A deep learning algorithm for 3D cell detection in whole mouse brain image datasets.

Understanding the function of the nervous system necessitates mapping the spatial distributions of its constituent cells defined by function, anatomy or gene expression. Recently, developments in tissue preparation and microscopy allow cellular populations to be imaged throughout the entire rodent brain. However, mapping these neurons manually is prone to bias and is often impractically time consuming. Here we present an open-source algorithm for fully automated 3D detection of neuronal somata in mouse whole-brain microscopy images using standard desktop computer hardware. We demonstrate the applicability and power of our approach by mapping the brain-wide locations of large populations of cells labeled with cytoplasmic fluorescent proteins expressed via retrograde trans-synaptic viral infection.

Pathogenic and targetable genetic alterations in 70 urachal adenocarcinomas.

Urachal cancer (UrC) is a rare but aggressive malignancy often diagnosed in advanced stages requiring systemic treatment. Although cytotoxic chemotherapy is of limited effectiveness, prospective clinical studies can hardly be conducted. Targeted therapeutic treatment approaches and potentially immunotherapy based on a biological rationale may provide an alternative strategy. We therefore subjected 70 urachal adenocarcinomas to targeted next-generation sequencing, conducted in situ and immunohistochemical analyses (including PD-L1 and DNA mismatch repair proteins [MMR]) and evaluated the microsatellite instability (MSI) status. The analytical findings were correlated with clinicopathological and outcome data and Kaplan-Meier and univariable/multivariable Cox regression analyses were performed. The patients had a mean age of 50 years, 66% were male and a 5-year overall survival (OS) of 58% and recurrence-free survival (RFS) of 45% was detected. Sequence variations were observed in TP53 (66%), KRAS (21%), BRAF (4%), PIK3CA (4%), FGFR1 (1%), MET (1%), NRAS (1%), and PDGFRA (1%). Gene amplifications were found in EGFR (5%), ERBB2 (2%), and MET (2%). We detected no evidence of MMR-deficiency (MMR-d)/MSI-high (MSI-h), whereas 10 of 63 cases (16%) expressed PD-L1. Therefore, anti-PD-1/PD-L1 immunotherapy approaches might be tested in UrC. Importantly, we found aberrations in intracellular signal transduction pathways (RAS/RAF/PI3K) in 31% of UrCs with potential implications for anti-EGFR therapy. Less frequent potentially actionable genetic alterations were additionally detected in ERBB2 (HER2), MET, FGFR1, and PDGFRA. The molecular profile strengthens the notion that UrC is a distinct entity on the genomic level with closer resemblance to colorectal than to bladder cancer.

Matrix metalloproteinase 7, soluble Fas and Fas ligand serum levels for predicting docetaxel resistance and survival in castration-resistant prostate cancer.

OBJECTIVE: To assess the predictive value of pre-chemotherapy matrix metalloproteinase 7 (MMP-7), soluble Fas (sFas) and Fas ligand (FasL) serum levels, as well as their changes during therapy. PATIENTS AND METHODS: Serum levels of MMP-7, Fas and FasL were determined by ELISA in 96 patients with castration-resistant prostate cancer (CRPC): 21 docetaxel-resistant patients who received one single series and 75 docetaxel-sensitive patients who received repeated series of docetaxel. In addition to the 96 pretreatment serum samples, 987 sera collected during chemotherapy were also analysed. RESULTS: Higher pretreatment serum MMP-7, sFas and prostate-specific antigen (PSA) levels were significantly associated with both docetaxel resistance (P = 0.007, P = 0.001, P < 0.001, respectively) and shorter cancer-specific survival (P < 0.001, P = 0.041, P < 0.001, respectively). High MMP-7 level remained an independent predictor of both docetaxel resistance (hazard ratio [HR] 2.298, 95% confidence interval [CI]: 1.354-3.899; P = 0.002) and poor cancer-specific survival (HR 2.11, 95% CI: 1.36-3.30; P = 0.001) in multivariable analyses. Greater increase in MMP-7 levels in the second treatment holiday and greater increase in PSA levels in the first and second treatment holidays were predictive of survival. CONCLUSIONS: Pretreatment serum MMP-7 levels may help to select patients with CRPC who are likely to benefit from docetaxel chemotherapy. Furthermore, MMP-7 levels alone or in combination with PSA levels could be used for therapy monitoring. Correlative studies embedded in clinical trials are necessary to validate these biomarkers for clinical decision-making.

