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Supplementary Data from Fibroblast Growth Factor Receptor 3 Mutation Analysis on Voided Urine for Surveillance of Patients with Low-Grade Non-Muscle–Invasive Bladder Cancer

Purpose: Mutations in the fibroblast growth factor receptor 3 (FGFR3) have been found in 70% of the low-grade non-muscle–invasive bladder cancer (NMI-BC) tumors. We aim to determine the potential of FGFR3 mutation analysis on voided urine to detect recurrences during surveillance of patients with low-grade NMI-BC.Experimental Design: FGFR3 mutation status of the study inclusion tumor was determined from 200 low-grade NMI-BC patients. Patients with an FGFR3-mutant inclusion tumor were selected for analysis and monitored by cystoscopy, and voided urine samples were collected. FGFR3 mutation analysis was done on 463 prospectively collected urines. Sensitivity and predictive value of the assay were determined for detection of concomitant recurrences. Longitudinal and Cox time-to-event analyses were done to determine the predictive value for detection of future recurrences.Results: Median follow-up was 3.5 years. The sensitivity of the assay for detection of concomitant recurrences was 26 of 45 (58%). Of the 105 positive urine samples, 85 (81%) were associated with a concomitant or a future recurrence. An FGFR3-positive urine was associated with a 3.8-fold (P < 0.0001) higher risk of having a recurrence in the Cox analysis. In contrast, only 41 of 358 (11%) FGFR3-negative urine samples were associated with a recurrence. Positive predictive value increased from 25% to 90% in patients having consecutive FGFR3-positive urine tests.Conclusions: FGFR3 mutation analysis on voided urine is a simple and noninvasive diagnostic method for detection of recurrences during surveillance of patients presenting with a low-grade FGFR3-mutant NMI-BC tumor. Clin Cancer Res; 16(11); 3011–8. ©2010 AACR.

PURPOSE OF REVIEW: To provide an overview of the recent literature on RNA-based molecular urine assays for the diagnosis and surveillance of non-muscle invasive bladder cancer (NMIBC). RECENT FINDINGS: Articles were eligible for inclusion if performance metrics sensitivity, specificity, and negative-predictive value (NPV) were reported or could be calculated. Only prospective studies published between 2020-2022 were included. Five out of fourteen studies addressed the primary diagnostic setting; the proportion of gross hematuria patients in all study populations was >50%. Only one study reported performance metrics within a microscopic hematuria subgroup. This study evaluated Xpert Bladder and reported a sensitivity: 73%, specificity: 84%, NPV: 99%, and PPV: 12%. Ten studies assessed test performance during surveillance for NMIBC. For the detection of high-grade (HG) and high-risk (HR) NMIBC, sensitivity, specificity, NPV, and PPV varied between 78-100%, 64-89%, 97.0-99.7%, and 9.2-39%. SUMMARY: Multiple RNA-based urine assays have been investigated for the detection of urothelial cancer in the primary or surveillance setting. However, studies included within this review have important limitations, hampering the interpretation of study results. As such, performance metrics should be interpreted with caution and further research is required to evaluate the clinical impact of RNA-based urine assays in daily practice.

Treatment of patients with urothelial carcinoma (UC) of the bladder or renal cancer has changed significantly during recent years and efforts towards biomarker-directed therapy are being investigated. Immune checkpoint inhibition (ICI) or fibroblast growth factor receptor (FGFR) directed therapy are being evaluated for non-muscle invasive bladder cancer (NMIBC) patients, as well as muscle-invasive bladder cancer (MIBC) patients. Meanwhile, efforts to predict tumor response to neoadjuvant chemotherapy (NAC) are still ongoing, and genomic biomarkers are being evaluated in prospective clinical trials. Currently, patients with metastatic UC (mUC) are usually treated with second-line ICI, while cisplatin-ineligible patients with programmed death-ligand 1 (PD-L1) positive tumors can benefit from first-line ICI. Platinum-relapsed UC patients harboring FGFR2/3 mutations can be treated with erdafitinib, while enfortumab vedotin has emerged as a novel third-line treatment option for mUC. In metastatic (clear cell) renal cell carcinoma (RCC), ICI was first introduced as second-line treatment after vascular endothelial growth factor receptor—tyrosine kinase inhibition (VEGFR-TKI). Currently, ICIs have also been introduced as first-line treatment in metastatic RCC. Although there is no evidence up to now for beneficial adjuvant treatment after surgery with VEGFR-TKIs in high-risk non-metastatic RCC, several trials are underway investigating the potential beneficial effect of ICIs in this setting.

PURPOSE: Currently, markers are lacking that can identify patients with high risk nonmuscle invasive bladder cancer who will fail bacillus Calmette-Guérin treatment. Therefore, we evaluated the prognostic value of T1 substaging in patients with primary high risk nonmuscle invasive bladder cancer. MATERIALS AND METHODS: Patients with primary high risk nonmuscle invasive bladder cancer who received ≥5 bacillus Calmette-Guérin induction instillations were included. All tumors were centrally reviewed, which included T1 substaging (microinvasion vs extensive invasion of the lamina propria). T1 patients were stratified into high risk or highest risk subgroups according to major urology guidelines. Primary end point was bacillus Calmette-Guérin failure, defined as development of a high grade recurrence. Secondary end points were high grade recurrence-free survival, defined as time from primary diagnosis to biopsy-proven high grade recurrence and progression-free survival. Time-to-event analyses were used to predict survival. RESULTS: A total of 264 patients with high risk nonmuscle invasive bladder cancer had tumor invasion of the lamina propria, of which 73% were classified as extensive invasion and 27% as microinvasion. Median followup was 68 months (IQR 43-98) and bacillus Calmette-Guérin failure was more common among patients with extensive vs microinvasive tumors (41% vs 21%, p=0.002). The 3-year high grade recurrence-free survival (defined as bacillus Calmette-Guerin failure) for patients with extensive vs microinvasive tumors was 64% vs 83% (p=0.004). In multivariate analysis, T1 substaging was an independent predictor of high grade recurrence-free survival (HR 3.2, p=0.005) and progression-free survival (HR 3.0, p=0.009). Patients with highest risk/microinvasive disease have an improved progression-free survival as compared to highest risk/T1e disease (p.adj=0.038). CONCLUSIONS: T1 substaging provides important prognostic information on patients with primary high risk nonmuscle invasive bladder cancer treated with bacillus Calmette-Guérin. The risk of bacillus Calmette-Guérin failure is higher in extensive vs microinvasive tumors. Substaging of T1 high risk nonmuscle invasive bladder cancer has the potential to guide treatment decisions on bacillus Calmette-Guérin vs alternative strategies at diagnosis.

