A luminal non-coding RNA-based genomic classifier confirms favourable outcomes in patients with clinically organ-confined bladder cancer treated with radical cystectomy.

OBJECTIVE: To further evaluate a genomic classifier (GC) in a cohort of patients undergoing radical cystectomy (RC), as long non-coding RNA (lncRNA)-based genomic profiling has suggested utility in identifying a distinct tumour subgroup corresponding to a favourable prognosis in patients with bladder cancer. PATIENTS AND METHODS: Transcriptome-wide expression profiling using Decipher Bladder was performed on transurethral resection of bladder tumour samples from a cohort of patients with high-grade, clinically organ-confined (cTa-T2N0M0) urothelial carcinoma (UC) who subsequently underwent RC without any neoadjuvant therapy (n = 226). The lncRNA-based luminal favourable status was determined using a previously developed GC. The primary endpoint was overall survival (OS) after RC. Secondary endpoints included cancer-specific mortality and upstaging at RC. RESULTS: In the study, 134 patients were clinical non-muscle-invasive bladder cancer (cTa/Tis/T1) and 92 patients were cT2. We identified 60 patients with luminal favourable subtype, all of which showed robust gene expression patterns associated with less aggressive bladder cancer biology. On multivariate analysis, patients with the luminal favourable subtype (vs without) were significantly associated with lower odds of upstaging to pathological (p)T3+ disease (odds ratio [OR] 0.32, 95% confidence interval [CI] 0.12-0.82; P = 0.02), any upstaging (OR 0.41, 95% CI 0.20-0.83; P = 0.01), and any upstaging and/or pN+ (OR 0.50, 95% CI 0.25-1.00; P = 0.05). Luminal favourable bladder cancer was significantly associated with better OS (hazard ratio 0.33, 95% CI 0.15-0.74; P = 0.007). CONCLUSIONS: This study validates the performance of the GC for identifying UCs with a luminal favourable subtype, harbouring less aggressive tumour biology.

Non-muscle-invasive micropapillary bladder cancer has a distinct lncRNA profile associated with unfavorable prognosis.

BACKGROUND: Molecular subtyping of bladder cancer has revealed luminal tumors generally have a more favourable prognosis. However, some aggressive forms of variant histology, including micropapillary, are often classified luminal. In previous work, we found long non-coding RNA (lncRNA) expression profiles could identify a subgroup of luminal bladder tumors with less aggressive biology and better outcomes. OBJECTIVE: In the present study, we aimed to investigate whether lncRNA expression profiles could identify high-grade T1 micropapillary bladder cancer with differential outcome. DESIGN, SETTING, AND PARTICIPANTS: LncRNAs were quantified from RNA-seq data from a HGT1 bladder cancer cohort that was enriched for primary micropapillary cases (15/84). Unsupervised consensus clustering of variant lncRNAs identified a three-cluster solution, which was further characterised using a panel of micropapillary-associated biomarkers, molecular subtypes, gene signatures, and survival analysis. A single-sample genomic signature was trained using lasso-penalized logistic regression to classify micropapillary-like gene-expression, as characterised by lncRNA clustering. The genomic classifier (GC) was tested on luminal tumors derived from the TCGA cohort (N = 202). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Patient and tumor characteristics were compared between subgroups by using X2 tests and two-sided Wilcoxon rank-sum tests. Primary endpoints were overall, progression-free and high-grade recurrence-free survival, calculated as the date of high-grade T1 disease at TURBT till date of death from any cause, progression, or recurrence, respectively. Survival rates were estimated using weighted Kaplan-Meier (KM) curves. RESULTS AND LIMITATIONS: Primary micropapillary HGT1 showed decreased FGFR3, SHH, and p53 pathway activity relative to tumors with conventional urothelial carcinoma. Many bladder cancer-associated lncRNAs were downregulated in micropapillary tumors, including UCA1, LINC00152, and MALAT1. Unsupervised consensus clustering resulted in a lncRNA cluster 1 (LC1) with worse prognosis that was enriched for primary micropapillary histology and the Luminal Unstable (LumU) molecular subtype. Interestingly, LC1 appeared to better identify aggressive HGT1 disease, compared to stratifying outcomes using primary histologic characteristics. A signature trained to identify LC1 cases showed good performance in the testing cohort, identifying seven cases with significantly worse survival (p < 0.001). Limitations include the retrospective nature of the study and the lack of a validation cohort. CONCLUSIONS: Using the lncRNA transcriptome we identified a subgroup of aggressive HGT1 bladder cancer that was enriched with micropapillary histology. These data suggest that lncRNAs can facilitate the identification of aggressive micropapillary-like tumors, potentially improving patient management.