Telomerase reverse transcriptase (TERT) promoter mutations are rare in urachal cancer.

High rates of telomerase reverse transcriptase (TERT) promoter mutations have recently been described in urothelial carcinoma (UC). Unlike UC in the bladder, adenocarcinomas account for the majority of urachal cancer (UrC) cases. As data in UrC is unclear, we analyzed TERT promoter mutations in a large cohort of UrC for its differential diagnostic, clinicopathological and prognostic significance. UrC cases from six academic centers were analyzed for c.-146C>T (C250T) and c.-124C>T (C228T) TERT promoter mutations by PCR and Sanger sequencing. Clinicopathological and survival data were collected. The cohort consisted of 15 men (56%) and 12 women (44%) with a median age of 50 years including 23 adenocarcinomas, two squamous cell carcinomas (SCC), one UC and one undifferentiated carcinoma. In one case of (mucinous) urachal adenocarcinoma a C228T mutation was detected (1/23; 4%), like in a case of SCC in addition to one C250T mutation in the UC case. TERT promoter mutations are very rare in urachal adenocarcinomas (unlike in UC) with differential diagnostic implications. Additionally, the low TERT promoter mutation rate in urachal adenocarcinomas is more comparable to colorectal adenocarcinomas than to UC, giving further support to recent genetic findings and therapeutic considerations.

Soluble syndecan-1 (SDC1) serum level as an independent pre-operative predictor of cancer-specific survival in prostate cancer.

BACKGROUND: PSA-screening detects many cases of clinically non-aggressive prostate cancer (PC) leading to significant overtreatment. Therefore, pre-operatively available prognostic biomarkers are needed to help therapy decisions. Syndecan-1 (SDC1) is a promising prognostic tissue marker in several cancers including PC but serum levels of shedded SDC1-ectodomain (sSDC1) have not been assessed in PC. METHODS: A total of 150 patients with PC were included in this study (n = 99 serum samples, n = 103 paraffin-embedded samples (FFPE), n = 52 overlap). SDC1 protein expression and cellular localization was evaluated by immunohistochemistry (IHC), while sSDC1 serum concentrations were measured by ELISA. Serum sSDC1 levels were compared to those of MMP7, which is known to be a protease involved in SDC1 ectodomain-shedding. Clinico-pathological and follow-up data were collected and correlated with SDC1 tissue and serum levels. Disease (PC)-specific (DSS) and overall-survival (OS) were primary endpoints. RESULTS: Median follow-up was 167 months in the serum- and 146 months in the FFPE-group. SDC1-reactivity was higher in non-neoplastic prostate glands compared to PC. In addition, cytoplasmatic, but not membranous SDC1 expression was enhanced in PC patients with higher Gleason-score >6 PC (P = 0.016). Soluble SDC1-levels were higher in patients with Gleason-score >6 (P = 0.043) and metastatic disease (P = 0.022) as well as in patients with progressed disease treated with palliative transurethral resection (P = 0.002). In addition, sSDC1 levels were associated with higher MMP7 serum concentration (P = 0.005). In univariable analyses, only sSDC1-levels exhibited a trend to unfavorable DSS (P = 0.077). In a multivariable pre-operative model, high pre-operative sSDC1-level (>123 ng/ml) proved to be an independent marker of adverse OS (P = 0.048) and DSS (P = 0.020). CONCLUSIONS: The present study does not confirm the prognostic relevance of SDC1-IHC. The significant higher sSDC1 serum levels in advanced cases of PC, suggest that SDC1 shedding might be involved in PC progression. Additionally, high sSDC1-level proved to be an independent factor of adverse OS and DSS in a multivariable pre-operative model, making evaluation of sSDC1-levels a promising tool for pre-operative risk-stratification and/or therapy monitoring. Prostate 76:977-985, 2016. © 2016 Wiley Periodicals, Inc.

Circulating and Tissue Expression Levels of YKL-40 in Renal Cell Cancer.