The risk of developing urothelial carcinoma of the bladder (UCB) in patients treated by radical nephroureterectomy (RNU) for an upper urinary tract urothelial carcinoma (UTUC) is 22% to 47% in the 2 years after surgery. Subject of debate remains whether UTUC and the subsequent UCB are clonally related or represent separate origins. To investigate the clonal relationship between both entities, we performed targeted DNA sequencing of a panel of 41 genes on matched normal and tumor tissue of 15 primary UTUC patients treated by RNU who later developed 19 UCBs. Based on the detected tumor‐specific DNA aberrations, the paired UTUC and UCB(s) of 11 patients (73.3%) showed a clonal relation, whereas in four patients the molecular results did not indicate a clear clonal relationship. Our results support the hypothesis that UCBs following a primary surgically resected UTUC are predominantly clonally derived recurrences and not separate entities., What's new? Patients treated by radical nephroureterectomy for upper urinary tract cancer have an increased risk of developing bladder carcinoma following surgery. It remains unclear, however, whether the upper urinary tract cancer and subsequent bladder carcinoma are clonally related or have separate origins. This targeted DNA sequencing study shows that almost 75% of patients have tumors that are clonally related, suggesting that seeding of tumor cells is the main mechanism of bladder carcinoma development following radical nephroureterectomy. This result underscores the need to minimalize the risk of seeding during surgery and/or diagnostic ureterorenoscopy plus biopsy, and to apply peri‐operative intravesical instillations with chemotherapy.

Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer (BC). FGFR3 mutations are common in noninvasive BC and associated with favorable BC prognosis. Overexpression was reported in up to 40% of FGFR3 wild-type muscle-invasive BC. We analyzed FGFR3 mutations, FGFR3, and p53 protein expression and assessed their prognostic value in a cohort of 1000 chemotherapy-naive radical cystectomy specimens. FGFR3 mutations were found in 11%, FGFR3 overexpression was found in 28%, and p53 overexpression was found in 69% of tumors. Among FGFR3 mutant tumors, 73% had FGFR3 overexpression versus 22% among FGFR3 wild-type tumors. FGFR3 mutations were significantly associated with lower pT stage, tumor grade, absence of carcinoma in situ, pN0, low-level p53, and longer disease-specific survival (DSS). FGFR3 overexpression was associated only with lower pT stage and tumor grade. Moreover, FGFR3 overexpression was not associated with DSS in patients with FGFR3 wild-type tumors. In conclusion, FGFR3 mutations identified patients with favorable BC at cystectomy. Our results suggest that FGFR3 mutations have a driver role and are functionally distinct from FGFR3 overexpression. Hence, patients with FGFR3 mutations would be more likely to benefit from anti-FGFR3 therapy. Ideally, further research is needed to test this hypothesis. Patient summary: Oncogenic fibroblast growth factor receptor 3 (FGFR3) mutations are very common in bladder cancer. In this report, we found that these FGFR3 mutations were associated with favorable features and prognosis of bladder cancer compared with patients with FGFR3 overexpressed tumors only. As a consequence, patients with FGFR3 mutant tumors would be more likely to benefit from anti-FGFR3 therapy than patients with FGFR3 protein overexpression only.

Histological tissue examination has been a longstanding practice for cancer diagnosis where pathologists identify the presence of tumors on glass slides. Slides acquired from laboratory routine may contain unintentional artifacts due to complications in surgical resection. Blood and damaged tissue artifacts are two common problems associated with transurethral resection of the bladder tumor. Differences in histotechnical procedures among laboratories may also result in color variations and minor inconsistencies in outcome. A digitized version of a glass slide known as a whole slide image (WSI) holds enormous potential for automated diagnostics. The presence of irrelevant areas in a WSI undermines diagnostic value for pathologists as well as computational pathology (CPATH) systems. Therefore, automatic detection and exclusion of diagnostically irrelevant areas may lead to more reliable predictions. In this paper, we are detecting blood and damaged tissue against diagnostically relevant tissue. We gauge the effectiveness of transfer learning against training from scratch. Best models give 0.99 and 0.89 F1 scores for blood and damaged tissue detection. Since blood and damaged tissue have subtle color differences, we assess the impact of color processing methods on the binary classification performance of five well-known architectures. Finally, we remove the color to understand its importance against morphology on classification performance.

Bladder cancer patients' stratification into risk groups relies on grade, stage and clinical factors. For non-muscle invasive bladder cancer, T1 tumours that invade the subepithelial tissue are high-risk lesions with a high probability to progress into an aggressive muscle-invasive disease. Detecting invasive cancerous areas is the main factor for dictating the treatment strategy for the patient. However, defining invasion is often subject to intra/interobserver variability among pathologists, thus leading to over or undertreatment. Computer-aided diagnosis systems can help pathologists reduce overheads and erratic reproducibility. We propose a multi-scale model that detects invasive cancerous areas patterns across the whole slide image. The model extracts tiles of different tissue types at multiple magnification levels and processes them to predict invasive patterns based on local and regional information for accurate T1 staging. Our proposed method yields an F1 score of 71.9, in controlled settings 74.9, and without infiltration 90.0.

BackgroundBCG is recommended as intravesical immunotherapy to reduce the risk of tumor recurrence in patients with non-muscle invasive bladder cancer (NMIBC). Currently, it is unknown whether intravesical BCG application induces trained immunity.MethodsThe aim of this research was to determine whether BCG immunotherapy induces trained immunity in NMIBC patients. We conducted a prospective observational cohort study in 17 NMIBC patients scheduled for BCG therapy and measured trained immunity parameters at 9 time points before and during a 1-year BCG maintenance regimen. Ex vivo cytokine production by peripheral blood mononuclear cells, epigenetic modifications, and changes in the monocyte transcriptome were measured. The frequency of respiratory infections was investigated in two larger cohorts of BCG-treated and non-BCG treated NMIBC patients as a surrogate measurement of trained immunity. Gene-based association analysis of genetic variants in candidate trained immunity genes and their association with recurrence-free survival and progression-free survival after BCG therapy was performed to investigate the hypothesized link between trained immunity and clinical response.ResultsWe found that intravesical BCG does induce trained immunity based on an increased production of TNF and IL-1β after heterologous ex vivo stimulation of circulating monocytes 6–12 weeks after intravesical BCG treatment; and a 37% decreased risk (OR 0.63 (95% CI 0.40 to 1.01)) for respiratory infections in BCG-treated versus non-BCG-treated NMIBC patients. An epigenomics approach combining chromatin immuno precipitation-sequencing and RNA-sequencing with in vitro trained immunity experiments identified enhanced inflammasome activity in BCG-treated individuals. Finally, germline variation in genes that affect trained immunity was associated with recurrence and progression after BCG therapy in NMIBC.ConclusionWe conclude that BCG immunotherapy induces trained immunity in NMIBC patients and this may account for the protective effects against respiratory infections. The data of our gene-based association analysis suggest that a link between trained immunity and oncological outcome may exist. Future studies should further investigate how trained immunity affects the antitumor immune responses in BCG-treated NMIBC patients