Patients with Muscle-Invasive Bladder Cancer with Nonluminal Subtype Derive Greatest Benefit from Platinum Based Neoadjuvant Chemotherapy.

PURPOSE: Neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC) in patients with nonmetastatic muscle-invasive bladder cancer (MIBC) confers an absolute survival benefit of 5%-10%. There is evidence that molecular differences between tumors may impact response to therapy, highlighting a need for clinically validated biomarkers to predict response to NAC. MATERIALS AND METHODS: Four bladder cancer cohorts were included. Inverse probability weighting was used to make baseline characteristics (age, sex and clinical tumor stage) between NAC-treated and untreated groups more comparable. Molecular subtypes were determined using a commercial genomic subtyping classifier. Survival rates were estimated using weighted Kaplan-Meier curves. Cox proportional hazards models were used to evaluate the primary and secondary study end points of overall survival (OS) and cancer-specific survival, respectively. RESULTS: A total of 601 patients with MIBC were included, of whom 247 had been treated with NAC and RC, and 354 underwent RC without NAC. With NAC, the overall net benefit to OS and cancer-specific survival at 3 years was 7% and 5%, respectively. After controlling for clinicopathological variables, nonluminal tumors had greatest benefit from NAC, with 10% greater OS at 3 years (71% vs 61%), while luminal tumors had minimal benefit (63% vs 65%) for NAC vs non-NAC. CONCLUSIONS: In patients with MIBC, a commercially available molecular subtyping assay revealed nonluminal tumors received the greatest benefit from NAC, while patients with luminal tumors experienced a minimal survival benefit. A genomic classifier may help identify patients with MIBC who would benefit most from NAC.

RNA-based urinary assays for non-muscle invasive bladder cancer.

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.

T1 Substaging of Nonmuscle Invasive Bladder Cancer is Associated with bacillus Calmette-Guérin Failure and Improves Patient Stratification at Diagnosis.

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.

Evaluation of the New American Urological Association Guidelines Risk Classification for Hematuria.

PURPOSE: Microhematuria is a prevalent condition and the American Urological Association has developed a new risk-stratified approach for the evaluation of patients with microhematuria. Our objective was to provide the first evaluation of this important guideline. MATERIALS AND METHODS: This multinational cohort study combines contemporary patients from 5 clinical trials and 2 prospective registries who underwent urological evaluation for hematuria. Patients were stratified into American Urological Association risk strata (low, intermediate or high risk) based on sex, age, degree of hematuria, and smoking history. The primary end point was the incidence of bladder cancer within each risk stratum. RESULTS: A total of 15,779 patients were included in the analysis. Overall, 727 patients (4.6%) were classified as low risk, 1,863 patients (11.8%) were classified as intermediate risk, and 13,189 patients (83.6%) were classified as high risk. The predominance of high risk patients was consistent across all cohorts. A total of 857 bladder cancers were diagnosed with a bladder cancer incidence of 5.4%. Bladder cancer was more prevalent in men, smokers, older patients and patients with gross hematuria. The cancer incidence for low, intermediate and high risk groups was 0.4% (3 patients), 1.0% (18 patients) and 6.3% (836 patients), respectively. CONCLUSIONS: The new risk stratification system separates hematuria patients into clinically meaningful categories with differing likelihoods of bladder cancer that would justify evaluating the low, intermediate and high risk groups with incremental intensity. Furthermore, it provides the relative incidence of bladder cancer in each risk group which should facilitate patient counseling regarding the risks and benefits of evaluation for bladder cancer.

A Genomic Classifier for Predicting Clinically Aggressive Luminal Bladder Tumors with Higher Rates of Pathological Up Staging.

