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To develop a new digital biomarker based on the analysis of primary tumour tissue by a convolutional neural network (CNN) to predict lymph node metastasis (LNM) in a cohort matched for already established risk factors.Haematoxylin and eosin (HE) stained primary tumour slides from 218 patients (102 N+; 116 N0), matched for Gleason score, tumour size, venous invasion, perineural invasion and age, who underwent radical prostatectomy were selected to train a CNN and evaluate its ability to predict LN status.With 10 models trained with the same data, a mean area under the receiver operating characteristic curve (AUROC) of 0.68 (95% confidence interval [CI] 0.678-0.682) and a mean balanced accuracy of 61.37% (95% CI 60.05-62.69%) was achieved. The mean sensitivity and specificity was 53.09% (95% CI 49.77-56.41%) and 69.65% (95% CI 68.21-71.1%), respectively. These results were confirmed via cross-validation. The probability score for LNM prediction was significantly higher on image sections from N+ samples (mean [SD] N+ probability score 0.58 [0.17] vs 0.47 [0.15] N0 probability score, P = 0.002). In multivariable analysis, the probability score of the CNN (odds ratio [OR] 1.04 per percentage probability, 95% CI 1.02-1.08; P = 0.04) and lymphovascular invasion (OR 11.73, 95% CI 3.96-35.7; P0.001) proved to be independent predictors for LNM.In our present study, CNN-based image analyses showed promising results as a potential novel low-cost method to extract relevant prognostic information directly from HE histology to predict the LN status of patients with prostate cancer. Our ubiquitously available technique might contribute to an improved LN status prediction.

Scientific evidence in medicine is based on data generated from research. Recently, the number of scientifically active physicians has decreased, which has led to the development of the Clinician Scientist Programs. To better structure and focus the research of young physicians, we aimed to investigate the impact of collaborations and other factors on the quality and output of scientific publications.The abstracts of three annual congresses of the German Society of Urology were systematically analysed regarding content, collaborations, and study design. Full-text publications and journals were identified through a MEDLINE® search. Impact factors (IFs) were identified using Journal Citation Reports™. To identify factors which predict publication and IFs, χ1,074 abstracts were reviewed. The publication rate of subsequent peer-reviewed full-text publications was 52.5%. Collaborations with at least one institution (odds ratio (OR) 2.02, 95% confidence interval (CI) 1.48-2.76, p0.0001), statistical analysis (OR 1.92, 95% CI 1.41-2.60, p0.0001), study design (prospective vs. retrospective: OR 1.43, 95% CI 1.06-1.93, p=0.021), and national collaborations (OR 1.43, 95% CI 1.04-1.98, p=0.029) increased the likelihood of publication in a peer-reviewed journal in a multivariable logistic regression analysis. Experimental design (OR 2.77, 95% CI 1.32-5.84, p=0.007), international collaborations (OR 2.26, 95% CI 1.23-4.15, p=0.009), oncologic topics (OR 1.94, 95% CI 1.23-3.07, p=0.005), prostate disease (OR 1.75, 95% CI 1.08-2.84, p=0.023), and statistical analysis (OR 1.68, 95% CI 1.06-2.64, p=0.026) were associated with a higher IF.Abstracts resulting from collaborative research projects had a higher likelihood of subsequent full-text publication and a higher IF. More full-text publications were reported when abstracts included a statistical analysis. Hence, intensive networking (e. g. at congresses and workshops) of researching physicians as well as statistical/biometrical classes could be key factors to improve academic success.

Background/Aims: Extracellular vesicles (EVs), including microvesicles and exosomes, deliver bioactive cargo mediating intercellular communication in physiological and pathological conditions. EVs are increasingly investigated as therapeutic agents and targets, but also as disease biomarkers. However, a definite consensus regarding EV isolation methods is lacking, which makes it intricate to standardize research practices and eventually reach a desirable level of data comparability. In our study, we performed an inter-laboratory comparison of EV isolation based on a differential ultracentrifugation protocol carried out in 4 laboratories in 2 independent rounds of isolation. Methods: Conditioned medium of colorectal cancer cells was prepared and pooled by 1 person and distributed to each of the participating laboratories for isolation according to a pre-defined protocol. After EV isolation in each laboratory, quantification and characterization of isolated EVs was collectively done by 1 person having the highest expertise in the respective test method: Western blot, flow cytometry (fluorescence-activated cell sorting [FACS], nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Results: EVs were visualized with TEM, presenting similar cup-shaped and spherical morphology and sizes ranging from 30 to 150 nm. NTA results showed similar size ranges of particles in both isolation rounds. EV preparations showed high purity by the expression of EV marker proteins CD9, CD63, CD81, Alix, and TSG101, and the lack of calnexin. FACS analysis of EVs revealed intense staining for CD63 and CD81 but lower levels for CD9 and TSG101. Preparations from 1 laboratory presented significantly lower particle numbers (p < 0.0001), most probably related to increased processing time. However, even when standardizing processing time, particle yields still differed significantly between groups, indicating inter-laboratory differences in the efficiency of EV isolation. Importantly, no relation was observed between centrifugation speed/k-factor and EV yield. Conclusions: Our findings demonstrate that quantitative differences in EV yield might be due to equipment- and operator-dependent technical variability in ultracentrifugation-based EV isolation. Furthermore, our study emphasizes the need to standardize technical parameters such as the exact run speed and k-factor in order to transfer protocols between different laboratories. This hints at substantial inter-laboratory biases that should be assessed in multi-centric studies.

Purpose Focal therapies (FTs) are investigated within prospective studies on selected patients treated for localized prostate cancer (PCa). Benefits are preservation of genitourinary function and reduced complications, but follow-up is elaborate and is associated with uncertainty as cancer-free survival appears to be lower compared to standard radical treatments. The aim of this study was to analyse patient-reported acceptance of FT and evaluate factors associated with treatment decision regret. Methods 52 patients who received focal high-intensity focused ultrasound for low- to intermediate-risk PCa between 2014 and 2019 within two prospective trials were eligible for a survey regarding PCa-related treatment regret and quality-of-life (Clark’s scale) and the following potential predictors: sociodemographic variables, Charlson Comorbidity Index, subjective aging (AARC-10 SF), and general health-related quality-of-life (SF-12). Cancer persistence/recurrence (multiparametric MRI and fusion biopsy after 12 months) and functional outcomes (EPIC-26 UI/UIO/S) data were also included in this study. Results The overall survey response rate was 92.3% (48/52 patients). Median follow-up was 38 months (interquartile range = 25–50 months). In total, ten patients (20.8%) reported treatment decision regret. In univariable analyses, a clinically meaningful increase in urinary incontinence showed a significant association (OR 4.43; 95% CI 0.99–20.53; p = 0.049) with regret. Cancer recurrence (OR 12.31; 95% CI 1.78–159.26; p = 0.023) and general health worry as a domain of Clark’s scale (OR 1.07; 95% CI 1.03–1.14; p Conclusion Acceptance of FT is comparable to standard treatments. Extensive follow-up including regular PSA testing does not cause additional regret but careful patient selection and information before FT is crucial.