SummaryThe recommended treatment for patients with high-risk non-muscle invasive bladder cancer (HR-NMIBC) is tumor resection followed by adjuvant Bacillus Calmette-Guérin (BCG) bladder instillations. However, only 50% of patients benefit from this therapy. In case of progression to advanced disease, patients must undergo a radical cystectomy with significant morbidity and have a poor clinical outcome. Identifying tumors least likely to respond to BCG can translate into alternative treatments, such as early radical cystectomy or novel targeted or immunotherapies. Here we present molecular profiling of 132 BCG-naive, HR-NMIBC patients, and 44 post-BCG recurrences (34 matched), which uncovered three distinct BCG Response Subtypes (BRS1-3). Patients with BRS3 tumors have reduced recurrence and progression-free survival compared to BRS1-2. BRS3 tumors expressed high EMT-basal markers and had an immunosuppresive profile, which was confirmed with spatial proteomics. Tumors which recurred post-BCG were enriched for BRS3. BRS stratification was validated in a second cohort of 151 BCG-naive HR-NMIBC patients and the molecular subtypes outperformed guideline recommended risk stratification based on clinicopathological variables. For clinical application, we validated that a commercially approved assay was able to accurately predict BRS3 tumors (AUROC 0.86). Our findings provide a potential clinical tool for improved identification of HR-NMIBC patients at the highest risk of progression, which can be used to select patients for early radical cystectomy or novel subtype-directed therapies.One Sentence SummaryMolecular subtypes are predictive of response to intravesical Bacillus Calmette-Guérin immunotherapy in non-muscle invasive bladder cancer.

Purpose: To evaluate the risk factors associated with positive surgical margins' (PSMs) location and their impact on disease-specific survival (DSS) in patients with bladder cancer (BCa) undergoing radical cystectomy (RC). Methods: We analyzed a large multi-institutional cohort of patients treated with upfront RC for non-metastatic (cT1-4aN0M0) BCa. Multivariable binomial logistic regression analyses were used to assess the risk of PSMs at RC for each location after adjusting for clinicopathological covariates. The Kaplan–Meier method was used to estimate DSS stratified by margins’ status and location. Log-rank statistics and Cox’ regression models were used to determine significance. Results: A total of 1058 patients were included and 108 (10.2%) patients had PSMs. PSMs were located at soft-tissue, ureter(s), and urethra in 57 (5.4%), 30 (2.8%) and 21 (2.0%) patients, respectively. At multivariable analysis, soft-tissue PSMs were independently associated with pathological stage T4 (pT4) (Odds ratio (OR) 6.20, p < 0.001) and lymph-node metastases (OR 1.86, p = 0.04). Concomitant carcinoma-in-situ (CIS) was an independent risk factor for ureteric PSMs (OR 6.31, p = 0.003). Finally, urethral PSMs were independently correlated with pT4-stage (OR 5.10, p = 0.01). The estimated 3-years DSS rates were 58.2%, 32.4%, 50.1%, and 40.3% for negative SMs, soft-tissue-, ureteric- and urethral PSMs, respectively (log-rank; p < 0.001). Conclusions: PSMs’ location represents distinct risk factors’ patterns. Concomitant CIS was associated with ureteric PSMs. Urethral and soft-tissue PSM showed worse DSS rates. Our results suggest that clinical decision-making paradigms on adjuvant treatment and surveillance might be adapted based on PSM and their location.

INTRODUCTION AND OBJECTIVE:Intravesical Bacillus Calmette-Guerin (BCG) is the recommended treatment option in high-risk non-muscle invasive bladder cancer (HR-NMIBC). However, disease recurrence oc...

Since December 2019, the emergence of a new Severe Acute Respiratory Syndrome- coronavirus (SARS-CoV-2) has led to a global coronavirus pandemic disease (COVID-19), with devastating consequences for all healthcare worldwide, including urological care. COVID-19 has led to concern among urological healthcare workers about viral presence, detection and routes of transmission during routine clinical practice. The potential presence of (active) virus in bodily fluids of COVID-19 patients remains a continuing topic of debate. Therefore, we highlight viral detection methods and review the presence of SARS-CoV-2 in urine, feces, and semen. Finally, we discuss how excretion of virus particles through urological bodily fluids might be pivotal to epidemiologic monitoring and control of the disease. © 2020 The Authors

Objectives: To determine the association between the FGFR3 mutation status and immuno-histochemistry (IHC) markers (p53 and Ki-67) in invasive bladder cancer (BC), and to analyze their prognostic value in a multicenter, multi-laboratory radical cystectomy (RC) cohort. Patients and methods: We included 1058 cN0M0, chemotherapy-naive BC patients who underwent RC with pelvic lymph-node dissection at 8 hospitals. The specimens were reviewed by uro-pathologists. Mutations in the FGFR3 gene were examined using PCR-SNaPshot; p53 and Ki-67 expression were determined by standard IHC. FGFR3 mutation status as well as p53 (cut-off>10%) and Ki-67 (cut-off>20%) expression were correlated to clinicopathological parameters and disease specific survival (DSS). Results: pT-stage was

Rationale: Several guidelines exist that address treatment of patients with nonmetastatic muscle-invasive bladder cancer (MIBC). However, most only briefly mention follow-up strategies for patients and hence the treating physician is often left to infer on what the preferred follow-up schema would be for an individual patient. Herein, we aim to synthesize recommendations for follow-up of patients with MIBC for easy reference. Methods: A multidisciplinary MIBC expert panel from the International Bladder Cancer Network was assembled to critically assess currently available major guidelines on surveillance of MIBC patients. Recommendations for follow-up were extracted and critically evaluated. Important considerations for guideline assessment included both aspects of oncological and functional follow-up-frequency of visits, the use of different imaging modalities, the role of cytology and molecular markers, and the duration of follow-up. Outcome: An International Bladder Cancer Network expert consensus recommendation was constructed for the follow-up of patients with MIBC based on the currently available evidence-based data.

Gemcitabine/cisplatin combination is the recommended neoadjuvant chemotherapy (NAC) for muscle-invasive bladder cancer (MIBC) patients undergoing surgical removal of the bladder (cystectomy). Patients without residual tumor at cystectomy ,complete pathological response (pCR), have a good 5-yr OS of 80%. However, due to NAC resistance, only 25% of the patients achieve a pCR. This study aims to increase the number of patients reaching pCR by discovery and functional interrogation of novel and pharmacologically targetable mechanisms causal to NAC resistance. We have identified an M1 aminopeptidase, NPEPPS, to regulate intracellular cisplatin import through volume regulated anion channels (VRACs). We show that genetic inhibition of NPEPPS increases the intracellular concentrations of cisplatin, hereby re-sensitizing gem/cis-resistant BC cell lines. On the other side, NPEPPS over-expression enhanced resistance to cisplatin. Pharmacological inhibition of NPEPPS with tosedostat results re-sensitization of gem/cis resistant BC cells in vitro and in vivo. Additional therapeutically validation was performed in gem/cis resistant patient-derived tumoroids. For this purpose, tumoroid cultures were generated from bladder cancer patients whom did not respond to pre-operative gem/cis, molecularly characterized and compared to the tumor of origin. Comparison of bladder cancer specific hotspot mutations, copy-number aberrations and H&E staining confirmed that patient-specific tumor traits were maintained in ex vivo tumoroid cultures. Moreover, tumoroid cultures generated from NAC-resistant patients were also resistant to cisplatin concentrations that exceeded physiological serum concentrations. Interestingly, addition of NPEPPS-inhibitor tosedostat sensitized these NAC resistant tumoroids to serum concentrations of cisplatin. These findings have potential for rapid translation into the clinic and invite trials investigating tosedostat to overcome chemoresistance. Citation Format: Mathijs P. Scholtes, Maryam Akbarzadeh, Dan Theodorescu, James C. Costello, Tokameh Mahmoudi, Tahlita C.M. Zuiverloon. NPEPPS regulates cisplatin-resistance and can be targeted to overcome treatment resistance in patient-derived bladder cancer tumoroids [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P177.