PURPOSE: Urothelial carcinoma of the luminal molecular subtype is associated with lower rates of pathological up staging from clinical stage T1-T2 to nonorgan confined (pT3 or greater and/or pN+) disease at radical cystectomy. However, approximately a third of luminal urothelial carcinoma cases were up staged to nonorgan confined disease, and these may be under treated if neoadjuvant chemotherapy is withheld. In this study we trained a genomic classifier to predict luminal nonorgan confined disease in patients diagnosed with clinically organ confined (cT1/T2) disease. MATERIALS AND METHODS: Specimens from transurethral resected high grade cT1-T2N0M0 urothelial carcinoma of the bladder that belonged to the luminal subtype (Seiler 2017) were randomly split into training (75) and testing (25) sets for the development of a single sample luminal up staging classifier using lasso/ridge-penalized logistic regression. All patients underwent radical cystectomy without neoadjuvant chemotherapy and the primary end point was up staging to nonorgan confined disease. A radical cystectomy cohort and a platinum treated neoadjuvant chemotherapy cohort were used to evaluate the luminal up staging classifier. RESULTS: Up staging to nonorgan confined disease occurred in 34% of luminal cases. The luminal up staging classifier predicted up staging in 32 of 34 cases, with 6 false-positives (AUC 0.96). The sensitivity for detection of luminal pN+ disease was 95% (20 of 21). Patients with predicted nonorgan confined luminal tumors had worse survival than those with organ confined luminal tumors (p=0.001). On multivariable analysis the luminal up staging classifier was a significant predictor of overall survival after adjusting for clinical variables available at transurethral resection. The luminal up staging classifier also predicted overall survival for aggressive luminal TCGA (The Cancer Genome Atlas) cases (n=83, p=0.043). In the neoadjuvant chemotherapy cohort the luminal up staging classifier predicted 9 up staging cases, all of which had excellent prognosis. CONCLUSIONS: A luminal up staging classifier was developed that distinguishes a subset of cT1-T2N0M0 luminal urothelial carcinoma cases at high risk for up staging to nonorgan confined disease at radical cystectomy and of death. Validation of this model in an independent, large patient cohort is necessary to determine how molecular stratification of luminal tumors could be used to guide treatment of these patients.

A Urine Based Genomic Assay to Triage Patients with Hematuria for Cystoscopy.

PURPOSE: Current clinical guidelines recommend cystoscopy in patients who present with hematuria to rule out a bladder tumor. We evaluated whether our previously developed urine assay was able to triage patients with hematuria for cystoscopy in a large prospective cohort. MATERIALS AND METHODS: A urine sample was collected before cystoscopy and mutation/methylation status of 6 genes was determined on cellular DNA. The existing diagnostic model was validated on this cohort. Logistic regression was applied to investigate other potential variables. The primary end point was the model performance as indicated by the AUC. Secondary end points were sensitivity, specificity and negative predictive value. Clinical usefulness was determined by the net benefit approach. RESULTS: In 838 patients biomarker status could be determined for all genes. Urothelial cancer was observed in 112 patients (98 of 457 in the gross and 14 of 381 in the microscopic hematuria group). Validation of the existing model resulted in an AUC of 0.93. Logistic regression analysis identified type of hematuria as a significant additional variable. Adding type of hematuria resulted in an AUC of 0.95 (96% sensitivity, 73% specificity, 99% negative predictive value). The assay identified all upper tract tumors not visible by cystoscopy (in 6). Net benefit analysis showed that the urine assay should be preferred over current clinical practice. Implementing the urine assay as a triage tool could lead to a 53% reduction in cystoscopies. CONCLUSIONS: The urine assay detected urothelial cancer with a very high accuracy and can be used to triage patients presenting with hematuria for cystoscopy.

Correction to: Genomic Subtyping in Bladder Cancer.

The original version of this article contained a mistake. The included Conflict of Interest statement was incorrect.

Genomic Subtyping in Bladder Cancer.

PURPOSE OF REVIEW: Molecular characterization of cancer allows us to understand oncogenesis and clinical prognosis as well as facilitates development of biomarkers and treatment. Our aim was to review the current literature on genomic characterization of bladder cancer, and how far we are in implementing genomics into clinical practice. RECENT FINDINGS: Bladder cancers are molecularly diverse tumors with a high mutational rate. On molecular level, bladder cancer can be categorized into at least six subtypes called luminal-papillary, luminal-unstable, luminal non-specified, basal-squamous, neuroendocrine-like, and stroma-rich. These subtypes have characteristic genomic and transcriptomic profiles and appear to have different prognoses. Several molecular subtypes have been identified in bladder cancer. Prospective trials are underway to validate the applicability of genomic subtypes for clinical decision making. Further integrative analyses of genomic alterations, gene expression, epigenetics, and proteomics need to be performed before genomic subtyping can be attained in clinical practice.