Zusammenfassung Hintergrund und Ziel der Arbeit Der Jahreskongress der Deutschen Gesellschaft für Urologie (DGU) spiegelt die urologische Forschungslandschaft im deutschsprachigen Raum wider. Ziel war es, durch die longitudinale Analyse der Kongressabstracts und hervorgehenden Vollpublikationen Trends aufzudecken. Material und Methoden Es erfolgte die systematische Analyse der Kongressabstracts des Jahreskongresses 2016 auf Inhalt, Studiendesign, Kooperationen, hervorgehenden Vollpublikationen sowie der veröffentlichenden Journalen. Hiernach erfolgte der Vergleich mit den Kongressen 2002 und 2009. Statistische Berechnungen erfolgten per χ2-, Mann-Whitney-U-, Cochran-Armitage- und Kruskal-Wallis-Test. Ergebnisse Auf den Kongressen 2002, 2009 und 2016 wurden 1073 Abstracts präsentiert. Abstracts zu Prostataerkrankungen (24,2 %, 29,7 %, 34,0 %; p = 0,0043), onkologische Abstracts (50,6 %, 57,9 %, 61,7 %; p = 0,003), multizentrische Studien (18,3 %, 28,6 %, 34,3 %; p p p = 0,009) und prospektive Arbeiten (62,1 %, 42,0 %, 36,0 %; p p p p = 0,15). Schlussfolgerung Die nationale und internationale Vernetzung der urologischen Forschungsgemeinschaft nimmt zu, prospektive Studien werden weniger präsentiert. Die Rate aus DGU-Abstracts hervorgehender Vollpublikationen zeigt sich über die drei Kongresse auf hohem Niveau. Die Veröffentlichungsrate in Open-access-Journalen ist bisher gering.

To investigate the predictors of recurrence and of de novo incontinence in patients treated by transurethral incision or resection for vesico-urethral anastomotic stenosis (VUAS) after radical prostatectomy. All patients undergoing endoscopic treatment for VUAS between March 2009 and October 2016 were identified in our multi-institutional database. Digital chart reviews were performed and patients contacted for follow-up. Recurrence was defined as any need for further instrumentation or surgery, and de-novo-incontinence as patient-reported outcome. Of 103 patients undergoing endoscopic VUAS treatment, 67 (65%) underwent transurethral resection (TR) and 36 (35%) transurethral incision (TI). TI was performed more frequently as primary treatment compared to TR (58% vs. 37%; p = 0.041). Primary and repeated treatment was performed in 46 (45%) and 57 patients (55%), respectively. Overall, 38 patients (37%) had a history of radiation therapy. There was no difference in time to recurrence for primary vs repeat VUAS treatment, previous vs no radiation, TR compared to TI (all p > 0.08). Regarding treatment success, no difference was found for primary vs. repeat VUAS treatment (50% vs. 37%), previous radiation vs. no radiation (42% vs. 43%), and TR vs. TI (37% vs. 53%; all p ≥ 0.1). Postoperative de novo incontinence was more common after TI vs. TR (31% vs. 12%; p = 0.032), no difference was observed for previous radiation therapy vs. no radiation therapy (18% vs. 18%; p > 0.9) or primary vs. repeat VUAS treatment (22% vs. 16%; p = 0.5). VUAS recurrence after endoscopic treatment is not predictable. Endoscopic treatment with TI showed a higher risk for de novo incontinence than TR, and previous irradiation and the number of treatments do not influence incontinence.

Fatty acid-binding protein 5 (FABP5), a transport protein for lipophilic molecules, has been proposed as protein marker in prostate cancer (PCa). The role of FABP5 gene expression is merely unknown. In two cohorts of PCa patients who underwent radical prostatectomy (n = 40 and n = 57) and one cohort of patients treated with palliative transurethral resection of the prostate (pTUR-P; n = 50) FABP5 mRNA expression was analyzed with qRT-PCR. Expression was correlated with clinical parameters. BPH tissue samples served as control. To independently validate findings on FABP5 expression, three microarray and sequencing datasets were reanalyzed (MSKCC 2010 n = 216; TCGA 2015 n = 333; mCRPC, Nature Medicine 2016 n = 114). FABP5 expression was correlated with ERG-fusion status, TCGA subtypes, cancer driver mutations and the expression of druggable downstream pathway components. FABP5 was overexpressed in PCa compared to BPH in the cohorts analyzed by qRT-PCR (radical prostatectomy p = 0.003, p = 0.010; pTUR-P p = 0.002). FABP5 expression was independent of T stage, Gleason Score, nodal status and PSA level. FABP5 overexpression was associated with the absence of TMPRSS2:ERG fusion (p

Objectives: Bladder neck contracture (BNC) is a bothersome complication following endoscopic treatment for benign prostatic hyperplasia (BPH). The objective of our study was to give a more realistic insight into contemporary endoscopic BNC treatment and to evaluate and identify risk factors associated with inferior outcome. Material and Methods: We identified patients who underwent transurethral treatment for BNC secondary to previous endoscopic therapy for BPH between March 2009 and October 2016. Patients with vesico-urethral anastomotic stenosis after radical prostatectomy were excluded. Digital charts were reviewed for re-admissions and re-visits at our institutions and patients were contacted personally for follow-up. Our non-validated questionnaire assessed previous urologic therapies (including radiotherapy, endoscopic, and open surgery), time to eventual further therapy in case of BNC recurrence, and the modality of recurrence management. Results: Of 60 patients, 49 (82%) and 11 (18%) underwent transurethral bladder neck resection and incision, respectively. Initial BPH therapy was transurethral resection of the prostate (TURP) in 54 (90%) and holmium laser enucleation of the prostate (HoLEP) in six (10%) patients. Median time from prior therapy was 8.5 (IQR 5.3–14) months and differed significantly in those with (6.5 months, IQR 4–10) and those without BNC recurrence (10 months, IQR 6–20, p = 0.046). Thirty-three patients (55%) underwent initial endoscopic treatment, and 27 (45%) repeated endoscopic treatment for BNC. In initially-treated patients, time since BPH surgery differed significantly between those with a recurrence (median 7.5 months, IQR 6–9) compared to those treated successfully (median 12 months, IQR 9–25, p = 0.01). In patients with repeated treatment, median time from prior BNC therapy did not differ between those with (4.5 months, IQR 2–12) and those without a recurrence (6 months, IQR 6–10, p = 0.6). Overall, BNC treatment was successful in 32 patients (53%). The observed success rate of BNC treatment was significantly higher after HoLEP compared to TURP (100% vs. 48%, p = 0.026). Type of BNC treatment, number of BNC treatment, and age at surgery did not influence the outcome. Conclusions: A longer time interval between previous BPH therapy and subsequent BNC incidence seems to favorably affect treatment success of endoscopic BNC treatment, and transurethral resection and incision appear equally effective. Granted the relatively small sample size, BNC treatment success seems to be higher after HoLEP compared to TURP, which warrants validation in larger cohorts.

Background: Perioperative cisplatin-based chemotherapy (CBC) can improve the outcome of patients with muscle-invasive bladder cancer (MIBC), but it is still to be defined which patients benefit. Mutations in DNA damage response genes (DDRG) can predict the response to CBC. The value of DDRG expression as a marker of CBC treatment effect remains unclear. Material and methods: RNA expression of the nine key DDRG (BCL2, BRCA1, BRCA2, ERCC2, ERCC6, FOXM1, RAD50, RAD51, and RAD52) was assessed by qRT-PCR in a cohort of 61 MICB patients (median age 66 y, 48 males, 13 females) who underwent radical cystectomy in a tertiary care center. The results were validated in the The Cancer Genome Atlas (TCGA) cohort of MIBC (n = 383). Gene expression was correlated with disease-free survival (DFS) and overall survival (OS). Subgroup analyses were performed in patients who received adjuvant cisplatin-based chemotherapy (ACBC) (Mannheim n = 20 and TCGA n = 75). Results: Low expression of RAD52 was associated with low DFS in both the Mannheim and the TCGA cohorts (Mannheim: p = 0.039, TCGA: p = 0.017). This was especially apparent in subgroups treated with ACBC (Mannheim: p = 0.0059, TCGA: p = 0.012). Several other genes showed an influence on DFS in the Mannheim cohort (BRCA2, ERCC2, FOXM1) where low expression was associated with poor DFS (p &lt, 0.05 for all). This finding was not fully supported by the data in the TCGA cohort, where high expression of FOXM1 and BRCA2 correlated with poor DFS. Conclusion: Low expression of RAD52 correlated with decreased DFS in the Mannheim and the TCGA cohort. This effect was especially pronounced in the subset of patients who received ACBC, making it a promising indicator for response to ACBC on the level of gene expression.

