Golimumab for the Prediction of Long-Term Clinical Remission in Patients with Ulcerative Colitis.

BACKGROUND AND AIMS: A considerable number of patients with ulcerative colitis (UC) who initially respond to golimumab (GLM), an anti-TNF-α antibody, gradually lose clinical response. Therapeutic drug monitoring has been proposed to optimize serum anti-TNF-α antibody concentrations before the loss of response; however, little is known about ideal serum GLM concentrations. We aimed to evaluate whether the serum GLM trough levels (TLs) early after the initiation of induction therapy affect the long-term outcomes in UC and to identify the early GLM TLs that should be targeted for better long-term outcomes. METHODS: Thirty-one patients were prospectively evaluated. The primary outcome was clinical remission at 54 weeks, and we measured the serum GLM TLs at weeks 6, 10, and 14. Receiver operating characteristic (ROC) curves were constructed to identify optimal GLM TL thresholds early after induction therapy that were associated with clinical remission at week 54. RESULTS: The GLM TL at week 14, but not at weeks 6 or 10, was significantly associated with clinical remission at week 54 (median [IQR] 1.6 [1.3-1.6] µg/mL vs. 0.9 [0.6-1.3] µg/mL; p = 0.04). The area under the ROC curve for GLM TLs at week 14 was 0.78. We identified a week-14 GLM TL of 1.1 µg/mL as the target threshold for achieving clinical remission at week 54. CONCLUSION: Our results demonstrate the value of early serum GLM TLs in predicting the long-term outcomes of GLM for patients with UC.

Prostate diseases and microbiome in the prostate, gut, and urine.

The microbiome in various organs involves a vast network that plays a key role in the health and wellness of the human body. With recent advances in biological technologies such as high-throughput sequencing, transcriptomics, and metabolomics, it appears that the microbial signature varies dynamically among individuals, creating various roles in metabolism, local and systemic inflammation, and host immunity. Urinary and genital organs, including the prostate, seminal vesicles, and urinary bladder, are reservoirs of several bacterial, viral, and fungal communities. Accumulating evidence has suggested profound roles for the gut, urinary, and intraprostate microbiomes in genitourinary benign and malignant diseases. This review article addresses microbiome-related evidence for three major diseases involved in prostate cancer: chronic prostatitis (CP), benign prostatic hyperplasia (BPH), and prostate cancer (PCa). Symptomatic CP is known as CP/chronic pelvic pain syndrome. CP is one of the most common prostate diseases in young men, accounting for 8% of all men visiting a urologic clinic. Although oral medication is the gold standard therapy for patients with BPH, approximately 13% of men present with clinical progression within 4 years after the initiation of treatment, with 5% requiring surgical intervention. The identification of proinflammatory cytokines and pathogens responsible for the clinical progression of BPH is still underway. Several topics regarding the association between PCa and the microbiome are discussed in this review as follows: i) intraprostatic microbiome and the risk of PCa, ii) gut microbiome and PCa, iii) gut microbiome and the risk of radiation-induced side effects, iv) isoflavone intake and equol-producing intestinal flora on PCa, and v) the inhibitory effect of daidzein and equol on tumor growth and progression of PCa. Further studies are required for a comprehensive understanding between the urogenital microbiome and prostate pathogenesis to facilitate the development of preventive and therapeutic approaches for prostate diseases.

Significant Improvement of Prognosis After the Advent of Immune Checkpoint Inhibitors in Patients with Advanced, Unresectable, or Metastatic Urothelial Carcinoma: A Propensity Score Matching and Inverse Probability of Treatment Weighting Analysis on Real-World Data.