Concordance of PD-L1 expression in matched urothelial bladder cancer specimens.

AIMS: Programmed death ligand 1 (PD-L1) expression has predictive value for response to immune-checkpoint inhibitor treatment in urothelial cancer patients. The consistency of PD-L1 expression among different specimen types, however, is unknown. The aim of this study is to compare PD-L1 expression in matched transurethral resections of the bladder (TURB), cystectomy specimens and lymph node metastases of urothelial cancer patients. METHODS AND RESULTS: We performed PD-L1 (SP142) immunohistochemistry on whole tissue slides of 115 urothelial carcinoma patients who had undergone TURB, followed by radical cystectomy and/or pelvic lymph node dissection. The PD-L1 assay was positive if PD-L1 expression in immune cells occupied ≥5% of the tumour area. PD-L1 was positive in 15 of 97 (15.5%) TURB, 17 of 98 (17.3%) cystectomies and nine of 49 (18.4%) lymph node metastases. Agreement of PD-L1 assay outcome between cystectomy and TURB (kappa = 0.34; P = 0.002) and cystectomy and lymph node metastasis (kappa = 0.35; P = 0.034) was fair; there was no agreement between TURB and lymph node metastasis (kappa = 0.045; P = 0.82). Discordance of PD-L1 outcome in matched TURB and cystectomy specimens occurred more frequently after neoadjuvant therapy (53.3% versus 25.4%; P = 0.03), and was not associated with other clinicopathological parameters. CONCLUSIONS: Urothelial bladder cancer patients showed fair agreement of PD-L1 assay outcome in cystectomies and matched TURB or lymph node specimens. PD-L1 expression was discordant more often after neoadjuvant therapy. Therefore, immune-checkpoint inhibitor studies should take into account specimen type and neoadjuvant therapy in assessing the predictive value of PD-L1 expression.

Upper tract urothelial cancer (UTUC) genomic profiling and correlation regarding benefit of platinum-based chemotherapy.

Upper tract urothelial cancer (UTUC) are rare subsets of urothelial cancer, which typically present with more aggressive course. Molecular markers stratifying urothelial tumors as luminal subtype and non-luminal subtype tumors have been proposed to select patients who may have greater or lesser benefit from neoadjuvant systemic therapy in bladder cancer, though not yet evaluated in UTUC. Here, a single-institution study retrospectively obtained clinical and genomic information in patients with UTUC and evaluated four patient tumors using the Decipher Bladder® assay and Foundation Medicine® test. All four patients had non-luminal molecular subtype including basal (N = 4) and mixed basal/claudin-low (N = 2) subtypes. The best clinical response achieved was stable disease in a patient who had basal/claudin-low subtype with residual ypT3 after neoadjuvant chemotherapy. For the remaining three patients, all were treated with platinum-based chemotherapy for eventual metastatic disease but all three showed progressive disease with limited overall survival, highlighting their aggressive course. The non-luminal subtype and lack of FGFR alteration may partly explain the poor overall outcomes while the real-world benefit of next generation sequencing for clinical use in UTUC patients require further clarification in a larger cohort study.

The Diagnostic Accuracy of Cystoscopy for Detecting Bladder Cancer in Adults Presenting with Haematuria: A Systematic Review from the European Association of Urology Guidelines Office.

