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We encountered a 9-year-old boy with a small bladder who had previously undergone multiple ureteroneocystostomies for unilateral refluxing megaureter. He underwent excision of the affected non-functioning kidney and ureterocystoplasty used the dilated regional ureter, in which the loop shaped urinary bladder was reconstructed without detubularization of the dilated ureter. The long-term postoperative course has been satisfactory. There have been no reports of ureterocystoplasty used a dilated ureter after multiple ureteroneocystostomies and none describing ureterocystoplasty in which the ureter was looped. This case is presented herein.

Shigeru Nakamura,1 Kazuya Tanabe,1 Taiju Hyuga,1,2 Taro Kubo,1 Satoru Inoguchi,1 Shina Kawai,1 Hideo Nakai1 1Department of Pediatric Urology, Jichi Medical University, Children’s Medical Center Tochigi, Shimotsuke, Japan; 2Department of Developmental Genetics, Institute of Advanced Medicine, Wakayama Medical University (WMU), Wakayama, JapanCorrespondence: Shigeru NakamuraDepartment of Pediatric Urology, Jichi Medical University, Children’s Medical Center, 3311-1, Yakushiji, Shimotsuke, Tochigi 329-0498, JapanTel +81-285-44-2111Fax +81-285-44-8452Email naka251148@gmail.comPurpose: To evaluate the treatment outcomes and postoperative complications associated with the systematic multi-site hydrodistention implantation technique (SMHIT) for primary vesicoureteral reflux (VUR) and to determine its mid-term efficacy and safety.Patients and Methods: We retrospectively reviewed the data for 17 ureters from 12 consecutive children, aged ≥ 1 year, with grade II–IV reflux and a history of febrile urinary tract infections (FUTI), who underwent a single-session of SMHIT. The primary outcome was the absence of postoperative FUTI (clinical success). The secondary outcome was improvement in reflux to grade 0–I on postoperative voiding cystourethrography (radiographic success).Results: Five and 7 children had bilateral and unilateral reflux, respectively. Reflux was categorized as grade II, III, and IV reflux in 2, 12, and 3 ureters, respectively. Seven of 10 (70%) toilet-trained children had bladder-bowel dysfunction (BBD) preoperatively. The SMHIT was performed for all patients, after which BBD improved. The mean postoperative follow-up period was 6 years and 9 months. The clinical success rate was 100%. Radiographic success was achieved in 16/17 ureters (94%) at 3– 4 months, 17/17 (100%) ureters at 1 year, and 17/17 (100%) ureters at 3 years postoperatively. Major complications did not develop postoperatively.Conclusion: When prioritizing treatment of concomitant BBD in children with primary VUR and avoiding dextranomer/hyaluronic acid injection therapy in contraindicated children according to the Food and Drug Administration recommendations, a single-session of SMHIT may be as effective and safe in the mid-term as performing open anti-reflux surgery.Keywords: dextranomer hyaluronic acid, Dx/HA, endoscopic treatment, vesicoureteral reflux

Background: Analysis of a microRNA (miRNA) expression signature of bladder cancer (BC) by deep-sequencing revealed that clustered miRNAs microRNA (miR)-451a, miR-144-3p, and miR-144-5p were significantly downregulated in BC tissues. We hypothesised that these miRNAs function as tumour suppressors in BC. The aim of this study was to investigate the functional roles of these miRNAs and their modulation of cancer networks in BC cells. Methods: The functional studies of BC cells were performed using transfection of mature miRNAs. Genome-wide gene expression analysis, in silico analysis, and dual-luciferase reporter assays were applied to identify miRNA targets. The association between miR-144-5p levels and expression of the target genes was determined, and overall patient survival as a function of target gene expression was estimated by the Kaplan–Meier method. Results: Gain-of-function studies showed that miR-144-5p significantly inhibited cell proliferation by BC cells. Four cell cycle-related genes (CCNE1, CCNE2, CDC25A, and PKMYT1) were identified as direct targets of miR-144-5p. The patients with high CCNE1 or CCNE2 expression had lower overall survival probabilities than those with low expression (P=0.025 and P=0.032). Conclusion: miR-144-5p functions as tumour suppressor in BC cells. CCNE1 and CCNE2 were directly regulated by miR-144-5p and might be good prognostic markers for survival of BC patients.