The recommended treatment for metastatic urothelial carcinoma (mUC) patients is platinum-based chemotherapy. Although initial response rates are moderate, the vast majority of patients experience a relapse due to chemoresistance and eventually succumb to their disease. Furthermore, platinum-based chemotherapy is toxic and approximately 30% of mUC patients are unfit for chemotherapy. Thus, there is a clear unmet need for novel, more efficacious treatment options in mUC with a safer toxicity profile. To propel the advancement of novel treatment options, we present a summary of key signaling pathways and molecular mechanisms that are known to be involved in bladder cancer tumorigenesis with a focus on promising candidate druggable molecular targets and innovative targeted therapies currently under clinical investigation. Targetable alterations were mainly described in fibroblast growth factor receptor (FGFR) and epidermal growth factor receptor (ErbB) tyrosine kinase receptor (RTK) families, downstream pathways, and chromatin remodelers, which are major bladder cancer driver genes. Drugs targeting the FGFR family members are emerging as personalized treatment options for selected mUC patients with tumor-specific FGFR alterations. The pan-FGFR inhibitor, erdafitinib, was first-in-class to receive U.S. Food and Drug Administration (FDA) approval in 2019, while inhibitors of ErbB family members have shown less potential. Antibody-drug conjugates (ADCs) are a class of targeted therapeutics that deliver cytotoxic drugs in close proximity to cancer cells by targeting RTKs or other transmembrane proteins. Enfortumab vedotin is the first-in-class ADC that was FDA approved for the treatment of locally advanced or mUC in 2019.

Intravesical instillations with Bacillus Calmette-Guérin (BCG) are the recommended treatment in T1 high-grade (HG) bladder cancer (BC) patients. However, even with BCG treatment, 50% of patients develop a HG recurrence or progression to advanced disease. Current risk stratification is insufficient to identify patients at risk of BCG failure. We aimed to identify molecular predictors of BCG failure in T1HG BC patients with the objective to improve clinical decision-making. Primary T1HG BC patients treated with ≥5/6 BCG induction instillations were retrospectively included. RNA-sequencing (50 million paired-end reads, ≥80% tumor) was performed on centrally reviewed tumors from: 1) BCG-naïve tumors and recurrent tumors from patients that failed BCG therapy (BCG non-responders), matched to an equal number of 2) patients who had an ongoing complete response until study inclusion (BCG responders). Endpoints were HG recurrence-free survival defined as BCG-failure (RFS) and progression-free survival (PFS), assessed by survival analyses. BCG-failure was defined as development of a biopsy-proven HG recurrence or progression to muscle-muscle invasion. Using RNA-seq, we sought to identify genes, pathways and signatures that stratified patients with respect to these endpoints. 132 BCG-naïve T1HG tumors were sequenced and 45 post-BCG tumors. Median follow-up for all patients was 75 months (IQR 48-106), 99 months for n=63 BCG-responders (IQR 74-120) and 49 months for n=69 BCG-non-responders (IQR 25-79). Three molecular subtypes were identified: BCG1 (32%), BCG2 (40%) and BCG3 (28%). BCG3 patients had a significantly worse RFS (HR 2.4 p Citation Format: Florus C. De Jong, Teemu D. Laajala, Robert F. Hoedemaeker, Sébastien Rinaldetti, Jolien T. Mensink, Angelique C. Van der Made, Deric K. Van der Schoot, Egbert R. Boevé, Ellen C. Zwarthoff, Joost L. Boormans, Dan Theodorescu, James C. Costello, Tahlita C. Zuiverloon. Transcriptomic analysis of BCG-treated T1HG bladder cancer patients identifies an EMT-basal subgroup with immune suppressive characteristics at high risk of BCG-failure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 614.

Intravesical instillations with Bacillus Calmette-Guérin (BCG) is the recommended treatment for T1 high-grade (HG) bladder cancer (BC) patients. Unfortunately, risk stratification is insufficient to identify patients at risk of BCG treatment failure. Another challenge is the worldwide BCG-shortage, which emphasizes the need for alternative therapeutic strategies. Thus, we aimed to investigate molecular signal transduction pathway activities using Philips' OncoSignal consumer-ready test in BCG treated T1HG BC in order to identify differentially expressed pathways that may predict clinical outcome or provide a rationale for alternative therapeutic strategies. Primary T1HG BC patients treated with ≥5/6 BCG induction instillations were retrospectively included. RNA-sequencing (50 million paired-end reads, containing ≥80% tumor) was performed on: 1) BCG-naïve tumors from patients with complete response to BCG at study inclusion (BCG-responders) and 2) BCG-naïve tumors with matching muscle-invasive bladder cancer (MIBC) recurrences during BCG therapy (BCG-failures). Philips OncoSignal analysis was performed to quantify functional signal transduction pathway activities. Molecular subtyping was performed using non-muscle invasive and muscle-invasive classifiers. Clinical and molecular subtyping results were combined with Philips OncoSignal to explore differences between BCG responders and BCG failures and between molecular subtypes. OncoSignal was performed in 20 tumors: 14 BCG-naïve (7 BCG-responders vs 7 BCG-failures) and 6 MIBC recurrences. Median follow-up for BCG-responders was 99 months (IQR 74-120); median follow-up for BCG-failures was 49 months (IQR 25-79), with a median time to progression of 14 months (IQR 10-41). Based on pilot data, MAPK and TGFβ pathway activity was significantly higher in tumors from BCG failures vs BCG-responders (both p Citation Format: Florus C. De Jong, Teemu D. Laajala, Robert F. Hoedemaeker, Sébastien Rinaldetti, Jolien T. Mensink, Angelique C. Van Der Made, Deric K. Van der Schoot, Egbert R. Boevé, Ellen C. Zwarthoff, Joost L. Boormans, Dan Theodorescu, James C. Costello, Tahlita C. Zuiverloon. Differential pathway analyses of BCG-treated T1HG bladder cancer using Philips OncoSignal: A pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 615.