CONTEXT: Haematuria can be macroscopic (visible haematuria [VH]) or microscopic (nonvisible haematuria [NVH]), and may be caused by a number of underlying aetiologies. Currently, in case of haematuria, cystoscopy is the standard diagnostic tool to screen the entire bladder for malignancy. OBJECTIVE: The objective of this systematic review is to determine the diagnostic test accuracy of cystoscopy (compared with other tests, eg, computed tomography, urine biomarkers, and urine cytology) for detecting bladder cancer in adults. EVIDENCE ACQUISITION: A systematic review of the literature was performed according to the Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy and Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) extension for diagnostic test accuracy studies' checklist. The MEDLINE, Embase, Cochrane CENTRAL, and Cochrane CDSR databases (via Ovid) were searched up to July 13, 2022. The population comprises patients presenting with either VH or NVH, without previous urological cancers. Two reviewers independently screened all articles, searched reference lists of retrieved articles, and performed data extraction. The risk of bias was assessed using Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2). EVIDENCE SYNTHESIS: Overall, nine studies were included in the qualitative analysis. Seven out of nine included trials covered the use of cystoscopy in comparison with radiological imaging. Overall, sensitivity of cystoscopy ranged from 87% to 100%, specificity from 64% to 100%, positive predictive value from 79% to 98%, and negative predictive values between 98% and 100%. Two trials compared enhanced or air cystoscopy versus conventional cystoscopy. Overall sensitivity of conventional white light cystoscopy ranged from 47% to 100% and specificity from 93.4% to 100%. CONCLUSIONS: The true accuracy of cystoscopy for the detection of bladder cancer within the context of haematuria has not been studied extensively, resulting in inconsistent data regarding its performance for patients with haematuria. In comparison with imaging modalities, a few trials have prospectively assessed the diagnostic performance of cystoscopy, confirming very high accuracy for cystoscopy, exceeding the diagnostic value of any other imaging test. PATIENT SUMMARY: Evidence of tests for detecting bladder cancer in adults presenting with haematuria (blood in urine) was reviewed. The most common test used was cystoscopy, which remains the current standard for diagnosing bladder cancer.

Alignment of molecular subtypes across multiple bladder cancer subtyping classifiers.

BACKGROUND: Treatment of patients with muscle-invasive bladder cancer (MIBC) includes cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC). Molecular subtypes have been associated with patient outcomes after NAC and RC, but the reported results have been highly inconsistent. OBJECTIVE: To evaluate the association of molecular subtypes from different classifiers with overall survival (OS) among patients with MIBC who underwent RC. MATERIALS AND METHODS: We analyzed gene expression data generated from transurethral resection of MIBC from a previously assembled and published meta-cohort, NACmeta (N = 601, 247 treated with NAC+RC and 354 RC without NAC), where extended follow-up was available. Molecular subtypes were assigned using the Genomic Subtyping Classifier (GSC), the Consensus Classifier, The Cancer Genome Atlas (TCGA) Classifier, and the Lund Classifier. For survival analysis, inverse probability weighting was used to balance the clinical NAC and non-NAC patient groups. RESULTS: A high consistency in gene expression patterns and nomenclature was observed between luminal-like subtypes, defined as GSC-Luminal, Consensus-Luminal Papillary (LumP), TCGA Luminal-Papillary (LumP) and Lund-UroA, but not for basal-like subtypes such GSC-Basal, Consensus Basal/Squamous, TCGA-Basal/Squamous and Lund-Basal/Squamous. Patients with luminal-like subtypes demonstrated no difference in 3-year OS when treated with or without NAC (P = 0.7 for GSC, P = 0.94 for Consensus, P = 0.87 for TCGA and P = 0.66 for Lund-UroA, respectively). CONCLUSION: Luminal-like molecular subtypes identify a subgroup of MIBC patients who do not appear to benefit from current NAC regimens, even for locally advanced disease. In addition, we were able to illustrate differences in subtyping nomenclature that are not reflected in the underlying biological definition of the subtypes. PATIENT SUMMARY: Muscle-invasive bladder cancer exhibits molecular diversity, and various classifications identify different groups who do not benefit from chemotherapy. On the other hand, there is a high inconsistency in the way cancer groupings are named.

A Genomic Urine Assay for Surveillance of Patients with Bladder Cancer Treated with Radiotherapy.