insulin signaling in the progression of a murine RCC model using metformin to treat hyperinsulinemia induced by a high-carbohydrate diet. METHODS: pT1-T4 tumor specimens from 99 patients with RCC were evaluated for IR expression by immunohistochemistry. Serum insulin and C-peptide levels were measured. The association between biomarker expression and serum concentration, prognostic factors, and clinical outcomes were assessed. C57BL/6 mice were randomized into four groups: low-carbohydrate diet, high-carbohydrate diet, low-carbohydrate diet þ metformin, and high-carbohydrate diet þ metformin groups. RENCA cells were injected subcutaneously. Body weight, glucose tolerance test, serum insulin level, and tumor volume were evaluated. Immunoblotting was performed to assess the insulin signaling pathway in the murine RCC model. RESULTS: The IR expression level was significantly lower in patients with tumor stage pT2-4 and with a preoperative serum Cpeptide level more than 5.14 ng/mL. High IR expression was an independent predictor of favorable progression-free survival after radical nephrectomy. In vivo progression of RENCA in the murine model was not significantly stimulated by hyperinsulinemia induced by a high-carbohydrate diet. However, in vivo progression of RENCA was significantly inhibited by metformin in both the lowand highcarbohydrate diet groups. IR expression was significantly higher in the tumors from the low-carbohydrate diet group and high-carbohydrate diet plus metformin group than that from the high-carbohydrate diet group. CONCLUSIONS: IR expression in RCC was inversely associated with cancer progression and serum insulin levels in both clinical and murine models. The anticancer effect of metformin in the murine RCC model might be derived from the direct effect of the agent on cancer cells rather than the effect of the lower insulin level. The lower expression of IR in the tumor from patients with a poor outcome might be the result of a higher serum insulin level, which is not strong cause of cancer progression.

INTRODUCTION AND OBJECTIVES: Generally in microRNA (miRNA) biogenesis, one strand (guide strand) of the duplex may potentially act as a functional miRNA and incorporated into the RNA-induced silencing complex and regulating target genes. In contrast, other strand (passenger strand: miRNA*) is disintegrated quickly. Our miRNA expression signature of bladder cancer (BC) by using deep-sequencing revealed that tumor-suppressive miRNA-145 and its passenger strand of miRNA-145* were significantly reduced in cancer tissues, suggesting these function as tumor suppressors in BC cells. The aim of the study was to investigate functional significance of miRNA-145* and its regulated molecular targets/ pathways in BC. METHODS: We evaluated the expression levels of miR-145/ miR-145* in 43 BC clinical specimens and 12 normal bladder epithelia by real-time PCR. The functional analysis of the miRNA and molecular targets/pathways searching strategies were described as our previous studies (J Urol,192:1822-1830,2014; FEBS Lett,588:3170-3179,2014; PLoS One,9:e84311,2014). RESULTS: Expression levels of miR-145 and miR-145* were significantly lower than that in normal bladder epithelia (P < 0.0001). Gain-of-function studies revealed that cell proliferation, migration, and invasion were significantly inhibited in both miR-145 and miRNA-145* transfection into BC cell lines (each, P < 0.001). Combination of gene expression analysis of miR-145*-transfectants and in silico analysis showed that several oncogenic genes (HK2, YES1, FOSL1 etc.) were identified as miR-145* regulation in BC cells. CONCLUSIONS: Our genome-wide miRNA expression signature showed that miR-145* as a tumor-suppressive miR-145 passenger strand frequently reduced in BC cells and acts as a tumorsuppressor. Elucidation of miR-145/145*-mediated novel cancer pathways provide new insights of the potential mechanisms of BC oncogenesis. Furthermore, tumor-suppressive miR-145/145* might be contributed to development of miRNA-based cancer therapeutics in the disease.

METHODS: We sequenced the whole genomes of the tumor and normal tissues from 60 RCC patients (T4N0M1) with well-documented clinical data to a mean depth of 30i A or greater. RESULTS: From the whole genome data, we identified somatic mutations and copy number alterations that may serve as the markers for and (or) possibly drive cancer progression. RCCs display two classes of structural variation: monogenomic and polygenomic tumors. We further deep-sequenced the whole genomes of several representative cases with polygenomic tumors to 120i A and more. Our studies identified the early genomic events that occur in the founder cells and shed light on the genome evolution of RCCs. By using the somatic mutations as the lineage markers, we traced the genetic record of the emergence of different sub-clones over time and found the genomic events that drive divergence of the founder cells into different subgroups. CONCLUSIONS: This study deciphers the fine genetic architecture of the RCC genomes and allows us to determine the extent of sub-clonal diversities within the bulk tumor mass of RCC, to build a polygenetic tree of the cancer to define when the divergence occurs, and to reveal what mutational processes are involved in the different stages of tumor evolution.