PURPOSE: The treatment landscape for advanced, unresectable, or metastatic urothelial carcinoma (aUC) has shifted substantially since the advent of immune checkpoint inhibitors (ICIs). We investigated the extent to which pembrolizumab therapy is superior to conventional chemotherapy as a second-line treatment. PATIENTS AND METHODS: A multicenter-derived database registered 454 patients diagnosed with aUC between 2008 and 2020. Of these, 94 patients (21%) who received second-line pembrolizumab and 75 (17%) who received second-line chemotherapy but never received third-line or later ICI therapy were included. We compared overall survival (OS) from the initial date of first-line chemotherapy between two groups by adjusting for prognostic factors through propensity score matching (PSM) and inverse probability of treatment weighting (IPTW). The IPTW-adjusted hazard ratio and 95% confidence interval were estimated using a multivariate Cox regression analysis. To identify patients who were more likely to benefit from second-line pembrolizumab than from chemotherapy, we performed a subgroup analysis for OS with an IPTW-adjusted model. RESULTS: The PSM-adjusted comparison showed a significant improvement in the prognosis with second-line pembrolizumab use (P = 0.01). The OS benefit with the advent of pembrolizumab was 8 months (18 months vs 26 months). Multivariable analyses using IPTW adjustment demonstrated that lymph node metastasis (P = 0.001), lung metastasis (P = 0.013), and bone metastasis (P = 0.003) were poor independent prognostic factors, and pembrolizumab use (P = 0.021) was a favorable independent prognostic factor. Subgroup analyses revealed that pembrolizumab was associated with survival benefits over chemotherapy in all subgroups, including young patients (age <70 years), those who received radical surgery, and those without visceral metastasis. CONCLUSION: We demonstrated a significant improvement in prognosis after the advent of pembrolizumab for patients with aUC. ICIs should not be restricted based on patient characteristics.

Inhibitory Effect of Orally Administered 5-Aminolevulinic Acid on Prostate Carcinogenesis in the FVB-Transgenic Adenocarcinoma of a Mouse Prostate (FVB-TRAMP) Model.

BACKGROUND: 5-aminolevulinic acid (5-ALA) is a constituent of mitochondrial electron carriers, heme and cytochrome c, which are crucial for aerobic energy metabolism and cell apoptosis. We investigated the chemopreventive efficacy of 5-ALA against prostate cancer using the FVB-transgenic adenocarcinoma of mouse prostate (FVB-TRAMP) model. METHODS: Samples were collected from 24 FVB-TRAMP mice at 12 and 20 weeks of age (named the first and second sets, respectively). Sixteen mice (from the first set) were randomly allocated into 3 treatment groups: 1) control (no treatment), 2) low dose of 5-ALA (30 mg/kg/day), and 3) high dose of 5-ALA (300 mg/kg/day). Similarly, 8 mice were divided into 2 treatment groups: 1) control and 2) high dose of 5-ALA (300 mg/kg/day). 5-ALA was orally administered to mice before cancer onset, from 6 weeks of age. RESULTS: In the control group, prostate cancer was pathologically detected in 33 and 50 % of mice at 12 and 20 weeks, respectively, while 25% of 12-week old mice in the low-dose group were affected and none of the high-dose group mice developed prostate cancer. Immunohistochemical analysis showed higher expression of cytochrome c oxidase subunit 4 (COX4) in the prostate gland of the high-dose group compared to the control (P = 0.018). Similarly, enzyme-linked immunosorbent assay using lysed prostate tissue revealed higher amounts of cytochrome c in the prostate of the high-dose group compared to the control (P = 0.021). Furthermore, western blot analysis showed higher level of cleaved caspase-3 in mice in the high-dose group diagnosed with high-grade prostatic intraepithelial neoplasia. CONCLUSION: Our results suggest that oral 5-ALA may support the functional expression of mitochondrial cytochrome c and COX4, leading to caspase 3-dependent apoptosis in carcinogenesis in FVB-TRAMP mice. Future clinical studies are warranted to confirm the chemopreventive value of 5-ALA in prostate carcinogenesis. 
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Clinical Impact of Tumor-Infiltrating Lymphocytes and PD-L1-Positive Cells as Prognostic and Predictive Biomarkers in Urological Malignancies and Retroperitoneal Sarcoma.