Background: Patients with muscle-invasive bladder cancer (MIBC) who receive radiotherapy with curative intent are followed by imaging, cystoscopy, and urine cytology. However, interpretation of cytology and cystoscopy is hampered by the impact of ionizing radiation on cells. Objective: To assess the diagnostic performance of a genomic urine assay to detect urinary tract recurrences in patients with MIBC treated by (chemo)radiation. Design setting and participants: Patients with nonmetastatic MIBC who underwent (chemo)radiation with curative intent from 2016 to 2020 were prospectively included. Follow-up consisted of cystoscopy and upper tract imaging. Prior to cystoscopy, a urine sample was analyzed to assess mutations in the genes FGFR3, HRAS, and TERT and methylation of OTX1, TWIST1, and ONECUT2. The treating physician was blinded for the assay result. Outcome measurements and statistical analysis: The primary endpoint was a urinary tract recurrence. Cross-sectional sensitivity, specificity, and negative predictive value (NPV) were analyzed using a previously developed logistic regression model for the detection of bladder cancer with this assay. The secondary endpoint was the risk of a future urinary tract recurrence following a positive test and negative cystoscopy/imaging, using a time-dependent Cox proportional hazard analysis. Results and limitations: A total of 143 patients were included, and 503 urine samples were analyzed. The median study duration was 20 mo (interquartile range [IQR] 10-33), and the median time to a recurrence was 16 mo (IQR 12-26). In 27 patients, 32 urinary tract recurrences were diagnosed, including three upper tract tumors. Of 32 recurrences, 18 (56%) had a concomitant urine test available. The diagnostic model had an area under the curve of 0.80 (95% confidence interval [CI] 0.69-0.90) with corresponding sensitivity, specificity, and NPV of 78 (95% CI 52-94), 77% (95% CI 73-81), and 99% (95% CI 97-100). When taking into account the anticipatory effect of the test, 28/32 (88%) recurrences were detected. A Cox regression analysis showed a hazard ratio of 14.8 for the development of a future recurrence (p < 0.001). A major limitation was the lack of a concomitant urine test result in 14/32 (44%) recurrences. Conclusions: A genomic urine assay detected urinary tract recurrences after (chemo)radiation in patients with MIBC, and a positive test was strongly associated with future recurrences. Although validation in a large cohort is warranted, the test has the potential to limit frequent cystoscopies. Patient summary: Radiotherapy is a bladder-sparing treatment in patients with bladder cancer. After treatment, these patients undergo visual inspection of the bladder by cystoscopy to detect possible recurrences. However, interpretation of cystoscopy is difficult due to the effects of radiation on the bladder lining. Hence, we analyzed the diagnostic value of a molecular urine test to detect recurrent disease in bladder cancer patients treated by radiotherapy, and we showed that the urine test has the potential to limit the number of cystoscopies.

A Molecular Urine Assay to Detect Recurrences During Surveillance of High-Risk Non-Muscle Invasive Bladder Cancer.

BACKGROUND: High-risk non-muscle invasive bladder cancer (HR-NMIBC) patients require long-term surveillance with cystoscopies, cytology and upper tract imaging. Previously, we developed a genomic urine assay for surveillance of HR-NMIBC patients with high sensitivity and anticipatory value. OBJECTIVE: We aimed to validate the performance of the assay in an unselected prospectively collected cohort of HR-NMIBC patients under surveillance. METHODS: We included patients from five centers and collected urine sample pairs (evening and morning urines) prior to cystoscopy. Mutation status (FGFR3/TERT) and methylation status (OTX1) was analyzed on DNA from voided urine specimens. A test was considered positive if≥1 alteration was detected in at least one urine sample. The primary endpoint was tumor recurrence. Sensitivity and specificity were determined. A generalized mixed effects model was used to adjust for within-patient correlation. Cox proportional hazard analyses with time-dependent covariates assessed the anticipatory effect of the urine assay. RESULTS: In total, 204 patients and 736 sample pairs were collected. Sixty-three recurrences were diagnosed for which we had concomitant assay results. On cross-sectional analyses, the assay detected 75% (95% CI 62.1% -84.7%) of recurrences, with a specificity of 70% (95% CI 66.4% -73.5%). Furthermore, mixed effects model analyses revealed OTX1 (p = 0.005) and TERT (p = 0.004) as significant predictors for disease recurrence. Median follow-up was 25.3 months (IQR 18.6-30.7). Twenty-nine tumors were diagnosed without concomitant urine samples, which included recurrences detected after urine collection ended. Longitudinal analyses showed that a positive urine assay predicted a recurrence over time (HR 3.5, p < 0.001). Furthermore, a recurrence during the study period was also a predictor for developing future recurrences (HR 2.1, p < 0.001). CONCLUSIONS: This study validates the performance of a previously developed urine assay in an unselected cohort of HR-NMIBC patients under surveillance. With a robust sensitivity/specificity and a strong anticipatory effect, this assay proves a useful adjunct ready for evaluation in a future randomized controlled trial.

A Urine-based Genomic Assay Improves Risk Stratification for Patients with High-risk Hematuria Stratified According to the American Urological Association Guidelines.