INTRODUCTION AND OBJECTIVES: Genomic characterization of urothelial carcinoma of the bladder (UCB) has begun to reveal significant intertumor heterogeneity when comparing samples from different subjects. As in other malignancies, intratumor heterogeneity, which may allow for tumor evolution and adaption, poses a significant challenge to personalized-medicine strategies. METHODS: To examine UCB tumor evolution and heterogeneity, we performed next-generation targeted sequencing on multiple temporally and spatially separated bladder tumors obtained at time of transurethral resection (TUR) and radical cystectomy (RC). Specimens were analyzed using a next-generation, targeted sequencing assay designed to identify point mutations, indels, and copy number alterations in 300 cancer-associated genes. RESULTS: Phylogenetic reconstruction revealed evidence of branched evolutionary growth. Evaluation of multiple tumors from individual subjects identified both shared and unique potential driver mutations. Evidence of convergent phenotypic evolution was detected through analysis of multiple distinct tumors from several subjects. For example, three separate tumors in one subject (see Figure) shared a common PIK3CA mutation (E453K) and had unique second mutations in PIK3CA (E542V, E545K, and E545Q, respectively). In another subject, distinct inactivatingmutations of EP300were identified in two temporally separated tumor samples. Macrodissection of single tumors into non-invasive and invasive components revealed significant intratumor heterogeneity; one case illustrates howanalysis of amuscleinvasive TURspecimen could result in undersampling and therebymiss the tumor clone that persisted at time of RC. CONCLUSIONS: We demonstrate branched evolution of UCB through genomic analyses of multiple temporally and spatially distinct bladder tumors from individual subjects. Macrodissection of individual tumor samples identified significant intratumor heterogeneity. These concepts may present major challenges to personalized-medicine approaches that rely on sampling of a single tumor at a specific timepoint in the evolution of a patient’s UCB.

INTRODUCTION AND OBJECTIVES: Cancer-stromal interaction is critical for cancer biology. Bladder cancer develops within the mucosa, and invades into muscle layer surrounded by abundant adipose tissue. Adipose tissue has stromal cell types, such as preadipocytes and mesenchymal stem cells (MSCs). Thus, the adipose tissue stromal cells (ATSCs) seem critical for bladder cancer biology. The aim of this study is to address the effects of ATSCs on the apoptosis, growth and invasion of the superficial and invasive types. METHODS: RT4 and RT112 were used as human superficial urothelial carcinoma type of bladder, while EJ and HT1376 were done as invasive type. ATSCs were isolated from subcutaneous adipose tissue of 1-week old male Wister rats. We examined the apoptosis, growth and invasion of cancer cells, using collagen gel invasion assay system, in which the tumor cells were co-cultured on ATSC-embedded gel layer. The cellular behavior, and the apoptosis-, growthand ivasion-related molecules were analyzed by morphometry, immunohistochemistry, Western blot and real-time RT-PCR. RESULTS: ATSCs promoted and inhibited the apoptosis and growth of superficial type cancer cells, respectively. In contrast, ATSCs inhibited and promoted the apoptosis and growth of invasive type cancer cells. ATSCs had no effect on the invasion of superficial type, whereas they promoted the invasion of invasive type. ATSCs promoted the expression of MAPK proteins (raf-1, MEK-1 and ERK1/ERK2) in all cancer cell types. In turn, superficial type promoted [alpha]-SMA-positive myofibroblastic differentiation in ATSCs, while invasive type did lipid dropletand S-100 protein-positive adipogenic differentiation in ATSCs. CONCLUSIONS: The data suggest the following issues: 1) ATSCs suppress the progression of superficial type urothelial carcinoma through the growth inhibition and apoptosis promotion, whereas ATSCs promote that of invasive type through the enhancement of the growth and invasion, and the apoptosis prohibition; 2) ATSCinduced MAPK pathway activation plays differential roles in biological behavior of superficial and invasive types; and 3) Superficial type induces myofibroblastic differentiation in ATSCs as cancer-associated phenotype, while invasive type does adipogenic differentiation in ATSCs. Source of Funding: none