Prognostic value of molecular markers, sub-stage and European Organisation for the Research and Treatment of Cancer risk scores in primary T1 bladder cancer.

UNLABELLED: 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-Guérin. 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.

A new and highly prognostic system to discern T1 bladder cancer substage.

BACKGROUND: Management of T1 bladder cancer (BCa) is controversial. OBJECTIVE: Evaluate the impact of substage on the clinical outcome of T1 BCa. DESIGN, SETTING, AND PARTICIPANTS: The T1 diagnosis of 134 first-diagnosis BCa patients from two university hospitals was confirmed. For the T1 substage, we used a new system that discerns T1-microinvasive (T1m) and T1-extensive-invasive (T1e) tumors. We then determined the invasion of the muscularis mucosae-vascular plexus (MM-VP): T1a (invasion above the MM-VP), T1b (invasion in the MM-VP), or T1c (invasion beyond the MM-VP). If the MM-VP was not present at the invasion front, the case was assigned to T1a or T1c. All patients were initially managed conservatively (with bacillus Calmette-Guérin). MEASUREMENTS: Multivariable analyses for progression and disease-specific survival (DSS). RESULTS AND LIMITATIONS: Median follow-up was 6.4 yr (interquartile range: 3.3-9.2 yr). Progression to ≥ T2 was observed in 40 patients (30%), and 19 patients (14%) died of BCa. The MM-VP was not present at the invasion front in 50 patients (37%). T1 substage was as follows: 40 T1m and 94 T1e; 81 T1a, 18 T1b, and 35 T1c. In multivariable analyses, substage (T1m/T1e) was significant for progression (p=0.001) and DSS (p=0.032), whereas substage according to T1a/T1b/T1c was not significant. Female gender (p=0.006) and carcinoma in situ (p=0.034) were also significant predictors of progression. The main limitation to the study is absence of a repeat transurethral resection. CONCLUSIONS: Substage according to the new system (T1m and T1e) was user-friendly, possible in 100% of cases, and very predictive of T1 BCa behavior. Future studies may ultimately lead to the incorporation of this new substaging system in the TNM classification system for urinary BCa.

FGFR3, HRAS, KRAS, NRAS and PIK3CA mutations in bladder cancer and their potential as biomarkers for surveillance and therapy.

BACKGROUND: Fifty percent of patients with muscle-invasive bladder cancer (MI-BC) die from their disease and current chemotherapy treatment only marginally increases survival. Novel therapies targeting receptor tyrosine kinases or activated oncogenes may improve outcome. Hence, it is necessary to stratify patients based on mutations in relevant oncogenes. Patients with non-muscle-invasive bladder cancer (NMI-BC) have excellent survival, however two-thirds develop recurrences. Tumor specific mutations can be used to detect recurrences in urine assays, presenting a more patient-friendly diagnostic procedure than cystoscopy. METHODOLOGY/PRINCIPAL FINDINGS: To address these issues, we developed a mutation assay for the simultaneous detection of 19 possible mutations in the HRAS, KRAS, and NRAS genes. With this assay and mutation assays for the FGFR3 and PIK3CA oncogenes, we screened primary bladder tumors of 257 patients and 184 recurrences from 54 patients. Additionally, in primary tumors p53 expression was obtained by immunohistochemistry. Of primary tumors 64% were mutant for FGFR3, 11% for RAS, 24% for PIK3CA, and 26% for p53. FGFR3 mutations were mutually exclusive with RAS mutations (p = 0.001) and co-occurred with PIK3CA mutations (p = 0.016). P53 overexpression was mutually exclusive with PIK3CA and FGFR3 mutations (p≤0.029). Mutations in the RAS and PIK3CA genes were not predictors for recurrence-free, progression-free and disease-specific survival. In patients presenting with NMI-BC grade 3 and MI-BC, 33 and 36% of the primary tumors were mutant. In patients with low-grade NMI-BC, 88% of the primary tumors carried a mutation and 88% of the recurrences were mutant. CONCLUSIONS/SIGNIFICANCE: The mutation assays present a companion diagnostic to define patients for targeted therapies. In addition, the assays are a potential biomarker to detect recurrences during surveillance. We showed that 88% of patients presenting with low-grade NMI-BC are eligible for such a follow-up. This may contribute to a reduction in the number of cystoscopical examinations.