Gemcitabine and cisplatin (GC) combination neoadjuvant chemotherapy (NAC) is standard of care for patients with muscle-invasive bladder cancer (MIBC). This combination is preferred due to its favorable toxicity profile. Approximately 25% of NAC treated patients will experience complete pathological response (pT0) at time of radical cystectomy (RC). NAC-treated patients with residual disease at RC have poorer prognosis compared to those who achieved pT0. Here we systematically defined the in vitro functional determinants of resistance to gemcitabine and cisplatin in bladder cancer. To accomplish this, we performed molecular profiling and functional genomic screening on a panel of bladder cancer cell lines (KU-19-19, T24, TCCSUP, 253J and 5637), each having the parental cell line with matched gemcitabine-, cisplatin-, and dual GC-resistant derivatives. We performed whole exome sequencing, RNA sequencing, and phospho-proteomic profiling on these 20 cell lines. To identify chemogenetic interactions that govern sensitivity or resistance to these agents, we performed whole genome CRISPR knockout screening in the GC-resistant cells both in the presence and absence of GC. We identified several novel genes that when inactivated are capable of sensitizing GC resistant cell lines to the two agents. The majority of shared chemosensitizing genes fall into several DNA damage response pathways. This is consistent with the established mechanism of action of GC chemotherapy and observations that have been made from clinical cohorts, where loss-of-function mutations in these pathways correlate with response to platinum-based chemotherapy. Notably, our work has identified several druggable candidates that could serve as potential targets for enhancing responses rates to GC. Interestingly, comparing these candidate genes and pathways with those differentially expressed in the RNA sequencing and proteomic profiling showed limited overlap. This work provides a comprehensive view of the functional determinants of GC sensitivity in bladder cancer. In support of and expanding on previous studies, we identified additional DNA damage response genes that when lost enhance sensitivity to GC. These genes should be further explored for their utility as predictive biomarkers of response to GC-based therapy in clinical cohorts. Importantly, a number of these could be targeted in combination with GC-based NAC to potentially improve response. Citation Format: Robert T. Jones, Tahlita C. Zuiverloon, Hedvig Vekony, Andrew Goodspeed, Teemu D. Laajala, Molishree Joshi, Colin Sempeck, Annie Jean, Megan Tu, Julia D. Wulfkuhle, Emanuel F. Petricoin, James C. Costello, Dan Theodorescu. Functional genomic screening in gemcitabine and cisplatin resistant bladder cancer cell lines identifies DNA repair pathways as mediators of chemosensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4091.

Neoadjuvant platinum-based chemotherapy (NAC) followed by radical cystectomy is the preferred first-line option for treating patients diagnosed with muscle-invasive bladder cancer (MIBC). One of the two most commonly utilized chemotherapy regimens for MIBC consists of combination therapy with gemcitabine and cisplatin (GC). Importantly, cisplatin-based chemotherapy leads to complete response (pT0) in approximately 25% of patients, while the remaining 75% are left with residual disease. For MIBC patients, the presence of residual disease at cystectomy is associated with significantly poorer prognosis. Previous efforts to screen tumor-derived specimens have identified a number of mutational biomarkers associated with NAC response; however, to date these efforts have been limited by the number of patients profiled and the molecular and mutational heterogeneity intrinsic to bladder cancers. Moreover, these efforts have primarily relied on a single technology (e.g., whole-exome sequencing or transcriptome profiling), and were not designed to look at GC-resistance patterns in a way that integrates these data types. Therefore, we sought to systematically define the molecular phenotypes associated with chemoresistant bladder cancer cells, and to functionally interrogate these features to better define essential genes and pathways that govern chemoresponsiveness. To accomplish this, we have extensively characterized a panel of six bladder cancer cell lines (KU-19-19, T24, TCCSUP, 253J, 5637, and RT112), each of which has three derivative cell lines with acquired resistance to gemcitabine, cisplatin, and GC combination. For all of these cell lines we have performed whole-transcriptome mRNA sequencing, whole-exome sequencing, and reverse phase protein microarray. Additionally, we have performed functional genomic screening using whole-genome CRISPR knockout libraries, in the combination GC-resistant derivative cell lines. Using these data we identified multiple important chemoresistance genes and pathways, both previously known and novel. Interestingly, even though we found heterogeneous expression patterns seen across different –omic platforms, the functional genomic screens in GC-resistant cell lines converged on many genes and pathways that are essential to DNA damage response. These data suggest that functional genomic screens are perhaps more likely to identify genes and pathways that are directly linked to each of these drugs' specific mechanisms of action, as opposed to context-specific molecular differences seen across cell lines. Importantly, these results could enable improved prediction and personalization of therapy through identifying biomarkers that predict chemosensitivity and resistance. This study also suggests that identifying more generalizable strategies to overcome chemoresistance could increase the number of bladder cancer patients experiencing deeper and more complete responses to these common chemotherapeutic drugs. Citation Format: Robert T. Jones, Tahlita C. M. Zuiverloon, Hedvig Vekony, Andrew Goodspeed, Teemu D. Laajala, Molishree Joshi, Colin Sempeck, Annie Jean, Megan Tu, Julia D. Wulfkuhle, Emanuel F. Petricoin, James C. Costello, Dan Theodorescu. Multi-omic interrogation of gemcitabine and cisplatin-resistant bladder cancer cell lines identifies unique and shared mediators of chemosensitivity and resistance [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 B11.

Objective: Fibroblast growth factor receptor 3 (FGFR3) is a potentially actionable target in bladder cancer (BC). FGFR3 mutations are associated with favorable prognosis in non-muscle invasive (NMI) BC and MIBC. Overexpression of FGFR3 was reported in up to 40% of FGFR3 wild-type MIBC. p53 alterations rarely coincide with FGFR3 mutations. We analyzed FGFR3 mutations and protein-expression of FGFR3 and p53 and assessed their prognostic value in a multicenter, multilaboratory setting. Methods: We included 1,000 cN0M0, chemotherapy-naive patients who underwent radical cystectomy (RC) with pelvic node dissection. Specimens were reviewed by eight uropathologists. At seven laboratories, FGFR3 mutation status was examined using PCR-SNaPshot. p53 and FGFR3 expression were determined by immunohistochemistry (IHC). FGFR3 mutation status, p53 and FGFR3 protein expression were correlated to each other, clinicopathologic parameters, and disease-specific survival (DSS). Results: FGFR3 mutations were found in 107/1,000 RCs (11%), of which 67 were S249C. Overexpression of FGFR3 was found in 279/1,000 (28%) of tumors. p53 overexpression (cut-off>10%) was found in 638/926 (69%) of available cases. Among FGFR3 mutant tumors, 73% had FGFR3 overexpression. Among FGFR3 wild-type tumors, 22% had FGFR3 overexpression. FGFR3 mutations were associated with lower pT-stage (P Conclusions: FGFR3 mutations identified patients with favorable BC with fewer p53 alterations at RC. FGFR3 overexpression was not associated with DSS in patients with FGFR3 wild-type tumors. Our results suggest that FGFR3 mutations (driver effect) have a distinct functional role than FGFR3 overexpression (passenger effect). Hence, patients with FGFR3 mutations may be more likely to benefit from anti-FGFR3 therapy than patients with only overexpression of FGFR3. Citation Format: Bas van Rhijn, Laura Mertens, Roman Mayr, Peter Bostrom, Mirari Marques, Geert van Leenders, Stefanie Gotz, Michiel van der Heijden, Michael Jewett, Francisco Real, Robert Stohr, Alexandre Zlotta, Markus Eckstein, Yanish Soorojebally, Max Burger, Wolfgang Otto, Francois Radvanyi, Damien Pouessel, Theo van der Kwast, Nuria Malats, Arndt Hartmann, Yves Allory, Deric van der Schoot, Ellen Zwarthoff, Tahlita Zuiverloon. FGFR3 mutations and their relation to FGFR3 expression and clinical outcome in a large radical cystectomy cohort: Implications for anti-FGFR3 bladder cancer treatment? [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 B23.