Over the past decade, an "immunotherapy tsunami", more specifically that involving immune checkpoint inhibitors (ICIs), has overtaken the oncological field. The interaction and cross-talk among tumor cells and several immune cells in the tumor microenvironment are dynamic and complex processes. As immune contexture can vary widely across different types of primary tumors and tumor microenvironments, there is still a significant lack of clinically available definitive biomarkers to predict patient response to ICIs, especially in urogenital malignancies. An increasing body of evidence evaluating urological malignancies has proven that tumor-infiltrating lymphocytes (TILs) are a double-edged sword in cancer. There is an urgent need to shed light on the functional heterogeneity in the tumor-infiltrating immune system and to explore its prognostic impact following surgery and other treatments. Notably, we emphasized the difference in the immunological profile among urothelial carcinomas arising from different primary origins, the bladder, renal pelvis, and ureter. Significant differences in the density of FOXP3-positive TILs, CD204-positive tumor-infiltrating macrophages, PD-L1-positive cells, and colony-stimulating factors were observed. This review discusses two topics: (i) the prognostic impact of TILs and (ii) predictive biomarkers for ICIs, to shed light on lymphocyte migration in four solid tumors, the urothelial carcinoma, renal cell carcinoma, prostate cancer, and retroperitoneal sarcoma.

Periprostatic fat thickness quantified by preoperative magnetic resonance imaging is an independent risk factor for upstaging from cT1/2 to pT3 in robot-assisted radical prostatectomy.

OBJECTIVES: To analyze the correlation between periprostatic fat thickness on multiparametric magnetic resonance imaging and upstaging from cT1/2 to pT3 in robot-assisted radical prostatectomy. METHODS: We retrospectively evaluated data from men with cT1/2 prostate cancer treated with robot-assisted radical prostatectomy at Nara Prefecture General Medical Center, Nara, Japan, between March 2013 and December 2017. We calculated the periprostatic fat thickness and subcutaneous thickness from preoperative multiparametric magnetic resonance imaging. We divided the cohort into two groups for analysis. Group 1 included patients upstaged from clinical to pathological stage, whereas group 2 included those without upstaging. RESULTS: Data on 220 patients meeting the inclusion criteria were included in the analysis. A total of 36 patients were upstaged from clinical T1 or T2 to pathological T3, whereas 184 patients were not upstaged. The upstaging was associated with prostate volume, Gleason score, prostate-specific antigen density, periprostatic fat thickness, Prostate Imaging Reporting and Data System score based on univariate analysis. Multivariate analysis showed prostate volume (P = 0.03, odds ratio 0.958, 95% confidence interval 0.921-0.996), Gleason score (P = 0.022, odds ratio 2.676, 95% confidence interval 1.153-6.213) and periprostatic fat thickness (P = 0.004, odds ratio 1.26, 95% confidence interval 1.079-1.471) as independent risk factors of upstaging. CONCLUSIONS: Prostate volume, Gleason score and periprostatic fat thickness on multiparametric magnetic resonance imaging are significantly associated with and independent risk factors for upstaging from cT1/2 to pT3 in patients undergoing robot-assisted radical prostatectomy.

Simple suturing of the bladder neck muscle layer at the vesicourethral anastomosis site to the dorsal vein complex during anterior reconstruction led to a better postoperative urinary continence after robot-assisted laparoscopic prostatectomy.

OBJECTIVES: To elucidate whether a modified technique for anterior reconstruction could improve urinary continence after robot-assisted laparoscopic radical prostatectomy (RALP). METHODS: Among 325 consecutive patients who underwent RALP at our hospital, 297 patients were included in this retrospective study, who had complete records including the status of postoperative urinary continence. Among these 297 patients, 194 underwent anterior reconstruction by suturing the lateral bladder wall to the arcus tendineus of the pectineal fascia without fixation of the vesicourethral anastomosis site to the dorsal vein complex (DVC) (lateral-suture group). In the remaining 103 patients, simple suturing of the bladder neck muscle layer at the vesicourethral anastomosis site with DVC to immobilize the vesicourethral anastomosis site (immobilized group) was performed. Those who did not required a pad was defined as continent. RESULTS: Operative and console times were significantly shorter in the immobilized group (242 vs. 268 min; p = 0.03, and 174 vs. 203 min; p = 0.009, respectively). Although there was no significant difference between the groups regarding the recovery of urinary continence within 3 months after RALP (21 vs. 22% at 1 month; p = 0.77, and 54 vs. 60% at 3 months; p = 0.33, respectively), more patients achieved urinary continence in the immobilized group than lateral-suture group after 6 months (71 vs. 83% at 6 months; p = 0.03 and 82 vs. 96% at 12 months; p = 0.001, respectively). CONCLUSIONS: Simple suture of the bladder neck muscle layer at the vesicourethral anastomosis site to DVC led to a better urinary continence status 6 months or later after RALP.