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.

Cystoscopy revisited as the gold standard for detecting bladder cancer recurrence: diagnostic review bias in the randomized, prospective CEFUB trial.

PURPOSE: We evaluated the influence of knowledge of urine test outcome on the accuracy of cystoscopy (diagnostic review bias) during surveillance in patients with low grade, nonmuscle invasive urothelial carcinoma. MATERIALS AND METHODS: We performed a prospective, single-blind, randomized, multicenter clinical trial of surveillance by microsatellite analysis urine test in 448 patients with nonmuscle invasive (pTa, pT1, G1, G2) urothelial carcinoma. Positive or negative urine test results were only communicated to the urologist in the intervention arm of 226 patients, in which cystoscopy was done if the test was positive, and at 3, 12 and 24 months. Urine test results were not communicated in the control arm of 222 patients who underwent standard 3-month cystoscopy. The primary outcome measure was the number of histologically proven bladder cancer recurrences. RESULTS: At a median 34-month followup 218 recurrences were detected in the intervention arm compared to 163 in the control arm (p <0.001). Of 131 cystoscopies done with knowledge of a positive urine test 42 recurrences were detected. Only 6 recurrences were found in the 120 cystoscopies done without information on the positive test result (chi-square p <0.001). There was no difference in recurrence detection when urine test results were negative in the intervention and control arms (18 of 260 patients or 7% and 18 of 326 or 6%, respectively, p = 0.45). CONCLUSIONS: Diagnostic review bias should be considered in the evaluation of point of care urine tests for bladder cancer monitoring. Awareness of a positive urine test result significantly improves the urothelial carcinoma detection rate using cystoscopy.

Pathological stage review is indicated in primary pT1 bladder cancer.

OBJECTIVE: To evaluate the effect of a pathology review on the clinical outcome of patients with primary pT1 bladder cancer (BC), as the clinical course of such patients is variable. PATIENTS AND METHODS: The slides of 164 primary (first diagnosis) pT1 bladder tumours from two university hospitals were reviewed by one pathologist for stage and grade (World Health Organization 1973 and 2004). Patients were initially managed conservatively with bacille Calmette-Guérin (BCG). Uni- and multivariate analyses compared the predictive value of age, gender, hospital, carcinoma in situ (CIS), tumour-size, reviewed grade and reviewed stage. RESULTS: With a mean follow-up of 6.4 years, there was disease progression in 48 (29%) patients and 26 (16%) died from BC. Associated CIS was found in 55 (34%) patients. After reviewing the slides, 24 (15%) tumours were downstaged to pTa, 134 (82%) remained pT1 and six (4%) were upstaged to > or =pT2. The grade review resulted in 74 G2, 90 G3, 37 low-grade and 127 high-grade lesions for the two systems used. In multivariate analyses, reviewed stage (both P < 0.001) and CIS (P = 0.017 and 0.023) had independent significance for progression and disease-specific survival, respectively. CONCLUSION: A stage review is indicated in pT1 BC, as almost 20% of pT1 tumours were up- or downstaged, and the reviewed stage predicted the patient's prognosis. Hence, pathology review identified patients with different prognoses who might benefit from other treatment strategies than BCG. We confirmed that CIS is an unfavourable sign in pT1 bladder cancer.

Non-muscle-invasive bladder cancer surveillance for which cystoscopy is partly replaced by microsatellite analysis of urine: a cost-effective alternative?

