Kirstin A. van der Keur

Microsatellite Analysis of Voided-Urine Samples for Surveillance of Low-Grade Non-Muscle-Invasive Urothelial Carcinoma: Feasibility and Clinical Utility in a Prospective Multicenter Study (Cost-Effectiveness of Follow-Up of Urinary Bladder Cancer Trial [CEFUB])

BACKGROUND Microsatellite analysis (MA) of voided-urine samples has been promoted as an alternative for cystoscopy surveillance (UCS) of patients with low-grade non-muscle-invasive papillary urothelial carcinoma (UC). OBJECTIVE To assess the feasibility and clinical utility of MA on voided-urine samples in a routine setting to detect or predict bladder cancer recurrences. DESIGN, SETTING, AND PARTICIPANTS We evaluated 228 patients monitored by MA of voided-urine samples and synchronous UCS who participated in a longitudinal prospective study in 10 hospitals. Follow-up started after diagnosis of a primary or recurrent pTa, pT1, grade 1 or grade 2 papillary UC. MEASUREMENTS Clinico-pathological parameters and fibroblast growth factor receptor 3 (FGFR3) gene mutation status of the inclusion tumour were determined. MA outcome was analysed in 1012 urine samples during a mean follow-up of 41 mo. Poor DNA quality prevented MA in 19% (197/1012) of the samples, leaving 815 visits for a cross-sectional analysis of sensitivity and specificity. We determined the predictive value (PPV) in a longitudinal analysis for 458 series with persistent MA results. Factors influencing diagnostic quality of MA were investigated. Kaplan-Meier analysis was performed to relate MA results to recurrence. RESULTS AND LIMITATIONS Cross-sectional sensitivity and specificity of MA for detection of a recurrence were 58% (49/84) and 73% (531/731), respectively. One pT1 grade 3 UC was missed. In a longitudinal analysis, the 2-yr risk to develop a recurrence reached 83% if MA outcome was persistently positive and 22% when MA was persistently negative. PPV of MA was higher with wild-type FGFR3 gene status and smoking habits. All four upper urinary tract tumours detected were preceded by a positive MA test. CONCLUSIONS Consecutive positive MA results are a strong predictor for future recurrences, but sensitivity needs to be improved, for example, by patient selection and testing of additional genetic markers in urine samples.

A methylation assay for the detection of non-muscle-invasive bladder cancer (NMIBC) recurrences in voided urine

Whats known on the subject? and What does the study add?

Liquid Biopsy Analysis of FGFR3 and PIK3CA Hotspot Mutations for Disease Surveillance in Bladder Cancer

BACKGROUND Disease surveillance in patients with bladder cancer is important for early diagnosis of progression and metastasis and for optimised treatment. OBJECTIVE To develop urine and plasma assays for disease surveillance for patients with FGFR3 and PIK3CA tumour mutations. DESIGN, SETTING, AND PARTICIPANTS Droplet digital polymerase chain reaction (ddPCR) assays were developed and tumour DNA from two patient cohorts was screened for FGFR3 and PIK3CA hotspot mutations. One cohort included 363 patients with non-muscle-invasive bladder cancer (NMIBC). The other cohort included 468 patients with bladder cancer undergoing radical cystectomy (Cx). Urine supernatants (NMIBC n=216, Cx n=27) and plasma samples (NMIBC n=39, Cx n=27) from patients harbouring mutations were subsequently screened using ddPCR assays. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Progression-free survival, recurrence-free survival, and overall survival were measured. Fishers exact test, the Wilcoxon rank-sum test and Cox regression analysis were applied. RESULTS AND LIMITATIONS In total, 36% of the NMIBC patients (129/363) and 11% of the Cx patients (44/403) harboured at least one FGFR3 or PIK3CA mutation. Screening of DNA from serial urine supernatants from the NMIBC cohort revealed that high levels of tumour DNA (tDNA) were associated with later disease progression in NMIBC (p=0.003). Furthermore, high levels of tDNA in plasma samples were associated with recurrence in the Cx cohort (p=0.016). A positive correlation between tDNA levels in urine and plasma was observed (correlation coefficient 0.6). The retrospective study design and low volumes of plasma available for analysis were limitations of the study. CONCLUSIONS Increased levels of FGFR3 and PIK3CA mutated DNA in urine and plasma are indicative of later progression and metastasis in bladder cancer. PATIENT SUMMARY Urine and plasma from patients with bladder cancer may be monitored for diagnosis of progression and metastasis using mutation assays.