Inhibition of Heparanase Expression Results in Suppression of Invasion, Migration and Adhesion Abilities of Bladder Cancer Cells.

Heparan sulfate proteoglycan syndecan-1, CD138, is known to be associated with cell proliferation, adhesion, and migration in malignancies. We previously reported that syndecan-1 (CD138) may contribute to urothelial carcinoma cell survival and progression. We investigated the role of heparanase, an enzyme activated by syndecan-1 in human urothelial carcinoma. Using human urothelial cancer cell lines, MGH-U3 and T24, heparanase expression was reduced with siRNA and RK-682, a heparanase inhibitor, to examine changes in cell proliferation activity, induction of apoptosis, invasion ability of cells, and its relationship to autophagy. A bladder cancer development mouse model was treated with RK-682 and the bladder tissues were examined using immunohistochemical analysis for Ki-67, E-cadherin, LC3, and CD31 expressions. Heparanase inhibition suppressed cellular growth by approximately 40% and induced apoptosis. The heparanase inhibitor decreased cell activity in a concentration-dependent manner and suppressed invasion ability by 40%. Inhibition of heparanase was found to suppress autophagy. In N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer mice, treatment with heparanase inhibitor suppressed the progression of cancer by 40%, compared to controls. Immunohistochemistry analysis showed that heparanase inhibitor suppressed cell growth, and autophagy. In conclusion, heparanase suppresses apoptosis and promotes invasion and autophagy in urothelial cancer.

γ-Klotho is correlated with resistance to docetaxel in castration-resistant prostate cancer.

The Klotho (KL) gene was first identified as a potent aging suppressor. The KL family currently comprises of three proteins: α-Klotho (KLA), ß-Klotho (KLB), and γ-Klotho (KLG). Many studies have shown that KLA and KLB participate in tumor progression or suppression, depending on the type of cancer; however, the relationship between KLG and prostate cancer has not yet been studied. Some studies have claimed that KL is correlated to sensitivity to chemotherapy. Here, we investigated the oncogenic potential of KLG in castration-resistant prostate cancer (CRPC). Immunohistochemical analysis using prostate biopsy specimens revealed that patients with high KLG expression in primary prostate cancer tissue had a significantly poor prognosis for overall survival. In addition, the prostate-specific antigen response rate after docetaxel (DTX) therapy in patients with high KLG expression was lower than that in patients with low KLG expression. To evaluate the potential of KLG as a therapeutic target in human prostate cancer, we generated a xenograft model of human CRPC cell line (PC-3) in male athymic mice. The animals were randomly divided into four groups as follows: i) control group (vehicle only); ii) DTX group (intraperitoneal administration); iii) small interfering RNA targeting KLG (KLG siRNA) group (intratumoral administration); and iv) a combination group (DTX plus KLG siRNA). After 3 weeks of treatment, the tumor weight and tumor Ki-67 labeling index were significantly lower in the KLG siRNA group and the combination group than in the control group. Sensitivity to DTX was increased upon treatment with KLG siRNA. These findings suggest that KLG expression in primary prostate cancer lesions is associated with resistance to DTX in CRPC and has potential as a diagnostic and therapeutic target for patients with CRPC.

Disabled Homolog 2 (DAB2) Protein in Tumor Microenvironment Correlates with Aggressive Phenotype in Human Urothelial Carcinoma of the Bladder.