PURPOSE: Blood levels of YKL-40 are elevated in various malignancies and other inflammatory diseases. Higher YKL-40 levels have consequently been shown to correlate with poor prognosis in several cancers. We investigated the prognostic value of circulating and tissue levels of YKL-40 in renal cell cancer. MATERIALS AND METHODS: Preoperative YKL-40 serum/plasma levels were determined in 222 surgically treated patients with renal cell cancer and in 35 controls. Postoperative serum samples were analyzed in 19 of the 222 renal cell cancer cases. Gene expression levels were assessed in 101 renal cell cancer frozen tissue samples using quantitative real-time reverse transcriptase-polymerase chain reaction. Finally immunohistochemical analysis was done in 37 renal cell cancer cases to assess tissue localization of YKL-40. Results were correlated with clinicopathological and followup data. RESULTS: YKL-40 serum but not tissue gene expression levels were higher in patients with renal cell cancer compared to controls (p = 0.050). Serum YKL-40 levels significantly increased following nephrectomy (p <0.001). High circulating YKL-40 concentrations were independently associated with shorter survival in the serum and plasma cohorts. YKL-40 gene expression did not correlate with patient prognosis. CONCLUSIONS: Preoperatively elevated circulating levels of YKL-40 predict survival in patients treated with nephrectomy for renal cell cancer independently of levels determined in serum or plasma. Tumor cells do not seem to be the main source of increased serum/plasma YKL-40 levels in patients with renal cell cancer.

Urachal Carcinoma of the Bladder: Impact of Clinical and Immunohistochemical Parameters on Prognosis.

PURPOSE: Urachal carcinoma of the bladder is a rare malignancy. Its histological phenotype is similar to that of primary bladder and colorectal adenocarcinoma. The aim of this study was to explore the expression and prognostic relevance of 6 select protein markers of urachal carcinoma of the bladder, including p53, Ki67, RHAMM, BGN, IMP3 and MMP-7, which were formerly shown to be prognostic in urothelial carcinoma and colorectal adenocarcinoma. MATERIALS AND METHODS: Clinical and followup data were obtained on a total of 26 patients with urachal carcinoma of the bladder treated at 2 university hospitals. Immunohistochemical analysis of p53, Ki67, RHAMM, BGN, IMP3 and MMP-7 expression was performed in samples from 15 patients. Clinicopathological parameters and immunohistochemical results were tested for prognostic value on univariable and multivariable analyses. RESULTS: Followup was 50 months. Five-year overall and progression-free survival was 46% and 32%, respectively. On multivariable analysis a positive resection margin was an independent predictor of poor overall survival (p = 0.025). RHAMM (p = 0.0431), IMP3 (p = 0.0052), Ki67 (p = 0.0006) and p53 (p = 0.0024) expression rates were significantly increased in urachal carcinoma of the bladder cells compared to normal urothelium. IMP3 was elevated in Sheldon tumor stage IIIA compared to IIIB or greater (p = 0.0048). None of the analyzed protein markers was associated with survival. CONCLUSIONS: The independent prognostic value of a positive resection margin underlines the importance of complete surgical removal of urachal carcinoma of the bladder combined with en bloc resection of the median umbilical ligament and umbilicus. Our results in a limited number of samples show that Ki67, p53, RHAMM and IMP3 expression is enhanced but has no prognostic significance in urachal carcinoma of the bladder.

The long noncoding RNA HOTAIR has tissue and cell type-dependent effects on HOX gene expression and phenotype of urothelial cancer cells.

BACKGROUND: Urothelial carcinoma (UC) is the fifth most common cancer in the developed world. Delineation of differentiation subtypes in UC highlighted the importance of aberrant differentiation. Understanding underlying mechanisms may facilitate diagnosis and development of efficient therapy strategies. It is well accepted that epigenetic mechanisms are involved. Long noncoding RNAs (lncRNAs), a new class of epigenetic factors, are thought to mediate molecular differences between cell types to control cellular identity. The present study focuses on the lncRNA HOTAIR, originating from the HOXC locus. Its overexpression induces an aggressive phenotype in many cancers and aberrant expression of homeotic HOX transcription factors, especially HOXD10, that regulate differentiation and tissue homeostasis. The aim of the present study was to determine the functional role of HOTAIR in UC with regard to aggressive phenotype, regulation of aberrant differentiation and altered HOX gene expression. METHODS: We determined RNA expression levels of HOTAIR and HOX genes in UC tissues and cell lines. Knockdown of HOTAIR and ectopic overexpression was performed to determine the effect on reported target genes in UC. Cell lines were stably transfected with HOTAIR to investigate changes in phenotype and HOX gene expression. RESULTS: HOTAIR was overexpressed in approximately half of UC tissues and cell lines. Effects of HOTAIR overexpression differed between cell lines. Whereas VM-CUB1 cells acquired the expected phenotype with increased proliferation, clonogenicity, anchorage independent growth, migratory activity and epithelial-to-mesenchymal transition, 5637 cells grew more slowly displaying induction of senescence and related immune response genes. Other UC lines showed intermediate effects. Expression profiling revealed divergent effects on HOX genes, cell cycle regulators and differentiation according with the phenotypic differences between HOTAIR-overexpressing VM-CUB1 and 5637 cells. CONCLUSIONS: Our data indicate that HOTAIR overexpression may affect differentiation state and aggressiveness of UC cells, but in a cell-type dependent manner. Our functional studies and the comparison of our expression data sets with those from other cancer cell types, which revealed minimal overlaps, indicate that effects of HOTAIR are strongly tissue-dependent and can even differ within one cancer type. Thus, HOTAIR functions and target genes cannot simply be transferred from one cancer type to the other.