Background Data from interventional studies suggest that a peritoneal flap after pelvic lymph node dissection (LND) during laparoscopic, robotic-assisted radical prostatectomy (RARP) may reduce the rate of symptomatic lymphoceles in transperitoneal approach. However, most of these studies are not conducted in a randomized controlled fashion, thus limiting their scientific value. A recent prospective, randomized, controlled trial (RCT) did not show superiority of a peritoneal flap while further trials are lacking. Therefore, the aim of the presented RCT will be to show that creating a peritoneal flap decreases the rate of symptomatic lymphoceles compared to the current standard procedure without creation of a flap. Methods/design PELYCAN is a parallel-group, patient- and assessor-blinded, phase III, adaptive randomized controlled superiority trial. Men with histologically confirmed prostate cancer who undergo transperitoneal RARP with pelvic LND will be randomly assigned in a 1:1 ratio to two groups—either with creating a peritoneal flap (PELYCAN) or without creating a peritoneal flap (control). Sample size calculation yielded a sample size of 300 with a planned interim analysis after 120 patients, which will be performed by an independent statistician. This provides a possibility for early stopping or sample size recalculation. Patients will be stratified for contributing factors for the development of postoperative lymphoceles. The primary outcome measure will be the rate of symptomatic lymphoceles in both groups within 6 months postoperatively. Patients and assessors will be blinded for the intervention until the end of the follow-up period of 6 months. The surgeon will be informed about the randomization result after performance of vesicourethral anastomosis. Secondary outcome measures include asymptomatic lymphoceles at the time of discharge and within 6 months of follow-up, postoperative complications, mortality, re-admission rate, and quality of life assessed by the EORTC QLQ-C30 questionnaire. Discussion The PELYCAN study is designed to assess whether the application of a peritoneal flap during RARP reduces the rate of symptomatic lymphoceles, as compared with the standard operation technique. In case of superiority of the intervention, this peritoneal flap may be suggested as a new standard of care. Trial registration German Clinical Trials Register DRKS00016794. Registered on 14 May 2019.

Introduction: Guideline recommendations are meant to help minimize morbidity and to improve the care of nonmuscle invasive bladder cancer (NMIBC) patients but studies have suggested an underuse of guideline-recommended care. The aim of this study was to evaluate the level of adherence of German and Austrian urologists to German guideline recommendations. Methods: A survey of 27 items evaluating diagnostic and therapeutic recommendations (15 cases of strong consensus and 6 cases of consensus) for NMIBC was administered among 14 urologic training courses. Survey construction and realization followed the checklist for reporting results of internet e-surveys and was approved by an internal review board. Results: Between January 2018 and June 2019, a total of 307 urologists responded to the questionnaire, with a mean response rate of 71%. The data showed a weak role of urine cytology (54%) for initial diagnostics although it is strongly recommended by the guideline. The most frequently used supporting diagnostic tool during transurethral resection of the bladder was hexaminolevulinate (95%). Contrary to the guideline recommendation, 38% of the participants performed a second resection in the case of pTa low-grade NMIBC. Correct monitoring of Bacille Calmette-Guérin (BCG) response with cystoscopy and cytology was performed by only 34% of the urologists. Conclusions: We found a discrepancy between certain guideline recommendations and daily routine practice concerning the use of urine cytology for initial diagnostics, instillation therapy with a low monitoring rate of BCG response, and follow-up care with unnecessary second resection after pTa low-grade NMIBC in particular. Our survey showed a moderate overall adherence rate of 73%. These results demonstrate the need for sharpening awareness of German guideline recommendations by promoting more intense education of urologists to optimize NMIBC care thus decreasing morbidity and mortality rates.

Background Growing evidence supports the pivotal role of long non-coding RNAs (lncRNAs) in the regulation of cancer development and progression. Their expression patterns and biological function in muscle invasive bladder cancer (MIBC) remain elusive. Methods Transcript levels of lncRNA miR-31 host gene (MIR31HG) and its splice variants were measured in our MIBC cohort (n = 102) by qRT-PCR, and validated in silico by the TCGA cohort (n = 370). Kaplan-Meier and multiple Cox regression analysis were conducted to evaluate the survival significance of MIR31HG and its splice variants. Functional experiments were performed to examine the proliferation and migration abilities of MIR31HG and its splice variants by knockdown approaches. Results In this study, a decreased expression of MIR31HG was found in bladder cancer cells and tissues, except in the basal subtype. Survival analysis showed that high expression of MIR31HG was associated with poor overall survival (OS) and disease-free survival (DFS) in patients with MIBC of basal subtype. Two splice variants of MIR31HG lacking exon 1 (MIR31HGΔE1) and exon 3 (MIR31HGΔE3) were identified to have specific expression patterns in different molecular subtypes of our MIBC cohort. MIR31HGΔE3 was highly expressed in basal subtype tumors. A high expression of MIR31HGΔE1 and MIR31HGΔE3 was associated with worse OS and DFS in our cohort. In vitro experiments revealed that knockdown of MIR31HG inhibits cell proliferation, colony formation, and migration in bladder cancer. Cell proliferation and migration assays after knockdown of splice variants of MIR31HG showed corresponding roles for the full-length transcript. Conclusions Our study demonstrates that MIR31HG and its splice variants could serve as biomarkers for the classification and prognosis prediction of patients with MIBC.

Objectives To report feasibility and outcome of salvage robotic-assisted laparoscopic radical prostatectomy (S-RALP) after focal therapy using high-intensity focused ultrasound (HIFU) treatment compared to primary robotic-assisted laparoscopic radical prostatectomy (pRALP). Methods In this bicentric trial patients undergoing S-RALP for detection of WHO2016/ISUP Grade Group 2 or 3 prostate cancer were previously treated in prospective focal HIFU trials. Perioperative data, complications, oncological and functional outcome were analysed. Patients who underwent pRALP were matched in a ratio 2(pRALP):1(S-RALP) according to preoperatively functional, oncological and clinical parameters. Results A total of 39 patients were included in the study (13S-RALP, 26pRALP). Median operative time in the S-RALP group was 260minutes (pRALP: 257minutes), blood loss was 230ml (pRALP: 300ml). Complications occurred in 46.2% (6/13) of S-RALP patients (pRALP: 26.9%), including four Clavien-Dindo III complications (pRALP: 2/26). In S-RALP adverse histological outcome (≥pT3a, pN+ or R1) was detected in 23.1% (3/13) (pRALP: 26.9%). There was one patient with PSA-persistence (pRALP: 2/26). Regarding functional outcomes there was no difference between the two groups observed (incontinence p=0.71, erectile function p=0.21). Conclusion S-RALP should be offered to patients with an early relapse after focal HIFU. The early oncological outcome is satisfactory and functional outcome one year postoperatively is similar to pRALP. However, S-RALP is associated with a higher rate of Clavien-Dindo III complications (mainly, placement of a drainage), of which patients should be informed beforehand.