OBJECTIVE To determine how good microsatellite analysis (MA) markers in voided urine samples should be to make a surveillance procedure cost-effective in which cystoscopy is partly replaced by MA for patients with non-muscle-invasive urothelial carcinoma (NMI-UC). PATIENTS AND METHODS We constructed a semi-Markov model with a time horizon of 2 years, and a man aged 65 years as reference case. Data were used from a randomized trial (including 448 patients with NMI-UC from 10 hospitals), and from other data sources. The costs and effects (probability of being in a specific health state) were compared for two surveillance strategies: (i) cystoscopy of the urinary bladder every 3 months (conventional arm), and (ii) semi-automated MA of voided urine samples to identify loss of heterozygosity every 3 months, with a control cystoscopy at 3, 12 and 24 months (test arm). Various sensitivity analyses were used to determine the sensitivity, specificity, and costs of MA of urine for which the test arm was as cost-effective as the conventional arm. RESULTS The probability of being without recurrence after 2 years of surveillance was similar (86.6% conventional arm vs 86.3% test arm) with currently available MA markers (sensitivity of 58% and specificity of 73%). However, the test arm led to higher costs ($4104 vs $3433 per head). The test arm would be as effective and cost the same as the conventional arm if the sensitivity of the currently available MA markers was increased at > or =61%, had a specificity of 73%, and decreased the costs of the MA test per follow-up sample from $158 to <$70. CONCLUSIONS Over 2 years, surveillance in which cystoscopy is partly replaced by currently available urinary MA to reduce patient burden can only provide a cost-effective alternative to the conventional surveillance if the MA urine test had a slightly higher sensitivity and its costs could be reduced.

The development of multiple bladder tumour recurrences in relation to the FGFR3 mutation status of the primary tumour.

Non-muscle invasive bladder cancers (NMI-BCs) represent 75% of bladder cancers upon presentation. After removal of the primary tumour by transurethral resection, multiple recurrences continue to develop in 70% of patients. Consequently, prolonged and costly surveillance by cystoscopy is required. Mutations in the FGFR3 oncogene are common in NMI-BCs and are associated with a lower chance of progression to muscle-invasive disease. Here we analysed the consistency of FGFR3 mutations in primary and recurrent tumours. This knowledge is of crucial importance if FGFR3 mutation analysis on urinary cells is to be used as an alternative for cystoscopical surveillance. To this end, we monitored the disease process and FGFR3 mutation status of primary and recurrent tumours in 118 patients with NMI-BC. During median follow-up of 8.8 years, these patients underwent 2133 cystoscopies and 80 patients developed 414 recurrences. FGFR3 mutations were equally prevalent in primary and recurrent tumours (63%). Patients can have different types of FGFR3 mutations in different tumours. Recurrence risk was not significantly different for patients with a mutant or wild-type primary tumour. Recurrence rates varied widely between patients but were constant for a patient and were unrelated to FGFR3 status. In the mutant patient group, in contrast to the wild-type group, recurrences continued to develop after 10 years. In 81% of the recurrences of patients with a mutant primary tumour, a mutation was found. Moreover, recurrences in this patient group were of lower stage and grade than those of patients with a wild-type primary tumour (p < 0.001). These results suggest that surveillance by FGFR3 mutation analysis on voided urine in combination with a reduced cystoscopy frequency of patients presenting with an FGFR3 mutant tumour is worth investigating.

Sexual function of patients under surveillance for bladder cancer.

OBJECTIVE To describe the prevalence of sexual dysfunction and evaluate risk factors in patients just diagnosed with non-muscle-invasive bladder cancer (NMI UC), who have the prospect of an intensive surveillance scheme by cysto-urethroscopy to detect tumour recurrences. PATIENTS AND METHODS We conducted a cross-sectional survey on 150 patients just diagnosed with primary or recurrent NMI UC. Patients were participating in a randomized clinical multicentre trial (CEFuB), comparing two surveillance schemes. Patients were asked to complete questionnaires at study entry 3 months before the start of the study-surveillance scheme (demographic characteristics, a validated visual analogue scale, and validated subset of questions on sexual function and performance derived from QLQ-BLS-24). The results were compared with those from an age-and gender-matched healthy population. RESULTS The response rate was 95% (142/150); 61% (87/142) of the respondents were sexually active in the previous 4 weeks after diagnosis, 66% (70/105) of men and 46% (17/37) of women. Although libido was not negatively affected, 54% (47/87) of the patients had a sexual dysfunction, and 23% (17/73) were afraid to inflict harm on their partner by sexual contact. Sexually active patients perceived a higher state of general health (P = 0.03). CONCLUSIONS The prevalence of sexual dysfunction in patients with NMI UC is very high (54%) compared with an age- and gender-matched healthy population (20-45%). No predictors for sexual dysfunction were found. These patients and partners would benefit from proper sexual information in the outpatient clinic.