Combinations of Urinary Biomarkers for Surveillance of Patients with Incident Nonmuscle Invasive Bladder Cancer: The European FP7 UROMOL Project

PURPOSE We determined a combination of markers with optimal sensitivity to detect recurrence in voided urine after resection of an incident low grade, nonmuscle invasive bladder tumor. MATERIALS AND METHODS A total of 136 patients with G1/G2 nonmuscle invasive bladder tumor were included in the study at transurethral resection of the incident tumor. At least 3 followup urine samples were required for patient selection. DNA was extracted from the incident tumor and cell pellets of subsequently collected urine samples. We performed FGFR3, PIK3CA and RAS mutation analysis, and microsatellite and methylation analysis on tissue and urine DNA samples. RESULTS We obtained 716 urine samples. The 136 patients experienced a total of 552 recurrences during a median 3-year followup. Sensitivity for detecting a recurrent tumor varied between 66% and 68% for the molecular tests after patient stratification based on tumor DNA analysis. A combination of markers increased sensitivity but decreased the number of patients eligible for a certain test combination. Combining urine cytology with FGFR3 analysis without stratifying for FGFR3 status of the incident tumor increased sensitivity from 56% to 76%. CONCLUSIONS A combination of markers increased the percentage of patients eligible for urine based followup and the sensitivity of recurrence detection. Adding FGFR3 analysis to urine cytology could be valuable for noninvasive followup of patients with nonmuscle invasive bladder cancer.

Genome-wide association study yields variants at 20p12.2 that associate with urinary bladder cancer.

Genome-wide association studies (GWAS) of urinary bladder cancer (UBC) have yielded common variants at 12 loci that associate with risk of the disease. We report here the results of a GWAS of UBC including 1670 UBC cases and 90 180 controls, followed by replication analysis in additional 5266 UBC cases and 10 456 controls. We tested a dataset containing 34.2 million variants, generated by imputation based on whole-genome sequencing of 2230 Icelanders. Several correlated variants at 20p12, represented by rs62185668, show genome-wide significant association with UBC after combining discovery and replication results (OR = 1.19, P = 1.5 × 10(-11) for rs62185668-A, minor allele frequency = 23.6%). The variants are located in a non-coding region approximately 300 kb upstream from the JAG1 gene, an important component of the Notch signaling pathways that may be oncogenic or tumor suppressive in several forms of cancer. Our results add to the growing number of UBC risk variants discovered through GWAS.

Validation of a DNA Methylation-Mutation Urine Assay to Select Patients with Hematuria for Cystoscopy

Purpose: Only 3% to 28% of patients referred to the urology clinic for hematuria are diagnosed with bladder cancer. Cystoscopy leads to high diagnostic costs and a high patient burden. Therefore, to improve the selection of patients for cystoscopy and reduce costs and over testing we aimed to validate a recently developed diagnostic urine assay. Materials and Methods: Included in study were 200 patients from a total of 3 European countries who underwent cystoscopy for hematuria, including 97 with bladder cancer and 103 with nonmalignant findings. Voided urine samples were collected prior to cystoscopy. DNA was extracted and analyzed for mutations in FGFR3, TERT and HRAS, and methylation of OTX1, ONECUT2 and TWIST1. Logistic regression was used to analyze the association between predictor variables and bladder cancer. Results: Combining the methylation and mutation markers with age led to an AUC of 0.96 (95% CI 0.92–0.99) with 93% sensitivity and 86% specificity, and an optimism corrected AUC of 0.95. The AUC was higher for T1 or greater tumors compared to Ta tumors (0.99 vs 0.93). The AUC was also higher for high grade tumors compared to low grade tumors (1.00 vs 0.93). Overall negative predictive value was 99% based on the 5% to 10% prevalence of bladder cancer in patients with hematuria. This would lead to a 77% reduction in diagnostic cystoscopy. Conclusions: Analyzing hematuria patients for the risk of bladder cancer using novel molecular markers may lead to a reduction in diagnostic cystoscopy. Combining methylation analysis (OTX1, ONECUT2 and TWIST1) with mutation analysis (FGFR3, TERT and HRAS) and patient age resulted in a validated accurate prediction model.

FGFR3, TERT and OTX1 as a Urinary Biomarker Combination for Surveillance of Patients with Bladder Cancer in a Large Prospective Multicenter Study

Purpose: Patients with nonmuscle invasive bladder cancer are followed with frequent cystoscopies. In this study FGFR3, TERT and OTX1 were investigated as a diagnostic urinary marker combination during followup of patients with primary nonmuscle invasive bladder cancer. Materials and Methods: In this international, multicenter, prospective study 977 patients with nonmuscle invasive bladder cancer were included. A total of 2,496 urine samples were collected prior to cystoscopy during regular visits. Sensitivity was estimated to detect concomitant recurrences. Kaplan‐Meier curves were used to estimate the development of future recurrences after urinalysis and a negative cystoscopy. Results: Sensitivity of the assay combination for recurrence detection was 57% in patients with primary low grade, nonmuscle invasive bladder cancer. However, sensitivity was 83% for recurrences that were pT1 or muscle invasive bladder cancer. Of the cases 2% progressed to muscle invasive bladder cancer. Sensitivity for recurrence detection in patients with primary high grade disease was 72% and 7% of them had progression to muscle invasive bladder cancer. When no concomitant tumor was found by cystoscopy, positive urine samples were more frequently followed by a recurrence over time compared to a negative urine sample (58% vs 36%, p <0.001). High stage recurrences were identified within 1 year after a positive urine test and a negative cystoscopy. Conclusions: Recurrences in patients with primary nonmuscle invasive bladder cancer can be detected by a combination of urine assays. This study supports the value of urinalysis as an alternative diagnostic tool in patients presenting with low grade tumors and as a means to identify high stage tumors earlier.