Comprehensive transcriptome expression analyses of bladder cancer revealed distinct lncRNA clusters with differential molecular and clinical characteristics. In this study, pivotal lncRNAs were assessed for their impact on survival and their differential expression between the molecular bladder cancer subtypes. FFPE samples from chemotherapy-naïve patients with muscle invasive bladder cancer (MIBC) were analyzed on the Nanostring nCounter platform for absolute quantification. An established 36-gene panel was used for molecular subtype classification into basal, luminal and infiltrated MIBC. In a second step, 14 pivotal lncRNAs were assessed for their molecular subtype attribution, and their predictive value in disease-specific survival. In silico validation was performed on a total of 487 MIBC patients (MDA, TGCA and Chungbuk cohort). Several pivotal lncRNAs showed a distinct molecular subtype attribution: e.g. MALAT1 showed a downregulation in the basal subtype (p = 0.009), TUG1 and CBR3AS1 showed an upregulation in the luminal subtype (p ≤ 0.001). High transcript levels of SNHG16, CBR3AS1 and H19 appeared to be predictive for a shorter disease-specific survival. Patients overexpressing putative oncogenes MALAT1 and TUG1 in MIBC tissue presented prolonged survival, suggesting tumor suppressive effects of both lncRNAs. The Nanostring nCounter proved to be a valid platform for the quantification of low-abundance transcripts including lncRNAs.

Central nervous System (CNS) disease in pediatric acute lymphoblastic leukemia (ALL) is a major concern, but still, cellular mechanisms of CNS infiltration are elusive. The choroid plexus (CP) is a potential entry site, and, to some extent, invasion resembles CNS homing of lymphocytes during healthy state. Given exosomes may precondition target tissue, the present work aims to investigate if leukemia-derived exosomes contribute to a permissive phenotype of the blood-cerebrospinal fluid barrier (BCSFB). Leukemia-derived exosomes were isolated by ultracentrifugation from the cell lines SD-1, Nalm-6, and P12-Ichikawa (P12). Adhesion and uptake to CP epithelial cells and the significance on subsequent ALL transmigration across the barrier was studied in a human BCSFB in vitro model based on the HiBCPP cell line. The various cell lines markedly differed regarding exosome uptake to HiBCPP and biological significance. SD-1-derived exosomes associated to target cells unspecifically without detectable cellular effects. Whereas Nalm-6 and P12-derived exosomes incorporated by dynamin-dependent endocytosis, uptake in the latter could be diminished by integrin blocking. In addition, only P12-derived exosomes led to facilitated transmigration of the parental leukemia cells. In conclusion, we provide evidence that, to a varying extent, leukemia-derived exosomes may facilitate CNS invasion of ALL across the BCSFB without destruction of the barrier integrity.

Current outcome prediction markers for localized prostate cancer (PCa) are insufficient. The impact of the lipid-modifying Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3B) in PCa is unknown. Two cohorts of patients with PCa who underwent radical prostatectomy (n = 40, n = 56) and benign prostate hyperplasia (BPH) controls (n = 8, n = 11) were profiled for SMPDL3B expression with qRT-PCR. Publicly available PCa cohorts (Memorial Sloane Kettering Cancer Centre (MSKCC, n = 131, n = 29 controls) and The Cancer Genome Atlas (TCGA, n = 497, n = 53 controls)) served for validation. SMPDL3B&rsquo, s impact on proliferation and migration was analyzed in PC3 cells by siRNA knockdown. In both cohorts, a Gleason score and T stage independent significant overexpression of SMPDL3B was seen in PCa compared to BPH (p &lt, 0.001 each). A lower expression of SMPDL3B was associated with a shorter overall survival (OS) (p = 0.005) in long term follow-up. A SMPDL3B overexpression in PCa tissue was confirmed in the validation cohorts (p &lt, 0.001 each). In the TCGA patients with low SMPDL3B expression, biochemical recurrence-free survival (p = 0.011) and progression-free interval (p &lt, 0.001) were shorter. Knockdown of SMPDL3B impaired PC3 cell migration but not proliferation (p = 0.0081). In summary, SMPLD3B is highly overexpressed in PCa tissue, is inversely associated with localized PCa prognosis, and impairs PCa cell migration.

Current outcome prediction markers for localized prostate cancer (PCa) are insufficient. The impact of the lipid-modifying Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3B) in PCa is unknown. Two cohorts of patients with PCa who underwent radical prostatectomy (n = 40, n = 56) and benign prostate hyperplasia (BPH) controls (n = 8, n = 11) were profiled for SMPDL3B expression with qRT-PCR. Publicly available PCa cohorts (Memorial Sloane Kettering Cancer Centre (MSKCC; n = 131, n = 29 controls) and The Cancer Genome Atlas (TCGA; n = 497, n = 53 controls)) served for validation. SMPDL3B’s impact on proliferation and migration was analyzed in PC3 cells by siRNA knockdown. In both cohorts, a Gleason score and T stage independent significant overexpression of SMPDL3B was seen in PCa compared to BPH (p < 0.001 each). A lower expression of SMPDL3B was associated with a shorter overall survival (OS) (p = 0.005) in long term follow-up. A SMPDL3B overexpression in PCa tissue was confirmed in the validation cohorts (p < 0.001 each). In the TCGA patients with low SMPDL3B expression, biochemical recurrence-free survival (p = 0.011) and progression-free interval (p < 0.001) were shorter. Knockdown of SMPDL3B impaired PC3 cell migration but not proliferation (p = 0.0081). In summary, SMPLD3B is highly overexpressed in PCa tissue, is inversely associated with localized PCa prognosis, and impairs PCa cell migration.

OBJECTIVE To investigate the role of forkhead box protein M1 (FOXM1) mRNA expression and its prognostic value in stage pT1 non-muscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS Clinical data and formalin-fixed paraffin-embedded tissues from transurethral resection of the bladder from patients with stage pT1 NMIBC, treated with an organ-preserving approach, were analysed retrospectively. Total RNA was isolated using commercial RNA extraction kits, and mRNA expression of FOXM1, MKI67, KRT20 and KRT5 was measured by single-step quantitative RT-PCR using RNA-specific TaqMan Assays. Statistical analysis was performed using Spearman's Rho, Wilcoxon or Kruskal-Wallis tests, Kaplan-Meier estimates of recurrence-free (RFS), progression-free (PFS) and cancer-specific survival (CSS) and Cox regression analysis. RESULTS Data from 296 patients (79.4% men, median age 72 years) were available for the final evaluation. Spearman correlation analysis showed that mRNA expression of FOXM1 was significantly correlated with MKI67 (ρ: 0.6530, P < 0.001) and with the luminal subtype, reflected by the positive correlation with KRT20 (ρ: 0.2113, P < 0.001). Furthermore, there was also a strong correlation of FOXM1 expression with adverse clinical and pathological variables, such as concomitant carcinoma in situ (P = 0.05), multifocal tumours (P = 0.005) and World Health Organization 1973 grade 3 disease (P < 0.001). Kaplan-Meier analysis showed overexpression of FOMX1 to be associated with worse PFS (P = 0.028) and worse CSS (P = 0.015). FOXM1 overexpression was also shown to be a predictive risk factor for CSS (hazard ratio 1.61 [1.13-2.34], L-R chi-squared: 7.19, P = 0.007). FOXM1 overexpression identified a subgroup of patients within the luminal subtype with worse RFS (P = 0.017), PFS (P < 0.001) and CSS (P = 0.015). Patients with low FOXM1 expression had better outcomes, irrespective of instillation therapy, whereas patients with high FOXM1 expression benefitted from intravesical chemotherapy with mitomycin C. CONCLUSION High FOXM1 expression was associated with adverse clinical and pathological features and worse outcomes, and predicted response to intravesical instillation therapy in patients with stage pT1 NMIBC.