Renal cell carcinoma and tumour thrombus in the inferior vena cava: clinical outcome of 98 consecutive patients and the prognostic value of preoperative parameters.

OBJECTIVES: To evaluate the outcome of patients after nephrectomy and removal of tumour thrombus and to assess the prognostic value of preoperative parameters. PATIENTS AND METHODS: Ninety-eight patients who were surgically treated between 2002 and 2011 were included. Patients' charts were reviewed, and patients with renal cell carcinoma (RCC) and concomitant tumour thrombus in the renal vein (RV) were compared with those with extended inferior vena cava (IVC) thrombus. Wilcoxon rank-sum test, Kaplan-Meier analysis and uni- and multivariate Cox regression analysis were used for statistical evaluation. RESULTS: Follow-up was 36 months (20-122 months), and 5-year disease-specific survival (DSS) and overall survival were 68.4 and 54.1 %, respectively. Patients with extended thrombus (levels 2-4) had higher intraoperative transfusion rates of concentrated red cells (CRC) and fresh-frozen plasma (FFP) compared with patients with thrombus confined to the RV (CRC: 5.8 vs. 1.5, p < 0.0001; FFP: 2.3 vs. 0.4, p = 0.0032). Surgery time (190 vs. 107 min, p < 0.0001), duration of hospitalisation (16 vs. 11 days, p = 0.0269), serum phosphate (3.64 vs. 3.29 mmol/l, p = 0.0369) and CRP levels (6.7 vs. 4.4 mg/dl, p = 0.0194) as well as aPTT were increased (33.7 vs. 29.6 s, p = 0.0059) in extended thrombus disease. In multivariate analysis, the presence of distant metastasis (p = 0.03) and lymphovascular invasion (p = 0.001), high platelet counts (p = 0.001) and high serum potassium levels (p = 0.032) proved to be independent prognostic factors. CONCLUSION: The surgical treatment of RCC with tumour thrombus in the RV or IVC has favourable results. Extended thrombus disease requires multidisciplinary approach. High serum potassium levels and platelet counts are associated with reduced DSS.

Prognostic value of tissue and circulating levels of IMP3 in prostate cancer.

Tissue levels of the oncofetal protein insulin-like growth factor 2 (IGF2) messenger RNA-binding protein 3 (IMP3) have been associated with poor prognosis in multiple human malignancies. However, its circulating levels have not yet been analyzed. Therefore, the aim of this study was to assess the prognostic value of both serum and tissue levels of IMP3 in prostate cancer (PC). IMP3 protein expression was analyzed in 124 PC and 13 benign prostate hyperplasia (BPH) patients using immunohistochemistry. Gene expression levels of IMP3 and its molecular target IGF2 were analyzed in 29 frozen and 26 paraffin-embedded PC tissues using real-time polymerase chain reaction and immunohistochemistry. Serum IMP3 levels were assessed in 94 PC and 20 BPH patients as well as in 20 controls using enzyme-linked immunosorbent assay. IMP3 immunostaining was present in 0% (0/13) of BPHs, 15% (15/101) of clinically localized PCs and 65% (15/23) of palliatively treated metastatic PCs (p < 0.001). Accordingly, serum IMP3 concentrations were significantly higher in PC compared to BPH patients which were higher than those in controls (p < 0.001 each). The highest concentrations were detected in metastatic PC patients (p = 0.036). In patients who underwent radical prostatectomy high IMP3 serum levels were independently associated with poor cancer-specific survival. IMP3 gene and protein expressions were not correlated with those of IGF2. In conclusion, we found enhanced IMP3 levels in tissue and serum samples of PC patients compared to non-PC men. Moreover, IMP3 was associated with metastasis and PC-specific survival. The tumor promoting effect of IMP3 appears to be independent from its regulatory role on IGF2 in PC.