Selection of Microsatellite Markers for Bladder Cancer Diagnosis without the Need for Corresponding Blood

Microsatellite markers are used for loss-of-heterozygosity, allelic imbalance and clonality analyses in cancers. Usually, tumor DNA is compared to corresponding normal DNA. However, normal DNA is not always available and can display aberrant allele ratios due to copy number variations in the genome. Moreover, stutter peaks may complicate the analysis. To use microsatellite markers for diagnosis of recurrent bladder cancer, we aimed to select markers without stutter peaks and a constant ratio between alleles, thereby avoiding the need for a control DNA sample. We investigated 49 microsatellite markers with tri- and tetranucleotide repeats in regions commonly lost in bladder cancer. Based on analysis of 50 blood DNAs the 12 best performing markers were selected with few stutter peaks and a constant ratio between peaks heights. Per marker upper and lower cut off values for allele ratios were determined. LOH of the markers was observed in 59/104 tumor DNAs. We then determined the sensitivity of the marker panel for detection of recurrent bladder cancer by assaying 102 urine samples of these patients. Sensitivity was 63% when patients were stratified for LOH in their primary tumors. We demonstrate that up-front selection of microsatellite markers obliterates the need for a corresponding blood sample. For diagnosis of bladder cancer recurrences in urine this significantly reduces costs. Moreover, this approach facilitates retrospective analysis of archival tumor samples for allelic imbalance.

In-depth investigation of the molecular pathogenesis of bladder cancer in a unique 26-year old patient with extensive multifocal disease: a case report.

BackgroundThe molecular characteristics and the clinical disease course of bladder cancer (BC) in young patients remain largely unresolved. All patients are monitored according to an intensive surveillance protocol and we aim to gain more insight into the molecular pathways of bladder tumors in young patients that could ultimately contribute to patient stratification, improve patient quality of life and reduce associated costs. We also determined whether a biomarker-based surveillance could be feasible.Case PresentationWe report a unique case of a 26-year-old Caucasian male with recurrent non-muscle invasive bladder tumors occurring at a high frequency and analyzed multiple tumors (maximal pTaG2) and urine samples of this patient. Analysis included FGFR3 mutation detection, FGFR3 and TP53 immunohistochemistry, mircosatellite analysis of markers on chromosomes 8, 9, 10, 11 and 17 and a genome wide single nucleotide polymorphism-array (SNP). All analyzed tumors contained a mutation in FGFR3 and were associated with FGFR3 overexpression. None of the tumors showed overexpression of TP53. We found a deletion on chromosome 9 in the primary tumor and this was confirmed by the SNP-array that showed regions of loss on chromosome 9. Detection of all recurrences was possible by urinary FGFR3 mutation analysis.ConclusionsOur findings would suggest that the BC disease course is determined by not only a patients age, but also by the molecular characteristics of a tumor. This young patient contained typical genetic changes found in tumors of older patients and implies a clinical disease course comparable to older patients. We demonstrate that FGFR3 mutation analysis on voided urine is a simple non-invasive method and could serve as a feasible follow-up approach for this young patient presenting with an FGFR3 mutant tumor.

Molecular markers increase precision of the European Association of Urology non-muscle invasive bladder cancer progression risk groups

Purpose: The European Association of Urology (EAU) guidelines for non–muscle-invasive bladder cancer (NMIBC) recommend risk stratification based on clinicopathologic parameters. Our aim was to investigate the added value of biomarkers to improve risk stratification of NMIBC. Experimental Design: We prospectively included 1,239 patients in follow-up for NMIBC in six European countries. Fresh-frozen tumor samples were analyzed for GATA2, TBX2, TBX3, and ZIC4 methylation and FGFR3, TERT, PIK3CA, and RAS mutation status. Cox regression analyses identified markers that were significantly associated with progression to muscle-invasive disease. The progression incidence rate (PIR = rate of progression per 100 patient-years) was calculated for subgroups. Results: In our cohort, 276 patients had a low, 273 an intermediate, and 555 a high risk of tumor progression based on the EAU NMIBC guideline. Fifty-seven patients (4.6%) progressed to muscle-invasive disease. The limited number of progressors in this large cohort compared with older studies is likely due to improved treatment in the past two decades. Overall, wild-type FGFR3 and methylation of GATA2 and TBX3 were significantly associated with progression (HR = 0.34, 2.53, and 2.64, respectively). The PIR for EAU high-risk patients was 4.25. On the basis of FGFR3 mutation status and methylation of GATA2, this cohort could be reclassified into a good class (PIR = 0.86, 26.2% of patients), a moderate class (PIR = 4.32, 49.7%), and a poor class (PIR = 7.66, 24.0%). Conclusions: We conclude that the addition of selected biomarkers to the EAU risk stratification increases its accuracy and identifies a subset of NMIBC patients with a very high risk of progression. Clin Cancer Res; 24(7); 1586–93. ©2018 AACR.