Background The cell surface interleukin 22 (IL-22) receptor complex is mainly expressed in epithelial and tissue cells like pancreatitis cells. Recent studies described that IL-22R was overexpressed in malignant diseases and was associated with a poor overall survival (OS). The role of IL-22RA1 gene expression in muscle invasive bladder cancer (MIBC) has not been investigated, yet. Objectives The aim of this study was to analyze the role of IL-22RA1 gene expression in patients with MIBC. Methods In a cohort of 114 patients with MIBC who underwent radical cystectomy, IL-22RA1 gene expression was analyzed with qRT-PCR and correlated with clinical parameters. Furthermore, Kaplan-Meier and Cox regression analysis were performed. For validation, an in silico dataset (TCGA 2017, n=407) was reanalyzed. Results IL-22RA1 gene expression was independent of clinicopathological parameters like age (P=0.2681), T stage (P=0.2130), nodal status (P=0.3238) and lymph vascular invasion (LVI, P=0.5860) in patients with MIBC. A high expression of IL-22RA1 was associated with a shorter OS (P=0.0040) and disease-specific survival (P=0.0385). Furthermore, a shorter disease-free survival (DFS) was also associated with a high expression of IL-22RA1 (P=0.0102). In the multivariable analysis, IL-22RA1 expression was an independent prognostic predictors regarding OS (P=0.0096, HR=0.48). In the TCGA cohort, IL-22RA1 expression was independent regarding to OS and DFS. Conclusion A high IL-22RA1 gene expression was associated with worse outcome. Furthermore, IL-22RA1 represented an independent predictor regarding OS in our cohort and therefore might be used for risk stratification in patients with MIBC.

Checkpoint inhibition has emerged as new therapeutic option in muscle-invasive bladder cancer. The objective of the present study was to evaluate the prognostic role of PD1 and PDL1 expression in non-muscle-invasive bladder cancer (NMIBC) and establish an objective measuring method using RNA quantification. We retrospectively analyzed clinical data and formalin-fixed paraffin-embedded tissues (FFPE) of patients with stage pT1 NMIBC who underwent transurethral resection of the bladder. mRNA expression of PD1, PDL1 and CD3 was measured by single step RT-qPCR and correlated to clinicopathological parameters, recurrence-free survival (RFS), progression-free survival (PFS) and carcinoma-specific survival (CSS). We have analyzed 334 patients with NMIBC at stage pT1 for mRNA analysis. Data from 296 patients (79% male, median age: 72 years) could be used. Spearman correlation revealed significant associations between mRNA expressions of PD1/PDL1 (ρ: 0.6024, p

// Sebastien Rinaldetti 1 , Ralph Markus Wirtz 2 , Thomas Stefan Worst 3 , Markus Eckstein 4 , Cleo Aaron Weiss 5 , Johannes Breyer 6 , Wolfgang Otto 6 , Christian Bolenz 7 , Arndt Hartmann 4 and Philipp Erben 3 1 Department of Hematology and Oncology, University Medical Center Mannheim, Medical Faculty Mannheim, University of Heidelberg, 68167 Mannheim, Germany 2 Stratifyer Molecular Pathology GmbH, 50935 Koln, Germany 3 Department of Urology, University Medical Center Mannheim, Medical Faculty Mannheim, University of Heidelberg, 68167 Mannheim, Germany 4 Institute of Pathology, University Erlangen-Nuremberg, 91054 Erlangen, Germany 5 Institute of Pathology, University Medical Center Mannheim, Medical Faculty Mannheim, University of Heidelberg, 68167 Mannheim, Germany 6 Department of Urology, University of Regensburg, 93053 Regensburg, Germany 7 Department of Urology, University of Ulm, 89075 Ulm, Germany Correspondence to: Sebastien Rinaldetti, email: sebastienrinal@hotmail.com Keywords: FOXM1, muscle invasive bladder cancer, KI67, subclassification, survival prediction Received: December 28, 2016 Accepted: April 10, 2017 Published: April 24, 2017 ABSTRACT Forkhead box M1 (FOXM1) is a late cell cycle gene that plays a crucial role in carcinogenesis and chemotherapeutic drug resistance. In this study, the impact of FOXM1 expression on patient outcome was investigated for the first time in formalin fixed and paraffin embedded (FFPE) samples of chemotherapy naive muscle-invasive bladder cancer (MIBC) patients. Expression analyses were performed on the Mannheim cohort (n=84) and validated on the independent Chungbuk cohort (n=61). In a Cox’ proportional hazards model, a distinct FOXM1 expression cut-off dividing both cohorts in a ‘high-risk’ and ‘low-risk’ group has been determined. Multivariate analyses showed that FOXM1 is an independent risk factor for outcome prediction superior to the TNM system. The FOXM1 ‘high-risk’ group had a 4- to 7-fold increased risk of death (p

Paracrine and long-range signaling via extracellular vesicles, such as exosomes and microvesicles, is deemed crucial for tumorigenesis, invasion and spread of solid tumors. The ESCRT machinery (endosomal sorting complexes required for transport) and Rab-proteins act as key players in vesicular trafficking and secretion. Yet, their role in prostate cancer (PCa) is unknown. Therefore, this study aimed to elucidate the relevance of these components in PCa. In silico reanalysis of genes with known involvement in vesicular trafficking and secretion in an existing microarray dataset revealed low expression of RAB27A, RAB27B and VPS36 to be predictive for reduced BCR-free survival in patients with localized PCa (p=0.033, 0.025 and 0.005). In the same microarray dataset underexpression of RAB27A, RAB27B and VPS36 was seen in distant metastases (p

Background: Little is known about the role of WNT signalling in pathological processes involving the urinary tract stroma. Here the impact of WNT signalling on bladder wall fibroblasts (BWFs) was studied using integrated expression profiling. Material and Methods: WNT ligand and downstream WNT pathway component expression was profiled in human BWFs using qRT-PCR. Highly expressed WNT2B was knocked down using siRNA in BWFs. The expression of 730 mRNAs and 800 miRNAs was analyzed on the nCounter MAX platform in #WNT2B and control transfected BWFs. qRT-PCR was used for validation in vitro and in matched scar and healthy bladder wall tissue samples of 12 patients with vesico-urethral anastomotic stricture (VUAS). Results: Thirteen genes and 9 miRNAs showed differential expression in #WNT2B cells. Among these were TNFSF10, a key apoptosis inductor, (0.22fold, p = 0.011) and miR-1246 (36.2fold, p = 0.031). miRNA target prediction indicated TNFSF10 to be regulated by miR-1246. qRT-PCR analysis confirmed differential expression of miR-1246 and TNFSF10 in #WNT2B BWFs. Furthermore, TNFSF10 was significantly underexpressed in VUAS tissue (p = 0.009). Conclusion: Perturbation of WNT signalling results in an altered expression of the apoptosis inductor TNFSF10. Similar changes are observed in VUAS. Further studies investigating the crosslink between WNT signalling and apoptosis regulation in the urinary tract stroma are warranted.