Clinical sequencing identifies potential actionable alterations in a high rate of urachal and primary bladder adenocarcinomas.

OBJECTIVE: Administration of targeted therapies provides a promising treatment strategy for urachal adenocarcinoma (UrC) or primary bladder adenocarcinoma (PBAC); however, the selection of appropriate drugs remains difficult. Here, we aimed to establish a routine compatible methodological pipeline for the identification of the most important therapeutic targets and potentially effective drugs for UrC and PBAC. METHODS: Next-generation sequencing, using a 161 cancer driver gene panel, was performed on 41 UrC and 13 PBAC samples. Clinically relevant alterations were filtered, and therapeutic interpretation was performed by in silico evaluation of drug-gene interactions. RESULTS: After data processing, 45/54 samples passed the quality control. Sequencing analysis revealed 191 pathogenic mutations in 68 genes. The most frequent gain-of-function mutations in UrC were found in KRAS (33%), and MYC (15%), while in PBAC KRAS (25%), MYC (25%), FLT3 (17%) and TERT (17%) were recurrently affected. The most frequently affected pathways were the cell cycle regulation, and the DNA damage control pathway. Actionable mutations with at least one available approved drug were identified in 31/33 (94%) UrC and 8/12 (67%) PBAC patients. CONCLUSIONS: In this study, we developed a data-processing pipeline for the detection and therapeutic interpretation of genetic alterations in two rare cancers. Our analyses revealed actionable mutations in a high rate of cases, suggesting that this approach is a potentially feasible strategy for both UrC and PBAC treatments.

High insulin-like growth factor mRNA-binding protein 3 (IMP3) protein expression is associated with poor survival in muscle-invasive bladder cancer.

UNLABELLED: What's known on the subject? and What does the study add? Insulin-like growth factor mRNA-binding protein 3 (IMP3) is an oncofetal protein found to be re-expressed in a series of human cancers including bladder cancer. In vitro analyses showed an invasion and proliferation promoting effect for IMP3. Further in vitro studies suggested that IMP3 is able to bind to the mRNAs of CD44 and insulin-like growth factor 2 (IGF2), enhancing their stability and expression. However, this molecular interaction has not yet been analysed in tumour samples. In the present study, we identified for the first time high IMP3 tissue protein expression as an independent predictor of poor patients' survival in muscle-invasive bladder cancer. Furthermore, there was no correlation between IMP3 and its molecular targets in bladder carcinoma specimens and concluded that the tumour-promoting effect of IMP3 is not related to its regulatory action on IGF2 and CD44. OBJECTIVE: To assess the prognostic value and molecular actions of the oncofetal protein insulin-like growth factor mRNA-binding protein 3 (IMP3) in muscle-invasive bladder cancer (BC). PATIENTS AND METHODS: IMP3 expression was analysed by immunohistochemistry, real-time polymerase chain reaction and Western blot analysis in 224 patients with BC. The molecular targets of IMP3; CD44, insulin-like growth factor 2 (IGF2) and its receptor the IGF1 receptor (IGF1-R) were also investigated. Expression levels were correlated with clinical follow-up data by using both univariate and multivariate Cox regression analyses. RESULTS: IMP3 mRNA and protein levels were significantly elevated in high-stage and high-grade muscle-invasive BC. In muscle-invasive BC IMP3 protein but not gene expression proved to be an independent predictor of disease-specific (hazard ratio [HR] 2.58, 95% confidence interval [CI] 1.28-4.56, P = 0.004) and overall survival (HR 2.07, 95% CI 1.12-3.82, P = 0.020). The expression levels of IGF2 and CD44 showed no correlation with that of IMP3. CONCLUSIONS: High IMP3 protein levels may identify patients with BC at high risk of disease progression and may therefore select patients for a more intensive therapy or for a strict follow-up. Its high expression in high-grade bladder carcinoma cells makes IMP3 for an attractive target for therapy. The tumour promoting effect of IMP3 is independent from its regulatory action on IGF2 and CD44 expression.

Clinical outcome and quality of life in octogenarian patients with muscle-invasive urothelial carcinoma of the bladder treated with radical cystectomy or transurethral resection of the bladder tumor: a retrospective analysis of 143 patients.