Disabled homolog-2 (DAB2) has been reported to be a tumor suppressor gene. However, a number of contrary studies suggested that DAB2 promotes tumor invasion in urothelial carcinoma of the bladder (UCB). Here, we investigated the clinical role and biological function of DAB2 in human UCB. Immunohistochemical staining analysis for DAB2 was carried out on UCB tissue specimens. DAB2 expression levels were compared with clinicopathological factors. DAB2 was knocked-down by small interfering RNA (siRNA) transfection, and then its effects on cell proliferation, invasion, and migration, and changes to epithelial-mesenchymal transition (EMT)-related proteins were evaluated. In our in vivo assays, tumor-bearing athymic nude mice subcutaneously inoculated with human UCB cells (MGH-U-3 or UM-UC-3) were treated by DAB2-targeting siRNA. Higher expression of DAB2 was associated with higher clinical T category, high tumor grade, and poor oncological outcome. The knock-down of DAB2 decreased both invasion and migration ability and expression of EMT-related proteins. Significant inhibitory effects on tumor growth and invasion were observed in xenograft tumors of UM-UC-3 treated by DAB2-targeting siRNA. Our findings suggested that DAB2 expression was associated with poor prognosis through increased oncogenic properties including tumor proliferation, migration, invasion, and enhancement of EMT in human UCB.

Tumor Location Based Segmentation in Upper-Tract Urothelial Carcinoma Impacts on the Urothelial Recurrence-Free Survival: A Multi-Institutional Database Study.

INTRODUCTION AND OBJECTIVES: The predictive impact of primary tumor location for patients with upper-tract urothelial carcinoma (UTUC) in the presence of concomitant urothelial bladder cancer, along with urothelial recurrence after the curative treatment is still contentious. We evaluated the association between precise tumor location and concomitant presence of urothelial bladder cancer and urothelial recurrence-free survival in patients with UTUC treated by radical nephroureterectomy with a bladder cuff. METHODS: A total of 1,349 patients with localized UTUC (Ta-4N0M0) from a retrospective multi-institutional cohort were studied. We queried four UTUC databases. This retrospective clinical series was of patients with localized UTUC managed by nephroureter-ectomy with a bladder cuff, for whom data were from the Nishinihon Uro-Oncology Collaborative Group registries. Patients with a history of chemotherapy or radiotherapy were excluded from the study. Associations between the location of the tumor and subsequent outcome following nephroureterectomy were assessed using COX multivariate analysis. The location of the tumor was verified by pathological samples. Urothelial recurrence was defined as tumor relapse in any local urothelium, and coded apart from distant metastasis. The median follow-up was 34 months. RESULTS: A total of 887 patients had an evaluation of the tumor location in which 475 patients had pelvic tumors (53.6%), 96 had ureteral tumors in the U1 segment (10.8%), 87 in the U2 segment (9.8%), and 176 in the U3 segment (19.8%). There were 52 patients who had multifocal tumors (5.9%) as follows: 8 (0.9%) in the pelvis and ureter, 11 (1.2%) in U1 + U2, 1 (0.1%) in U1 + U3, 27 (3.0 %) in U2 + U3, and 6 (0.7%) in U1 + U2 + U3. In all, 145 (16.3%) had concomitant bladder tumors. Logistic regression analysis of gender, age, hydronephrosis, cytology, performance status, grade, lymphovascular invasion, pT, pN, and tumor focality showed that tumor location was associated with the presence of concomitant bladder cancer (p = 0.004, HR = 1.265). When the tumor location was stratified into 8 segments, including multifocal tumors, only the U3 segment remained as a predictor for the presence of concomitant bladder cancer (p = 0.002, HR = 2.872). Kaplan-Meier analysis for unifocal disease showed that lower ureter tumors (a combination of U2 and U3) had a worse prognosis for urothelial recurrence than pelvic tumors or upper ureteral tumors (U1) (p < 0.001 for lower ureteral tumors versus pelvic tumors, p = 0.322 for upper ureteral tumor versus pelvic tumor by log rank). Multivariate analysis showed that lower ureter remained as a prognostic factor for urothelial recurrence after adjusting for gender, age, hydronephrosis, urine cytology, lymphovascular invasion, pT, and pN (p < 0.001, HR = 1.469), and a similar tendency was found when the analysis was run for patients without concomitant bladder tumors (p = 0.003, HR = 1.446). Patients with lower ureteral tumors had a higher prevalence of deaths (HR = 2.227) compared to patients with upper ureter tumors. CONCLUSIONS: This multi-institutional study showed that the primary tumor locations were independently associated with the presence of concomitant bladder tumors and subsequent urothelial recurrence.