BACKGROUND: According to the recent American Urological Association (AUA) guideline on hematuria, patients are stratified into groups with low, intermediate, and high risk of urothelial carcinoma (UC). These risk groups are based on clinical factors and do not incorporate urine-based tumor markers. OBJECTIVE: To evaluate whether a urine-based genomic assay improves the redefined AUA risk stratification for hematuria. DESIGN, SETTING, AND PARTICIPANTS: We selected patients with complete biomarker status, as assessed on urinary DNA, from a previously collected prospective Dutch hematuria cohort (n = 838). Patients were stratified into the AUA risk categories on the basis of sex, age, and type of hematuria. Biomarker status included mutation status for the FGFR3, TERT, and HRAS genes, and methylation status for the OTX1, ONECUT2, and TWIST1 genes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was the diagnostic model performance for different hematuria risk groups. Further analyses assessed the pretest and post-test UC probability in the hematuria subgroups using a Fagan nomogram. RESULTS AND LIMITATIONS: Overall, 65 patients (7.8%) were classified as low risk, 106 (12.6%) as intermediate risk, and 667 (79.6%) as high risk. The UC incidence differed significantly between the gross hematuria (21%, 98/457) and microscopic hematuria (4%, 14/381) groups (p < 0.001). All cancer cases were in the high-risk group, which had UC incidence of 16.8% (112/667). Application of the diagnostic model revealed robust performance among all risk groups (area under the receiver operating characteristic curve 0.929-0.971). Depending on the risk group evaluated, a negative urine assay was associated with post-test UC probability of 0.3-2%, whereas a positive urine assay was associated with post-test UC probability of 31-42%. CONCLUSIONS: This study shows the value that a urine-based genomic assay adds to the AUA guideline stratification for patients with hematuria. It seems justified to safely withhold cystoscopy for patients with AUA low risk who have a negative urine assay. In addition, evaluation should be expedited for patients with AUA intermediate or high risk and a positive urine assay. PATIENT SUMMARY: Patients who have blood in their urine (hematuria) can be classified as having low, intermediate, or high risk of having cancer in their urinary tract. We found that use of a urine-based genetic test improves the accuracy of predicting which patients are most likely to have cancer. Patients with a negative test may be able to avoid invasive tests, while further tests could be prioritized for patients with a positive test.

Long-term efficacy of hyperthermic intravesical chemotherapy for BCG-unresponsive non-muscle invasive bladder cancer.

BACKGROUND: The recommended treatment for patients with Bacillus Calmette-Guérin (BCG) unresponsive non-muscle invasive bladder cancer (NMIBC) is radical cystectomy (RC). However, many patients refuse, or are unfit for RC. Therefore, alternative bladder-sparing treatment modalities are needed for BCG-unresponsive NMIBC. In this study we sought to assess the long-term efficacy of hyperthermic intravesical chemotherapy (HIVEC) as alternative to radical cystectomy in BCG-unresponsive non-muscle invasive bladder cancer patients. METHODS AND MATERIALS: Retrospectively collected data from 56 patients with BCG-unresponsive NMIBC who received ≥5 HIVEC instillations between October 2014 and March 2020 was analyzed. All patients met the BCG-unresponsive criteria according to the current EAU guideline on NMIBC 2020. Patients were followed-up with cystoscopy and/or bladder biopsies, urine cytology and annually CT-urography. The Primary outcome was the high grade (HG) recurrence-free survival (RFS), defined as the time from the first HIVEC instillation until histologically confirmed intravesical recurrence or last follow-up. The Kaplan Meier method was used to estimate survival outcomes. Secondary outcomes were: complete response rate (CR), adverse events (AE), assessed by the Common Terminology Criteria for Adverse Events v5.0 (CTCAE) and tumor progression to muscle invasive disease or distant metastases. RESULTS: The median follow-up was 32.2 months (IQR 13.7-44.8). The 1- and 2-year HG-RFS was 53% (SE:6.8) and 35% (SE:6.9), respectively. The CR for patients with CIS was 70% (21/30) at 6 months. Overall, 80% of the population developed an AE, only 1 was classified as CTCAE ≥3. Limitation of this study was the small sample size. CONCLUSION: HIVEC resulted in a 2-year HG-RFS of 35% for BCG-unresponsive NMIBC patients without severe side-effects and therefore HIVEC seems to be an alternative treatment option for patients who refuse or are unfit for RC.