PURPOSE: To compare clinical outcome and quality of life (QoL) in octogenarian patients with muscle-invasive urothelial carcinoma (MIBC) either treated by radical cystectomy (RC) or transurethral resection of the tumor (TURBT). METHODS: We identified octogenarian patients with MIBC in our institutions since 2005. Clinical treatment outcomes and QoL were analyzed. Uni- and multivariable Cox regression analyses, two-tailed Wilcoxon test, Mann-Whitney test and Fisher's exact test were assessed as appropriate. QoL was evaluated using FACT-G (Functional Assessment of Cancer Therapy-General) questionnaire. RESULTS: 143 patients were identified (RC: 51 cases, TURBT: 92 cases). Mean follow-up was 14 months (0-100 months). Median overall survival (OS) was 12 months in the RC group and 7 months in the TURBT group. TURBT and low preoperative hemoglobin were independent risk factors for reduced cancer-specific survival (CSS) (TURBT: p = 0.019, Hb: p = 0.008) and OS (TURBT: p = 0.026, Hb: p = 0.013) in multivariable analyses. Baseline QoL was low throughout the whole cohort. There was no difference in baseline FACT-G scoring comparing RC and TURBT (FACT-G total score (median): RC 43.7/108 vs. TURBT 44.0/108, p = 0.7144). Increased FACT-G questionnaire scoring was assessed for RC patients (median percentage score change RC 22.9%, TURBT 2.3%, p < 0.0001). CONCLUSION: RC and TURBT are feasible treatment options for MIBC in octogenarian patients. In our cohort, RC was associated with increased CSS, OS and QoL. QoL in general was low throughout the whole cohort. Interdisciplinary decision-making has to be improved for these critically ill patients.

Stone extraction with loop ureteral catheter versus ureteroscopy in small distal ureteral stones-retrospective comparison of 547 consecutive patients.

BACKGROUND: To evaluate whether stone extraction with a loop ureteral catheter (LUC) in distal ureteral stones is associated with a higher frequency of ureteral strictures compared to treatment with primary ureteroscopic stone removal (p-URS) or ureteroscopic laser lithotripsy (l-URS). METHODS: Five hundred and forty-seven consecutive patients were primarily endourologically treated for distal ureteral stones in our department between 2005 and 2019 and included in the study protocol. Data was retrospectively obtained from the patients' charts and medical reports as well as from office-based urologists. Data analysis was performed using Fisher's exact test, Mann-Whitney test or Student's t-test as appropriate. A level of P<0.05 was assigned statistical significance. RESULTS: Four hundred and twelve patients were treated by URS (p-URS n=304, l-URS n=108) and another 135 by LUC stone extraction. Median follow-up was 41 [2-159] months. There was no difference between the groups concerning age, gender, proportion of patients with ureteral stenting, operating time, hospitalization or readmission rates. The number of ureteric strictures was small in all procedures [n=3 (1.0%) in p-URS, n=2 (1.9%) in l-URS and n=2 (1.5%) in LUC] and there was no difference between the groups concerning this serious complication (p-URS vs. LUC: P=0.6465; l-URS vs. LUC: P=0.9999). CONCLUSIONS: In small distal stones, LUC stone extraction still is an alternative to URS procedures in stone management with comparable results concerning postinterventional ureteral strictures. In experienced hands, it still has its value in accurately selected patients.

Nuclear Localization of Robo is Associated with Better Survival in Bladder Cancer.

The Slit-Robo pathway has shown to be altered in several malignant diseases. However, its role in bladder cancer is poorly understood. Therefore, we aimed to assess the tissue expression of Robo1 and Robo4 as well as their ligand Slit2 in different stages of bladder cancer to explore possible changes of Slit-Robo signalling during the progression of bladder cancer. Robo1, Robo4 and Slit2 gene expressions were analyzed in 92 frozen bladder cancer tissue samples by using reverse transcription quantitative real-time PCR. Immunohistochemical analyses were performed on 149 formalin-fixed and paraffin-embedded bladder cancer tissue samples. Results were correlated with the clinical and follow-up data by performing both univariable and multivariable analyses. Robo1 and Robo4 nuclear staining intensitiy was significantly higher in low stage and low grade bladder cancer. Elevated Robo1 nuclear staining was associated with better disease-specific survival (DSS) (p = 0.045). Similarly, stronger Robo4 nuclear staining tended to be associated with longer DSS (p = 0.061). We found higher Robo1 and Slit2 gene expression levels in advanced stages of bladder cancer (p = 0.007 and p < 0.001). High Slit2 gene expression was correlated with significantly shorter DSS (p < 0.005), while Robo1 and Robo4 gene expressions were not associated with patients' prognosis. Our results demonstrate that the nuclear expression of Robo1 and Robo4 is associated with a favourable prognosis suggesting that its translocation into the nucleus represent a posttranslational regulation process which may exhibit an antitumor effect in bladder cancer.