[Migration of Hem-o-Lok Clips into the Urinary Bladder Used during Robot-Assisted Laparoscopic Prostatectomy].

Recently, robot-assisted laparoscopic prostatectomy (RALP) has become a widely accepted surgical alternative for the treatment of prostate cancer. The intravesical migration of clips is a rare surgical complication of RALP. From March2013 to July 2018, 320 patients underwent RALP at our hospital. Migration of a Hem-o-Lok clip into the urinary bladder occurred in 4 of the 320 patients (1.3%). We analyzed these 4 patients in terms of subjective symptoms, intra- and post-operative findings, site of the migrated clip, and its treatment. The mean duration from RALP to the diagnosis was 13.8 months (2-26 months). The main symptoms due to migrated clips were : narrowed urinary stream, perineal pain, gross hematuria, and painful urination. In all cases, the size of the migrated clip was medium-large, and the events developed on the side contralateral to the first assistant. The clips were transurethrally removed using a Holmium-laser in 2 patients, and spontaneous excretion was observed in 1. The remaining patient has been asymptomatic and is being conservatively observed. In order to prevent the migration of clips used during RALP, the size of the clips and direction of the clip tail may be important. The first assistant should place the clips carefully, especially on the contralateral side.

Evaluation of pro­ and anti­tumor effects induced by three colony­stimulating factors, G­CSF, GM­CSF and M­CSF, in bladder cancer cells: Is G­CSF a friend of bladder cancer cells?

Cytotoxic chemotherapy is the standard treatment for patients with advanced bladder cancer. However, this treatment can cause transient and prolonged neutropenia, which can result in fatal infection. Three recombinant human colony­stimulating factors (CSFs), granulocyte CSF (G­CSF), granulocyte­macrophage CSF (GM­CSF), and macrophage CSF (M­CSF), are currently available to reduce the duration and degree of neutropenia. The present study investigated the pro­ and anti­tumor effects of these three CSFs and the changes in molecular profiles. Xenograft tumors in athymic mice were generated by subcutaneously inoculating the human bladder cancer cell lines MGH­U3 and UM­UC­3. A total of 2 weeks after cell inoculation, mice were randomly divided into four groups (control, G­CSF, GM­CSF and M­CSF) and treated thrice a week for 2 weeks. Tumor growth during monitoring and tumor weight at the time of euthanization were significantly higher in mice treated with G­CSF and lower in mice treated with GM­CSF compared with the control mice. Tumors were examined by immunostaining with antibodies against proteins associated tumor proliferation (Ki­67), angiogenesis [CD31 and vascular endothelial growth factor (VEGF)], anti­immunity (CD204) and epithelial­mesenchymal transition (EMT; E­cadherin). Immunohistochemical staining revealed that tumor proliferation, angiogenesis, recruitment of M2 macrophages and EMT were promoted by G­CSF, whereas lymphangiogenesis and recruitment of M2 macrophages were inhibited by GM­CSF. Treatment­associated changes in serum pro­ and anti­tumoral cytokines and chemokines were evaluated by enzyme­linked immunosorbent assay (ELISA)­based arrays. In the ELISA for serum, the levels of cytokines associated with angiogenesis (interleukin­6 and VEGF), and EMT (transforming growth factor­ß1 and ­ß2) were elevated in mice treated with G­CSF. Treatment with GM­CSF and M­CSF also affected the level of these cytokines characteristically. The current results indicate that administration of exogenous G­CSF to patients with bladder cancer promotes tumor growth through promotion of cell proliferation, angiogenesis, recruitment of M2 macrophages and enhancement of EMT through the modulation of the tumor microenvironment.