Background: Limited data are available for the use of agents in metastatic castration-resistant prostate cancer (mCRPC) beyond the third-line. We provide data during treatment with cabazitaxel (CAB), helping to improve the informed-consent process. Patients and Methods: We retrospectively reviewed patients treated with fourth-line or beyond CAB for mCRPC after failure of previous therapies with docetaxel, abiraterone acetate, enzalutamide and/or radium-223. The progression-free survival (PFS) and the overall survival (OS) were estimated using the Kaplan-Meier method and compared to published data based on a structured literature review. The hospitalization rate was recorded. Factors influencing 6-months OS were analyzed. Results: Fifteen patients were identified at 4 institutions and included in the analysis. The median PFS was 104 days (range 47-397 days). The median time to death was 10 months (range 2-16). PFS and OS data are in accordance with 17 published patients so far. During the therapy, eleven (73%) of the patients were hospitalized. Prostate-specific antigen (PSA, 500 units; hazards ratio [HR] 1.491, 95% CI 1.000-2.0175), white blood cell count (HR 0.425, 95% CI 0.108-0.952), hemoglobin (HR 0.6014, 95% CI 0.2942-1.0758), and alkaline phosphatase (100 units; HR 1.0964, 95% CI 1.000-1.2859) correlate with 6-months OS. Conclusions: CAB beyond the third-line is often accompanied by hospitalization. PFS is a significant proportion of the median time of OS. The baseline laboratory might be a good indicator for the decision between CAB and best-supportive care.

Im Januar 2015 wurde im Rahmen der Grundung der GeSRU Academics die Forschergruppe „Blasenkarzinom Experimentell“ initiiert. Eine der grundlegenden Herausforderungen, um in einem Netzwerkverbund produktiv arbeiten zu konnen, ist das Herausarbeiten thematischer und methodischer Expertise als Voraussetzung fur gemeinsame Projekte. Daher war es der Gruppe ein erstes Anliegen, die Forschungsschwerpunkte einzelner Mitglieder zu bundeln und darzustellen, um im Anschluss aktuelle Fragestellungen zu formulieren. Im folgenden Ubersichtsbeitrag werden drei Themenkomplexe erlautert, die eine hohe Aufmerksamkeit an verschiedenen Standorten in Deutschland erfahren und daher in Verbundprojekte munden sollen.

Background: Muscle-invasive bladder cancer (MIBC) represents approximately two thirds of invasive urothelial bladder cancers (UBC) and has high morbidity and mortality. Despite intensive efforts to improve patient treatment and outcome, two thirds of patients with UBC will have a recurrence or disease progression within 5 years. We conducted this study to gain further insights in the immunologic tumor microenvironment (TIME). Material and Methods: Stromal tumor-infiltrating lymphocytes (sTILs) were scored continuously on HE slides in a cohort of 135 patients with MIBC treated by radical cystectomy (adjuvant chemotherapy n= 34) according to current recommendations (Salgado et al., 2015). In parallel, we assessed intrinsic subtypes by 21-gene Nanostring signature adapted from the MDACC-subtyping approach. Tertiary lymph structures were assessed by whole slide immunohistochemistry of CD3, CD8, CD68, and CD79a. Spatial immune profiling was carried out on regionally (tumor center, invasive margin) designed TMAs by CD3, CD8, CD56 (NK-cells), CD68, PD-1, and PD-L1 and revealed spatial organized immune phenotypes. Results were validated in 407 MIBC of the TCGA cohort by hierarchical clustering analysis, immune cell population analysis via CIBERSORT, and sTIL-scoring on digitalized HE-slides. Furthermore, tumor mutational burden, neoantigen load, and mutational patterns as well as mutational signatures were correlated with immune phenotypes in the TCGA cohort. Results: We demonstrate that quantity and spatial distribution of sTILs within the tumor immune microenvironment (TIME) predict stages of tumor inflammation, subtypes, and patient survival and correlate with expression of immune checkpoints in an analysis of 542 MIBC. High sTILs indicate an inflamed subtype with 80% 5-year disease-specific survival. A lack of immune infiltrates identifies an uninflamed subtype with a survival rate of less than 25%. A separate immune-evading phenotype with upregulated immune checkpoints was associated with poor survival. Within the TIME are tertiary lymph node structures (TLS), which can mediate antitumor activity via active immune cells. High TLS amounts and close tumor distance correlated significantly with an inflamed phenotype and favorable survival. The uninflamed and evasion phenotypes showed lowest TLS numbers and farthest tumor distances and shortest survival. High inflammation also correlated with increased neoantigen load, high TMB, and specific mutational patterns (TCGA-MSig1, TCGA-MSig3/4). Patients treated with adjuvant chemotherapy showed a favorable prognosis dependent on high sTILs. Conclusion: Determination of sTILs and tumor subtypes may stratify therapy success and patient survival. Considering sTILs can easily be quantified using simple morphologic parameters such as hematoxylin-eosin, sTILs can be implemented for predicting patient survival and outcome after adjuvant platinum-containing chemotherapy in a routine manner. This abstract is also being presented as Poster A03. Citation Format: Markus Eckstein, Carolin Pfannstiel, Katherine B. Chiappinelli, Danijel Sikic, Sven Wach, Ralph M. Wirtz, Adrian Wullweber, Helge Taubert, Johannes Breyer, Wolfgang Otto, Thomas Worst, Maximilian Burger, Bernd Wullich, Christian Bolenz, Nicole Fuhrich, Carol Geppert, Veronika Weyerer, Robert Stoehr, Simone Bertz, Bastian Keck, Franziska Erlmeier, Philipp Erben, Arndt Hartmann, Pamela Strissel, Reiner Strick. Tumor immune microenvironment drives prognostic relevance correlating with bladder cancer subtypes [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr PR04.

Muscle-invasive bladder cancer (MIBC) represents approximately two-thirds of invasive urothelial bladder cancers (UBC) and has high morbidity and mortality. Men are over 3-fold more frequently affected by UBC than women. Despite intensive efforts to improve patient treatment and outcome, two-thirds of patients with UBC will have a recurrence or disease progression within 5 years. We demonstrated that the quantity and spatial distribution of stromal tumor-infiltrating lymphocytes (sTIL) within the tumor immune microenvironment (TIME) predict stages of tumor inflammation, subtypes, and patient survival and correlate with expression of immune checkpoints in an analysis of 542 patients with MIBC. High sTILs indicated an inflamed subtype with an 80% 5-year DSS, and a lack of immune infiltrates identified an uninflamed subtype with a survival rate of less than 25%. A separate immune evading phenotype with upregulated immune checkpoints associated with poor survival. Within the TIME are tertiary lymphoid structures (TLS), which can mediate antitumor activity via immune cells. High TLS amounts and close tumor distance correlated significantly with an inflamed phenotype and favorable survival. The uninflamed and evasion phenotypes showed lowest TLS numbers, farthest tumor distances, and shortest survival. High inflammation also correlated with increased neoantigen load and mutational burden. Patients treated with adjuvant chemotherapy showed a favorable prognosis, which was dependent on high sTILs. Determination of sTILs and tumor subtypes may stratify therapy success and patient survival, and considering sTILs can easily be quantified using simple morphologic parameters, like hematoxylin and eosin, sTILs can be implemented for predicting patient survival in a routine manner.