Introduction: High-risk non-muscle invasive bladder cancer (HR-NMIBC) patients represent a challenging group as they have the highest risk of treatment failure despite transurethral tumor resection (TUR) and adjuvant intravesical Bacillus Calmette-Guérin (BCG) instillations. Treatment failure is associated with an increased risk of progression and unnecessary exposure to toxicity of BCG, while continuing BCG shortage demands selective use of limited resources. The current risk stratification is insufficient at predicting BCG failure in HR-NMIBC patients. Therefore, we aim to investigate whether pathologic depth of tumor invasion (i.e., T1 substaging) is able to improve patient stratification. Methods: HR-NMIBC patients who received a TUR and at least induction BCG (5 of 6 instillations) were included at four European centers between 2000−2016. Clinicopathologic data were collected and all diagnostic TURs, re-TURs, and subsequent TURs of tumor recurrences were reviewed, which included T1 substaging (micro vs. extensive [pT1e]). Primary endpoint was BCG failure according to EAU guidelines. Progression was defined as muscle-invasive and/or metastatic disease. Logistic regression analysis and Cox regression models were used to identify predictive markers of BCG failure on diagnostic TUR and first recurrence. Results: N=513 HR-NMIBC patients were included: median age 70 years, 78% men. Median follow-up was 52 months. In total, 316 (62%) patients had pT1 disease, 144 (28%) pTa, and 53 (10%) pTis. BCG failure occurred in 28% (18% progression, 6% metastasis). Median time to progression was 18 months. Strongest predictors of BCG failure on diagnostic TUR were pT1e substaging (HR: 2.1; 95% CI 1.1–4.0; p=0.023), concomitant carcinoma in situ (CIS) (HR: 3.0; 95% CI 1.5–5.7; p=0.003), and lymphovascular invasion (LVI) (HR: 3.3, 95% CI 1.0–11, p=0.05). pT1e substaging combined with either concomitant CIS or LVI increased the risk of BCG failure (HR: 3.6; 95% CI 1.8–6.9; p Conclusion: Substaging has the potential to improve the current risk stratification model by identification of HR-NMIBC patients at the highest risk of BCG failure and is specifically able to identify patients at high risk of progression. Substaging can be performed by every pathologist with minimal additional training required. Combined with other pathologic markers, substaging can be used as a stratification tool for early cystectomy or to include patients in clinical trials. Citation Format: Florus C. de Jong, Robert F. Hoedemaeker, Jolien T.M. Mensink, Vebjørn Kvikstad, Egbert R. Boevé, Deric K.E. van der Schoot, Ellen C. Zwarthoff, Tahlita C.M. Zuiverloon. Pathologic T1 substaging improves the identification of high-risk non-muscle invasive bladder cancer patients at risk of BCG failure [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 B06.

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Background Bladder cancer (BC) cell lines are indispensable in basic and preclinical research. Currently, an up-to-date and comprehensive overview of available BC cell lines is not available. Objective To provide an overview and resources on the origin, pathological and molecular characteristics of commonly used human, murine and canine BC cell lines. Methods A PubMed search was performed for relevant articles published between 1980 and 2017 according to the following MeSH terms: cell line; cell line, tumor; urinary bladder neoplasms; carcinoma, transitional cell. The Cellosaurus database was searched, using the term "bladder" and/or "urothelial carcinoma". We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Results We provide information on 157 human, murine and canine BC cell lines. 103 human BC cell lines have molecular data available, of which 69 have been profiled by at least one "omic" technology. We outline how these cell lines are currently being used for in vitro and in vivo experimental models. These results allow direct comparison of BC cell lines to patient samples, providing information needed to make informed decisions on the most genomically appropriate cell line to answer research questions. Furthermore, we show that cross-contamination remains an issue and describe guidelines for prevention. Conclusions In the BC field, multiple human, murine and canine BC cell lines have been developed and many have become indispensable for in vitro and in vivo research. High-throughput -omic technologies have dramatically increased the amount of molecular data on these cell lines. We synthesized a comprehensive overview of these data as a resource for the BC scientific community.

Survival of patients with muscle-invasive bladder cancer is poor and new therapies are needed. Currently, none of the targeted agents that are approved for cancer therapy have been approved for the treatment of bladder cancer and the few clinical trials that have been performed had limited success, often owing to a lack of efficacy and toxic effects. However, many other novel targeted agents have been investigated in animal models of bladder cancer. EGFR, FGFR-3, VEGF, mTOR, STAT3, the androgen receptor and CD24 are molecular targets that could be efficiently inhibited, resulting in reduced tumour growth, and that have been investigated in multiple independent studies. Several other targets, for example COX-2, IL-12, Bcl-xL, livin and choline kinase a, have also been observed to inhibit tumour growth, but these findings have not been replicated to date. Limitations of several studies include the use of cell lines with mutations downstream of the target, providing resistance to the tested therapy. Furthermore, certain technologies, such as interfering RNAs, although effective in vitro, are not yet ready for clinical applications. Further preclinical research is needed to discover and evaluate other possible targets, but several validated targets are now available to be studied in clinical trials.

Recent advances in next-generation sequencing techniques have greatly improved our understanding of the genomic alterations in bladder cancer. Cisplatin-based chemotherapy provides a viable treatment option in the neoadjuvant, adjuvant and metastatic setting in a selected group of patients, but chemoresistance is a major problem. The underlying mechanisms of treatment resistance are poorly understood and elucidating these pathways will subsequently lead to improved patient selection, less unnecessary drug-related toxicity, improved patient outcome and decreased healthcare costs. This review provides an overview of mechanisms of chemoresistance and describes the current knowledge on how the genomic landscape influences therapy outcome in muscle-invasive bladder cancer patients.

BACKGROUND: Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. OBJECTIVES: To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). DESIGN, SETTING, AND PARTICIPANTS: A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and 21 mo, respectively). Voided urine from patients with suspicion of incident UBC (n=174), or under surveillance after diagnosis of non-muscle-invasive UBC (n=194), was tested using a SNaPshot assay. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival. RESULTS AND LIMITATIONS: In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p=0.0002). There was no association between TERT mutations and mRNA expression (p=0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature. CONCLUSIONS: Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences. Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression.