Comparison of cancer detection rates by transrectal prostate biopsy for prostate cancer using two different nomograms based on patient's age and prostate volume.

BACKGROUND: The aim of this study is to evaluate the efficacy of two different Nara Urological Research and Treatment Group (NURTG) nomograms allocating 6-12 biopsy cores based on age and prostate volume. MATERIALS AND METHODS: From April 2006 to July 2014, a total of 1,605 patients who underwent initial prostate biopsy were enrolled. Based on a nomogram taking the patient's age and prostate volume into consideration, 6-12 biopsy cores were allocated. Two types of nomogram were used, for the former group (before March 2009) and latter group (March 2009 onward). Cancer detection rates in all patients and those with prostate-specific antigen values in the gray zone (4.0-10 ng/mL) were compared. Predictive parameters for detection of prostate cancer in gray-zone patients were also investigated. RESULTS: The cancer detection rates in all patients and those in the gray zone were 48% and 38% in the former group and 54% and 41% in the latter group, respectively. The cancer detection rate in all patients was significantly higher in the latter group compared with the former group, but detection in gray-zone patients did not show a significant difference between the two groups (P=0.011 and P=0.37, respectively). Multivariate analysis indicated that age, digital rectal examination, prostate volume, transrectal ultrasonography findings, and volume/biopsy ratio were significant predictive parameters in gray-zone patients. The clinically insignificant cancer detection rate was significantly lower in the latter group compared with the former group (P=0.0008). CONCLUSION: The latter nomogram provided more acceptable detection rates of clinically significant and insignificant cancer than the former one, and we consider that an initial maximum 12-core transrectal ultrasound-guided needle biopsy may be sufficient for prostate cancer diagnosis.

Expression of peptide transporter 1 has a positive correlation in protoporphyrin IX accumulation induced by 5-aminolevulinic acid with photodynamic detection of non-small cell lung cancer and metastatic brain tumor specimens originating from non-small cell lung cancer.

BACKGROUND: Recently, 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX fluorescence was reported to be a useful tool during total surgical resection of high-grade gliomas. However, the labeling efficacy of protoporphyrin IX fluorescence is lower in metastatic brain tumors compared to that in high-grade gliomas, and the mechanism underlying protoporphyrin IX fluorescence in metastatic brain tumors remains unclear. Lung cancer, particularly non-small cell lung cancer (NSCLC), is the most common origin for metastatic brain tumor. Therefore, we investigated the mechanism of protoporphyrin IX fluorescence in NSCLC and associated metastatic brain tumors. METHODS: Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) was employed to evaluate the protein and mRNA levels of five transporters and enzymes involved in the porphyrin biosynthesis pathway: peptide transporter 1 (PEPT1), hydroxymethylbilane synthase (HMBS), ferrochelatase (FECH), ATP-binding cassette 2 (ABCG2), and heme oxygenase 1 (HO-1). The correlation between protein, mRNA, and protoporphyrin IX levels in NSCLC cells were evaluated in vitro. Immunohistochemistry was used to determine proteins that played a key role in intraoperative protoporphyrin IX fluorescence in clinical samples from patients with NSCLC and pathologically confirmed metastatic brain tumors. RESULTS: A significant correlation between PEPT1 expression and protoporphyrin IX accumulation in vitro was identified by western blotting (P = 0.003) and qRT-PCR (P = 0.04). Immunohistochemistry results indicated that there was a significant difference in PEPT1 between the intraoperative protoporphyrin IX fluorescence-positive and protoporphyrin IX fluorescence-negative groups (P = 0.009). CONCLUSION: Expression of PEPT1 was found to be positively correlated with 5-ALA-induced protoporphyrin IX accumulation detected by photodynamic reaction in metastatic brain tumors originating from NSCLC.