PURPOSE: The proto-oncogene forkhead box M1 (FOXM1) is associated with poor survival in many cancers. The impact of FOXM1 expression on progression-free survival (PFS) of non-muscle invasive bladder cancer (NMIBC) has not yet been investigated. The differential expression of FOXM1 between the different molecular NMIBC subtypes has further been assessed.METHODS: Transcript levels of FOXM1 and MKI67 were determined in 460 NMIBC patients (UROMOL cohort) by RNA-Seq and validated in silico by the Chungbuk and Lund cohort (n = 277). FOXM1 and MKI67 cutoffs were identified by the minimal p value method. Variables were evaluated by multivariable Cox regression analyses in order to identify independent predictors.RESULTS: FOXM1 is an independent predictor for PFS superior to current histological, clinical and molecular staging methods. Patients with high FOXM1 expression have a 6- to 8-fold higher risk of progression in multivariable analysis (p CONCLUSION: FOXM1 is a highly prognostic marker for disease progression of NMIBC superior to current histological, clinical and molecular staging methods and MKI67. It is mainly expressed in the Class 2 and genomically unstable molecular bladder cancer subtypes. Its role in drug resistance development makes FOXM1 valuable biomarker for NMIBC risk stratification.

Background Recently, the Food and Drug Administration (FDA)/European Medicines Agency (EMA) restricted first-line use of atezolizumab and pembrolizumab in patients with metastasised urothelial carcinoma by defining distinct programmed cell death ligand-1 cut-offs. We analysed the diagnostic performance of all FDA/EMA-approved programmed cell death ligand-1 assays with emphasis on new restrictions for first-line treatment with atezolizumab and pembrolizumab. Patients and methods Two hundred fifty-one urothelial carcinomas were analysed on tissue microarrays with four cores of each tumour. Stains were performed in certified laboratories on Ventana Benchmark Ultra and Dako Link 48 autostainers. Stains were read on an assay-by-assay basis by two trained pathologists. Overall percentage agreement (OPA) was calculated across the preset cut-offs. Positive percentage agreement (PPA) and negative percentage agreement (NPA) were calculated across different scoring algorithms. Venn diagrams were constructed to illustrate discordance according to the recent FDA/EMA guidelines. Results The Dako 28-8, 22c3 and the Ventana SP263 assays showed high interassay correlation (r-range 0.83–0.91). Interassay correlation between the Ventana SP142 and the three other assays was moderate (r-range 0.66–0.75). OPA of 93.3% was achieved between the Dako 28-8, 22c3 and Ventana SP263 assays. OPA including the SP142 was 84.1%. Pooled PPA and NPA of different scoring algorithms was 89.4% and 95.3% for the Dako 28-8, 22c3 and the SP263 assays, respectively. With the SP142 assay, pooled PPA was 59.1%. The SP142 assay identifies fewer eligible patients for first-line treatment with atezolizumab/pembrolizumab. Conclusion Dako 28-8, 22c3 and SP263 assays show interchangeable performance. The SP142 assay shows divergent staining results. Interassay variability leads to different detection rates of eligible patients for first-line treatment with atezolizumab and pembrolizumab.

Background In the era of individualized oncological therapy in bladder cancer, FGFR3 mutations, FGFR2/FGFR3 gene fusions and FGFR mRNA expression as potential oncological targets and their association to anti-PD-1/anti-PD-L1 (IO) treatment outcomes in patients with advanced urothelial cancer of the bladder (UCB) were studied in a German patient cohort. Methods Within a cohort of 72 patients with metastatic UCB from 5 academic centers in Germany (collected between 2016 and 2018) FGFR3 mutations, FGFR2 and FGFR3 gene fusions as well as FGFR expression in formalin-fixed, paraffin embedded (FFPE) tumour samples and their association to survival were examined using SNaPshot PCR, RT-qPCR as well as Next Generation Sequencing. Statistical analyses comprised Kaplan-Meier survival analyses, Spearman rank correlations, non-parametric testing. Results In 17% of all patients FGFR3 mutations or gene fusions could be detected. Patients with FGFR3 alterations did not have better outcome after immunoncology (IO) treatment, but rather tended to have inferior disease specific survival. All alterations of FGFR3 resulted in overexpression of FGFR3 mRNA. Combination of FGFR mutation analysis and FGFR mRNA assessment improved IO outcome prediction. Overexpression of FGFR3 mRNA was negatively associated with PDL1 expression (mRNA and protein level). High FGFR2 mRNA expression in primary tumors predicted better disease specific survival of UCB patients receiving IO therapy, whereas high FGFR3 mRNA expression was associated with tumor specific death in patients exhibiting of low FGFR2 mRNA expressions (p Conclusions The assessment of FGFR mRNA by standardized RT-qPCR identified a high risk UCB patient cohort, which does have inferior disease specific survival despite IO treatment and does overexpress FGFR3 mRNA. The assessment of FGFR mRNA levels by using this standardized, locally applicable FGFR test system could identify an FGFR inhibitor target population with poor response to IO treatment which is twice the size as currently detected by FGFR genomic alterations alone. Legal entity responsible for the study BRIDGE Consortium e.V. Funding Janssen. Disclosure F. Roghmann: Advisory / Consultancy: Janssen; Travel / Accommodation / Expenses: Novartis; Advisory / Consultancy: Roche; Travel / Accommodation / Expenses: Ipsen. All other authors have declared no conflicts of interest.

Background Immunotherapy (IO) with antibodies against PD1 or PD-L1 has been approved for 1st and 2nd line treatment of metastasized urothelial bladder cancer (mUC). Here we evaluated efficacy in a retrospective Real-World-Phase IV trial based on molecular subtypes and PD-L1 status. Methods Cancer specific survival from start of IO treatment to death was retrospectively analyzed in a multicenter cohort of 65 patients having received palliative immune checkpoint inhibition for mUC in 1st and 2nd line setting. Intrinsic molecular subtypes were assessed by CK5, CK20, GATA3, FOXA1 and CD44 immunohistochemistry and RT-qPCR of KRT5 and KRT20. PD-L1 status was determined using the 28-8 Dako-assay. Stromal tumor infiltrating lymphocytes were assessed on HE slides (Salgado et al, 2015). Survival analyses were performed by applying clinically relevant cut-offs and using Kaplan-Meier analysis and logistic regression for cancer specific survival (CSS). Results Altogether, 43% of tumors presented with basal differentiation, 57% were luminal. As expected, basal tumors exhibited significantly higher levels of PD-1 and PD-L1 gene expression, higher amounts of sTILs and PD-L1+ immune and tumor cells. Hierarchical clustering of all immunological biomarkers revealed three cluster: “Inflamed high” (n = 13, 20%), “Inflamed low” (n = 22, 33.8%), and “Uninflamed” (n = 30, 46.2%). There were no significant survival differences for any of these groups (CSS). However, patients with good performance status (ECOG 0) and low PD-L1 expression showed significantly better CSS compared to those with ECOG0 / PD-L1 high and ECOG1 or ECOG2, respectively (30.3 months vs. 16.33, 12.07 and 15.13 months; p = 0.0047). Conclusions Immunological determinants of mUC show comparable distributions and correlations with subtypes of localized curatively treated MIBC patients. Interestingly, data indicate improved CSS after PD-1/PD-L1 treatment in patients with good performance and low PD-L1 expression, which warrants validation in larger cohorts. Clinical trial identification DRKS00016925. Legal entity responsible for the study STRATIFYER Molecular Pathology GmbH. Funding Janssen-Cilag GmbH. Disclosure R. Wirtz: Leadership role: CEO & CSO, STRATIFYER Molecular Pathology GmbH. All other authors have declared no conflicts of interest.