Serum and tissue syndecan-1 levels in renal cell carcinoma.

BACKGROUND: The proteoglycan syndecan-1 is involved in cell proliferation, adhesion and angiogenesis. It was shown to be involved in cancer progression in different tumor entities. So far, the role of syndecan-1 in renal cell carcinoma (RCC), one of the most common diseases in urologic oncology, was little described. Purpose of the present study was to obtain serum concentrations and tissue expression levels of syndecan-1 in a cohort of patients diagnosed with RCC. METHODS: Clinical and follow-up data were obtained from 413 RCC patients. SDC1 levels were determined in serum samples of 100 patients by enzyme-linked immunosorbent assay and tissue SDC1 expression was measured by immunohistochemistry (IHC) in 343 cases. Results were correlated with clinicopathological and follow-up data. RESULTS: Five and ten years overall and cancer specific survival were 67% and 56% [overall survival (OS)] and 79% and 76% [cancer-specific survival (CSS)]. In female patients and locally advanced disease (≥T3), tissue SDC1 expression was decreased (female 85.6% vs. male 71.1% low tissue SDC1 expression, P=0.0153 and ≤T2 70.0% vs. ≥T3 87.2% low tissue SDC1 expression, P=0.0055) compared to male patients and organ confined disease. Locally advanced tumor stage, presence of lymph node or distant metastases, high Fuhrman grading and clear cell carcinoma as histopathological subtype were independent prognostic factors for reduced CSS and OS. There was no impact of serum SDC1 (sSDC1) serum concentration or SDC1 tissue protein expression on OS, CSS or recurrence free survival (RFS) in uni- or multivariable analysis. CONCLUSIONS: sSDC1 concentration or SDC1 tissue protein expression levels had no influence on patients' prognosis in the present cohort of patients diagnosed with RCC.

Prevalence of APC and PTEN Alterations in Urachal Cancer.

Urachal carcinoma (UrC) is a rare tumor with remarkable histological and molecular similarities to colorectal cancer (CRC). Adenomatous polyposis coli (APC) is the most frequently affected gene in CRC, but the prevalence and significance of its alterations in UrC is poorly understood. In addition, loss of phosphatase and tensin homologue (PTEN) was shown to be associated with therapy resistance in CRC. Our primary aim was to assess specific genetic alterations including APC and PTEN in a large series of UrC samples in order to identify clinically significant genomic alterations. We analyzed a total of 40 UrC cases. Targeted 5-gene (APC, PTEN, DICER1, PRKAR1A, TSHR, WRN) panel sequencing was performed on the Illumina MiSeq platform (n = 34). In addition, ß-catenin (n = 38) and PTEN (n = 30) expressions were assessed by immunohistochemistry. APC and PTEN genes were affected in 15% (5/34) and 6% (2/34) of cases. Two of five APC alterations (p.Y1075*, p.K1199*) were truncating pathogenic mutations. One of the two PTEN variants was a pathogenic frameshift insertion (p.C211fs). In 29% (11/38) of samples, at least some weak nuclear ß-catenin immunostaining was detected and PTEN loss was observed in 20% (6/30) of samples. The low prevalence of APC mutations in UrC represents a characteristic difference to CRC. Based on APC and ß-catenin results, the Wnt pathway seems to be rarely affected in UrC. Considering the formerly described involvement of PTEN protein loss in anti-EGFR therapy-resistance its immunohistochemical testing may have therapeutic relevance.

Timing and targeting of cell-based VEGF165 gene expression in ischemic tissue.