We determined whether FGFR3 mutation analysis of voided urine samples would be cost-effective to partly replace cystoscopy in the surveillance of patients treated for nonmuscle invasive urothelial carcinoma.In this decision analytical study we analyzed data on 70 Dutch patients with FGFR3 positive primary tumors and a median followup of 8.8 years. Surveillance strategies were compared in a Markov model. Modified surveillance consisted of FGFR3 mutation analysis of voided urine samples every 3 months, and cystoscopy at 3, 12 and 24 months. Standard surveillance was defined as cystoscopy every 3 months and minimal surveillance was defined as cystoscopy at 3, 12 and 24 months. Analysis was stratified for 3 risk profiles, including surveillance after 1) the primary tumor, 2) the first to third recurrence and 3) the fourth recurrence or more. Sensitivity analysis was performed to evaluate the impact of variations in cost, sensitivity and specificity.The probability of no recurrence after 2 years of surveillance after a primary tumor was higher for modified surveillance than for standard and minimal surveillance, eg after primary tumors (95.7% vs 95.0% and 93.9%, respectively). The total cost of surveillance after the primary tumor was lower for minimal and modified surveillance (€2,254 and €2,558, respectively) than for standard surveillance (€5,861). Results were robust to changing inputs over plausible ranges and consistent for each of the 3 risk profiles.Surveillance in which cystoscopy is partly replaced by FGFR3 mutation analysis of urine seems a safe, effective and cost-effective surveillance strategy. Further validation in larger cohorts is required.

Study Type - Prognosis (case series) Level of Evidence 4 What's known on the subject? and What does the study add? The stakes are high when making treatment decisions in T1 bladder cancer (BC). Conservative management may lead to progression and possibly death from BC. Conversely, radical cystectomy could be over-treatment of non-progressive disease. The problem for clinicians is that reliable prognostic indices are lacking. We performed a head-to-head comparison of two substaging systems, European Organisation for the Research and Treatment of Cancer (EORTC) risk scores and four molecular markers in T1 carcinomas of the bladder treated conservatively with BCG. T1 sub-stage according to a new system (micro-invasive [T1m] and extensive-invasive [T1e]) was the most important clinical variable for predicting progression to carcinoma invading bladder muscle. The performance of the EORTC risk scores was disappointing for this T1 sub-group. Molecular markers were not significant in multivariable analysis for predicting progression. Future studies may lead to the incorporation of sub-stage (T1m/T1e) in the TNM classification system for urinary BC to guide clinical decision-making in T1 BC. OBJECTIVE To evaluate the prognostic significance of four molecular markers, sub-stage and European Organisation for the Research and Treatment of Cancer (EORTC) risk scores in primary T1 bladder cancer (BC) treated with adjuvant bacille Calmette-Guerin. PATIENTS AND METHODS The slides of 129 carcinomas of the bladder from two university hospitals were reviewed and the T1 diagnosis was confirmed. T1 sub-staging was done in two separate rounds, using a new system that identifies micro-invasive (T1m) and extensive-invasive (T1e) T1BC, and then according to invasion of the muscularis mucosae (T1a/T1b/T1c). The EORTC risk scores for recurrence and progression were calculated. Uni- and multivariable analyses for recurrence and progression were performed using clinicopathological variables, T1 sub-stage, EORTC risk scores and molecular markers (fibroblast growth factor receptor 3 gene mutation and Ki-67, P53, P27 expression). RESULTS The median follow-up was 6.5 years. Forty-two patients remained recurrence-free (33%). Progression to T2 or metastasis was observed in 38 (30%) patients. In multivariable analysis for recurrence, multiplicity was significant. In multivariable analysis for progression, female gender, sub-stage (T1m/T1e) and carcinoma in situ (CIS) were significant. Molecular markers were significant in univariable and in multivariable analyses for recurrence. EORTC risk scores were not significant. CONCLUSIONS CIS, female gender and sub-stage (T1m/T1e) were the most important variables for progression. The additional value of molecular markers was modest. Sub-stage (T1m/T1e) could potentially be incorporated in future tumour-node-metastasis classifications.

Background: Multiple clinical risk factors and genetic profiles have been demonstrated to predict progression of non-muscle invasive bladder cancer; however, no easily clinical applicable gene signature has been developed to predict disease progression independent of disease stage and grade. Methods: We measured the intra-patient variation of an 88-gene progression signature using 39 metachronous tumours from 17 patients. For delineation of the optimal quantitative reverse transcriptase PCR panel of markers, we used 115 tumour samples from patients in Denmark, Sweden, UK and Spain. Results: Analysis of intra-patient variation of the molecular markers showed 71% similar classification results. A final panel of 12 genes was selected, showing significant correlation with outcome. In multivariate Cox regression analysis, we found that the 12-gene signature was an independent prognostic factor (hazard ratio=7.4 (95% confidence interval: 3.4–15.9), P Conclusion: Intra-patient marker variation in metachronous tumours is present. Therefore, to increase test sensitivity, it may be necessary to test several metachronous tumours from a patient’s disease course. A PCR-based 12-gene signature significantly predicts disease progression in patients with non-muscle invasive bladder cancer. Background:Multiple clinical risk factors and genetic profiles have been demonstrated to predict progression of non-muscle invasive bladder cancer; however, no easily clinical applicable gene signature has been developed to predict disease progression independent of disease stage and grade.Methods:We measured the intra-patient variation of an 88-gene progression signature using 39 metachronous tumours from 17 patients. For delineation of the optimal quantitative reverse transcriptase PCR panel of markers, we used 115 tumour samples from patients in Denmark, Sweden, UK and Spain.Results:Analysis of intra-patient variation of the molecular markers showed 71% similar classification results. A final panel of 12 genes was selected, showing significant correlation with outcome. In multivariate Cox regression analysis, we found that the 12-gene signature was an independent prognostic factor (hazard ratio=7.4 (95% confidence interval: 3.4-15.9), P