Background: Aggressive variant transformation in metastatic castration-resistant prostate cancer (mCRPC) represents an under-recognized phenomenon. There is an urgent need for non-invasive biomarkers to detect these variants and identify treatment alternatives. Methods: A prospective observational pilot study in mCRPC patients receiving treatment with cabazitaxel (CAB) was conducted. Neuromediators were sequentially evaluated and their impact on disease endpoints calculated. Targeted next-generation sequencing (NGS) of cell-free DNA (cfDNA) was also performed in a highly pretreated subset of patients. Results: 23 patients were included. Estimated effects indicate that neuron-specific enolase (NSE) levels at baseline may be correlated with overall survival (NSE unit 18.3 ng/ml: HR1.262 (95% confidence interval (CI) 0.985-1.616)) and that chromogranin A (CGA) may be correlated with progression-free survival (CGA unit 98.1 ng/ml: HR1.341 (95% CI 1.011-1.778)). cfDNA analysis revealed mutations annotated in prostate cancer (PCA) and small cell cancers (SCC). 1 patient showed elevated neuromediators along with annotated mutations in PCA and SCC, potentially indicating aggressive variant cancer. In 3 patients KIT mutations (e.g. pM541L, pV654A) known to be tissue-based biomarkers with level 1 evidence for the treatment with imatinib and sunitinib were found. Conclusions: Sequential analysis of neuromediators and targeted NGS of cfDNA provide insight for the estimation of tumor heterogeneity under therapy with CAB.

INTRODUCTION: Gene expression analyses have identified similarities between bladder and breast cancer, where clinical risk stratification is based on Her2, ESR1, PGR and Ki67 expression. The aim of the study was to assess the respective marker gene expression in patients treated with radical cystectomy for muscle-invasive bladder cancer (MIBC) and to evaluate the applicability of breast cancer subtypes for MIBC risk stratification. MATERIALS & METHODS: 102 patients treated with radical cystectomy for MIBC were assessed. Using routine FFPE tissue and an IVD validated kit, mRNA expression was measured by single step RT-qPCR. Partition test were employed to define cut-off values for high or low marker gene expression. Association of expression with outcome was assessed using Kaplan-Meier analysis and multivariate cox regression analysis. Finally, we performed validation of our results in the MD-Anderson cohort (n=57). RESULTS: Cancer specific survival (CSS) was impaired in patients with high gene expression of Her2 ( P =0.0009) and ESR1 ( P =0.04). In the multivariate regression model Her2 expression remained significant for the prediction of CSS (HR=2.11, CI 1.11-4.21, P =0.024). Furthermore, molecular stratification by breast cancer subgroups was significant ( P =0.023) for CSS prediction. Especially the differentiation between Her2 -positive and Luminal A (HR=4.41, CI 1.53-18.71, P =0.004) and Luminal B (HR=1.96, CI 0.99-4.08, P =0.053) respectively was an independent prognostic parameter for CSS. External validation resulted in comparable risk stratification with differences in fractional subgroups distribution. CONCLUSION: Gene expression of Her2, ESR1, PGR, Ki67 and corresponding breast cancer subtypes allow a risk-stratification in MIBC, whereby Her2 overexpressing tumors reveal a particularly poor prognosis.

// Sebastien Rinaldetti 1 , Eugen Rempel 2, 3 , Thomas Stefan Worst 3, 4 , Markus Eckstein 5 , Annette Steidler 4 , Cleo Aaron Weiss 6 , Christian Bolenz 7 , Arndt Hartmann 5 and Philipp Erben 4, * 1 Department of Hematology and Oncology, Medical Faculty Mannheim, University of Heidelberg, 68167 Mannheim, Germany 2 German Cancer Research Center (DKFZ), Division of Signalling and Functional Genomics, 69120 Heidelberg, Germany 3 Department of Stem Cell Biology, Centre of Organismal Studies, University Heidelberg, 69120 Heidelberg, Germany 4 Department of Urology, Medical Faculty Mannheim, University of Heidelberg, 68167 Mannheim, Germany 5 Institute of Pathology, University Erlangen-Nuremberg, 91054 Erlangen, Germany 6 Institute of Pathology, University Medical Centre Mannheim, 68167 Mannheim, Germany 7 Department of Urology, University of Ulm, 89075 Ulm, Germany * On behalf of the BRIDGE Consortium Correspondence to: Sebastien Rinaldetti email: sebastienrinal@hotmail.com Keywords: bladder cancer subtypes; survival prediction; drug targets; gene expression; biomarkers Received: September 19, 2017 Accepted: April 27, 2018 Published: May 25, 2018 ABSTRACT Background: Transcriptome expression studies identified distinct muscle invasive bladder cancer (MIBC) subtypes closely related with breast cancer subclasses. Here we developed a sensitive quantification method for MIBC subclassification (luminal, basal, p53-like). In addition, the subtype specific expression of drug targets has been investigated. Methods: Absolute quantification (nCounter) of a 64-gene panel was performed on MIBC patients (n=47) treated exclusively with radical cystectomy (RC). In conjunction of 170 MIBCs from 3 independent cohorts, a minimal set of consensus genes has been established. Survival of the consensus subtypes has been assessed by multivariate analysis. Relevant drug targets were tested for their subtype specificity in a clustering independent assessment. Results: A reduced 36-gene panel stably clustered into 3 subtypes throughout the cohorts (luminal, basal, infiltrated). Patients treated by RC only, showed worst 8-year disease specific survival (DSS) for the luminal subtype in contrast to the infiltrated subtype (17% vs. 73%, p=0.011). In multivariate analyses, the risk stratification based on luminal versus not-luminal MIBC proved to be an independent predictor for DSS superior to the TNM system in patients with RC. Drug targets (e.g. ERBB2, FGFR, AR, PDGFRB ) showed a distinct subtype attribution. The subtypes based on this nCounter screening could further be validated by the TCGA cohort. Conclusion: This MIBC subtype screening predicted survival and allowed an analysis of subtype specific drug targets, thus being a powerful tool for the translation of personalized MIBC treatment concepts.

In prostate cancer and other malignancies sensitive and robust biomarkers are lacking or have relevant limitations. Prostate specific antigen (PSA), the only biomarker widely used in prostate cancer, is suffering from low specificity. Exosomes offer new perspectives in the discovery of blood-based biomarkers. Here we present a proof-of principle study for a proteomics-based identification pipeline, implementing existing data sources, to exemplarily identify exosome-based biomarker candidates in prostate cancer. Exosomes from malignant PC3 and benign PNT1A cells and from FBS-containing medium were isolated using sequential ultracentrifugation. Exosome and control samples were analyzed on an LTQ-Orbitrap XL mass spectrometer. Proteomic data is available via ProteomeXchange with identifier PXD003651. We developed a scoring scheme to rank 64 proteins exclusively found in PC3 exosomes, integrating data from four public databases and published mass spectrometry data sets. Among the top candidates, we focused on the tight junction protein claudin 3. Retests under serum-free conditions using immunoblotting and immunogold labeling confirmed the presence of claudin 3 on PC3 exosomes. Claudin 3 levels were determined in the blood plasma of patients with localized (n = 58; 42 with Gleason score 6–7, 16 with Gleason score ≥8) and metastatic prostate cancer (n = 11) compared with patients with benign prostatic hyperplasia (n = 15) and healthy individuals (n = 15) using ELISA, without prior laborious exosome isolation. ANOVA showed different CLDN3 plasma levels in these groups (p = 0.004). CLDN3 levels were higher in patients with Gleason ≥8 tumors compared with patients with benign prostatic hyperplasia (p = 0.012) and Gleason 6–7 tumors (p = 0.029). In patients with localized tumors CLDN3 levels predicted a Gleason score ≥ 8 (AUC = 0.705; p = 0.016) and did not correlate with serum PSA. By using the described workflow claudin 3 was identified and validated as a potential blood-based biomarker in prostate cancer. Furthermore this workflow could serve as a template to be used in other cancer entities.