BACKGROUND: Therapeutic angiogenesis has become a key technology in experimental and clinical medicine. Only few data are available on the effects of timing and targeting of therapeutic proteins after cell-based gene transfer. This work investigates such effects after temporary expression of vascular endothelial growth factor 165 (VEGF(165)), the most commonly used angiogenic protein for therapeutic purposes. METHODS: We established a cell-based gene-transfer model using fibroblasts to temporarily produce VEGF(165). Cells were implanted into 40 rats. Protein expression and angiogenic effects were measured by PCR, immunohistology, and microangiography. To determine an improvement for survival of ischemically challenged tissue, cells were implanted in an ischemic flap model at different locations and time points. RESULTS: After implantation of modified cells, a temporary increase was found in the target tissue for VEGF(165), endothelial cell counts, and capillary network formations. Four wk later, histological alterations in the target tissue area were not different from controls. Implantation of modified cells into flap plus wound margin 1 wk before surgery showed significant improvement of tissue survival demonstrated by planimetric measurements and blood vessels counting in the target tissue. CONCLUSION: In our model, temporary expression of VEGF(165) induces therapeutically relevant angiogenesis and improves blood supply only if applied 1 wk before ischemia. It is essential to include the surrounding area for induction of angiogenesis in this model. In contrast, the angiogenic effects are not effective in the target area and its surrounding tissue, if therapeutic gene expression is started during onset of ischemia or 2 wk before ischemia in this model.

Biomarkers in Urachal Cancer and Adenocarcinomas in the Bladder: A Comprehensive Review Supplemented by Own Data.

Urachal cancer (UrC) is a rare but aggressive cancer. Due to overlapping histomorphology, discrimination of urachal from primary bladder adenocarcinomas (PBAC) and adenocarcinomas secondarily involving the bladder (particularly colorectal adenocarcinomas, CRC) can be challenging. Therefore, we aimed to give an overview of helpful (immunohistochemical) biomarkers and clinicopathological factors in addition to survival analyses and included institutional data from 12 urachal adenocarcinomas. A PubMed search yielded 319 suitable studies since 1930 in the English literature with 1984 cases of UrC including 1834 adenocarcinomas (92%) and 150 nonadenocarcinomas (8%). UrC was more common in men (63%), showed a median age at diagnosis of 50.8 years and a median tumor size of 6.0 cm. No associations were noted for overall survival and progression-free survival (PFS) and clinicopathological factors beside a favorable PFS in male patients (p = 0.047). The immunohistochemical markers found to be potentially helpful in the differential diagnostic situation are AMACR and CK34ßE12 (UrC versus CRC and PBAC), CK7, ß-Catenin and CD15 (UrC and PBAC versus CRC), and CEA and GATA3 (UrC and CRC versus PBAC). Serum markers like CEA, CA19-9 and CA125 might additionally be useful in the follow-up and monitoring of UrC.

Diagnostic and prognostic value of long noncoding RNAs as biomarkers in urothelial carcinoma.

Many long noncoding RNAs (lncRNAs) are deregulated in cancer and contribute to oncogenesis. In urothelial carcinoma (UC), several lncRNAs have been reported to be overexpressed and proposed as biomarkers. As most reports have not been confirmed independently in large tissue sets, we aimed to validate the diagnostic and prognostic value of lncRNA upregulation in independent cohorts of UC patients. Thus, expression of seven lncRNA candidates (GAS5, H19, linc-UBC1, MALAT1, ncRAN, TUG1, UCA1) was measured by RT-qPCR in cell lines and tissues and correlated to clinicopathological parameters including follow-up data (set 1: N n = 10; T n = 106). Additionally, publicly available TCGA data was investigated for differential expression in UC tissues (set 2: N n = 19; T n = 252,) and correlation to overall survival (OS). All proposed candidates tended to be upregulated in tumour tissues, with the exception of MALAT1, which was rather diminished in cancer tissues of both data sets. However, strong overexpression was generally limited to individual tumour tissues and statistically significant overexpression was only observed for UCA1, TUG1, ncRAN and linc-UBC1 in tissue set 2, but for no candidate in set 1. Altered expression of individual lncRNAs was associated with overall survival, but not consistently between both patient cohorts. Interestingly, lower expression of TUG1 in a subset of UC patients with muscle-invasive tumours was significantly correlated with worse OS in both cohorts. Further analysis revealed that tumours with low TUG1 expression are characterized by a basal-squamous-like subtype signature accounting for the association with poor outcome. In conclusion, our study demonstrates that overexpression of the candidate lncRNAs is found in many UC cases, but does not occur consistently and strongly enough to provide reliable diagnostic or prognostic value as an individual biomarker. Subtype-dependent expression patterns of lncRNAs like TUG1 could become useful to stratify patients by molecular subtype, thus aiding personalized treatments.