Context: Currently, bacillus Calmette-Guerin (BCG) intravesical instillations are standard treatment for patients with high-grade non-muscle-invasive bladder cancer; however, no markers are available to predict BCG response. Objective: To review the contemporary literature on markers predicting BCG response, to discuss the key issues concerning the identification of predictive markers, and to provide recommendations for further research studies. Evidence acquisition: We performed a systematic review of the literature using PubMed and Embase databases in the period 1996-2010. The free-text search was extended by adding the following keywords: recurrence, progression, survival, molecular marker, prognosis, TP53, Ki-67, RB, fibronectin, immunotherapy, cytokine, interleukin, natural killer, macrophage, PMN, polymorphism, SNP, single nucleotide polymorphism, and gene signature. Evidence synthesis: If thresholds for the detection of urinary interleukin (IL)-8, IL-18, and tumour necrosis factor apoptosis-inducing ligand levels are standardised, measurement of these cytokines holds promise in the assessment of BCG therapy outcome. Studies on immunohistochemical markers (ie, TP53, Ki-67, and retinoblastoma) display contradictory results, probably because of the small patient groups that were used and seem unsuitable to predict BCG response. Exploring combinations of protein levels might prove to be more helpful to establish the effect of BCG therapy. Single nucleotide polymorphisms, either in cytokines or in genes involved in DNA repair, need to be investigated in different ethnicities before their clinical relevance can be determined. Measurement of urinary IL-2 levels seems to be the most potent marker of all the clinical parameters reviewed. Conclusions: IL-2 levels are currently the most promising predictive markers of BCG response. For future studies focusing on new biomarkers, it is essential to make more use of new biomedical techniques such as microRNA profiling and genomewide sequencing. (C) 2011 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Purpose: FGFR3 mutations occur in 70% of nonmuscle invasive bladder tumors. Although urine based FGFR3 mutation analysis can detect recurrence, its sensitivity may be limited if samples have few or no tumor cells. We determined whether test sensitivity depends on tumor size and the time point of urine collection, and how to increase sensitivity. Materials and Methods: A total of 440 urine samples from 18 patients with a suspicious bladder lesion at cystoscopy were collected during 6 days before surgery. Eight patients (300 samples) had an FGFR3 mutant tumor, including 4 each with a tumor greater than 3 and less than 1.5 cm. Polymerase chain reaction based FGFR3 analysis was done on all tumors and urine samples. Results: FGFR3 mutations were detected in 257 of the 300 urine samples (86%) from patients with an FGFR3 mutant tumor. Assay sensitivity was 100% for tumors greater than 3 cm and 75% for tumors less than 1.5 cm. It increased to 100% in patients with a less than 1.5 cm tumor when samples were pooled during 24 hours. Sensitivity was not influenced by the time of urine collection. All urine samples from patients with an FGFR3 wild-type tumor were negative for FGFR3 mutation. Conclusions: The sensitivity of tumor detection increased with tumor size. FGFR3 assay sensitivity depends on the number of shed tumor cells and improves by increasing urine volume. These findings suggest that there is an upper limit to the sensitivity of the FGFR3 assay when 1 urine sample is analyzed. This may also apply to other DNA or RNA based assays.

What's known on the subject? and What does the study add? Multiple studies report on the detection of methylation in voided urine samples as a possible approach for the follow-up of non-muscle invasive bladder cancer patients. Previous studies analyze methylation gene panels in a mixture of primary and recurrent tumours. As primary tumours are larger than recurrent tumours and thus easier to detect in urine, validation of methylation markers in urine samples from patients with primary tumours will result in a test sensitivity that does not reflect the true sensitivity of the assay. This study is the first to select a subset of genes specifically methylated in non-muscle invasive bladder cancer recurrences and validates the gene panel in two independent sets of urine samples from recurrent patients, thus simulating the disease course according to the clinical presentation. OBJECTIVE • To develop a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay for the detection of non-muscle invasive bladder cancer (NMIBC) recurrences in voided urine. PATIENTS AND METHODS • Genes frequently methylated in NMIBC tumours (n= 37) were selected to develop a BC-specific MS-MLPA assay. • Genes methylated in blood from patientswith BC (n= 29) and genes methylated in urine from patients with no history of BC (n= 46) were excluded. • A four-gene panel with the highest predictive value was selected from the initial assay. This four-gene panel was tested and validated on urine from patients with a histologically confirmed recurrence (n= 68 test set; n= 49 validation set) and urine samples from patients without BC (n= 91, test set) and urine from recurrence-free BC (rec-free BC) patients (n= 60, validation set). • A model was developed to predict the probability of having a recurrence based on methylation of the four-gene panel and a threshold probability with the highest sensitivity and specificity was determined. • The outcome of the model was validated on BC urine samples (n= 65) and on urine samples from rec-free BC patients (n= 29). RESULTS • The BC MS-MLPA assay consisted of 23 methylation probes. The selected four-gene panel included: APC_a, TERT_a, TERT_b, and EDNRB. This panel reached an area under the receiver operating characteristic curve (AUC) of 0.82 (test set) and AUC 0.69 (validation set). Sensitivity and specificity for the detection of a concomitant tumour were 63.3% and 58.3% respectively (test set) and 72.3% and 55.2%, respectively (validation set). CONCLUSIONS • We have developed a methylation detection assay specifically for the detection of recurrences in patients with NMIBC in voided urine. • The findings are promising and improvement of this test could eventually contribute to a more individualized patient friendly surveillance.

Purpose: Mutations in the fibroblast growth factor receptor 3 (FGFR3) have been found in 70% of the low-grade non-muscle–invasive bladder cancer (NMI-BC) tumors. We aim to determine the potential of FGFR3 mutation analysis on voided urine to detect recurrences during surveillance of patients with low-grade NMI-BC. Experimental Design: FGFR3 mutation status of the study inclusion tumor was determined from 200 low-grade NMI-BC patients. Patients with an FGFR3-mutant inclusion tumor were selected for analysis and monitored by cystoscopy, and voided urine samples were collected. FGFR3 mutation analysis was done on 463 prospectively collected urines. Sensitivity and predictive value of the assay were determined for detection of concomitant recurrences. Longitudinal and Cox time-to-event analyses were done to determine the predictive value for detection of future recurrences. Results: Median follow-up was 3.5 years. The sensitivity of the assay for detection of concomitant recurrences was 26 of 45 (58%). Of the 105 positive urine samples, 85 (81%) were associated with a concomitant or a future recurrence. An FGFR3-positive urine was associated with a 3.8-fold (P < 0.0001) higher risk of having a recurrence in the Cox analysis. In contrast, only 41 of 358 (11%) FGFR3-negative urine samples were associated with a recurrence. Positive predictive value increased from 25% to 90% in patients having consecutive FGFR3-positive urine tests. Conclusions: FGFR3 mutation analysis on voided urine is a simple and noninvasive diagnostic method for detection of recurrences during surveillance of patients presenting with a low-grade FGFR3-mutant NMI-BC tumor. Clin Cancer Res; 16(11); 3011–8. ©2010 AACR.

Contains fulltext : 170971.pdf (Publisher’s version ) (Closed access) RATIONALE: Assessment of patients with asymptomatic microhematuria (aMh) has been a challenge to urologists for decades. The aMh is a condition with a high prevalence in the general population and also an established diagnostic indicator of bladder cancer. Acknowledging aMh needs to be assessed within a complex context, multiple guidelines have been developed to identify individuals at high risk of being diagnosed with bladder cancer. MATERIAL & METHODS: This structured review and consensus of the International Bladder Cancer Network (IBCN) identified and examined 9 major guidelines. These recommendations are partly based on findings from a long-term study on the effects of home dipstick testing, but also on the assumption that early detection of malignancy might be beneficial. RESULTS: Despite similar designs, these guidelines differ in a variety of parameters including definition of aMh, rating of risks, use of imaging modalities, and the role of urine cytology. In addition, recommendations for further follow-up after negative initial assessment are controversial. In this review, different aspects for aMh assessment are analyzed based upon the evidence currently available. DISCUSSION: We question whether adherence to the complicated algorithms as recommended by most guidelines is practical for routine use. Based upon a consensus, the authors postulate a need for better tools. New concepts for risk assessment permitting improved risk stratification and prepone cystoscopy before refined imaging procedures (computed tomography scan and magnetic resonance imaging) are suggested.