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.

Prediction of progression of non-muscle-invasive bladder cancer by WHO 1973 and 2004 grading and by FGFR3 mutation status: a prospective study.

OBJECTIVES: The clinical management of non-muscle-invasive urothelial cell carcinoma of the bladder (UCC) is challenging, as it has a marked tendency to recur and to progress. Aim of this study was to investigate the prognostic value of the WHO 1973 and 2004 grading systems and biomarkers FGFR3, CK20 and Ki-67. METHODS: In a prospective study, tumours from 221 patients were studied for the expression of CK20 and Ki-67 by immunohistochemistry, and FGFR3 status by SNaPshot mutation detection. Staging and grading were performed according to the WHO classification systems of 1973 and 2004. RESULTS: : Median follow-up was 35 mo. Recurrence occurred in 72 of 221 patients. None of the parameters was able to predict disease recurrence. CK20, Ki-67, FGFR3 mutation, molecular grade using FGFR3 mutation analysis and Ki-67, and histological grading and staging were significantly associated with disease progression in stage. In multivariable analyses, WHO 1973 and 2004 grading systems remained statistically significant and independent predictors of progression, with p=0.005 for WHO 1973 and p=0.004 for 2004. FGFR3 status was able to discriminate progressors from nonprogressors in a subset of patients with high-grade UCC (p=0.009). CONCLUSIONS: This is the first prospective study comparing the WHO 1973 and 2004 grading systems. We show that both grading systems contribute valuable independent information. Therefore, it should be considered whether a better grading system could be developed that incorporates essential elements from both. The combination of WHO 2004 grading with FGFR3 status allows a better risk stratification for patients with high-grade non-muscle-invasive UCC.

Patients' perceived burden of cystoscopic and urinary surveillance of bladder cancer: a randomized comparison.

OBJECTIVE: To compare, in patients with non-muscle-invasive low-grade (pTa/pT1, G1/G2) urothelial cell carcinoma of the urinary bladder, the perceived burden of flexible cystoscopy or surveillance by microsatellite analysis (MA) in voided urine, as such patients are normally recommended to adhere to regular cysto-urethroscopic surveillance (CUS). PATIENTS AND METHODS: In all, 220 participants of a randomized trial comparing CUS and surveillance by MA were asked to complete questionnaires 1 week after cystoscopy or urine sample collection. We assessed the discomfort and pain reported during CUS, experiences with MA, and physical symptoms, medical consumption and general functioning in the week after CUS/urine sampling. RESULTS: We analysed data from 732 questionnaires (197 patients) completed after CUS and 184 (67 patients) after collecting urine. The introduction of the cystoscope was reported to cause discomfort in 39% and pain in 35% of the responses to the questionnaires; the waiting time for the results of MA was reported as burdensome in 19%. Painful micturition was significantly more frequent in the week after CUS than after MA (30% and 12%, respectively). The frequency of fever (1% and 2%) and haematuria (7% and 6%) was similar in both groups. Older patients reported significantly less pain and discomfort from cystoscopy, and this was not related to having more previous cystoscopies. CONCLUSION: CUS caused pain and discomfort in about a third of patients. The burden of MA appeared fully attributable to the waiting time for the test result. The present results are a further motivation in the search for less invasive surveillance tests.

A simple and fast method for the simultaneous detection of nine fibroblast growth factor receptor 3 mutations in bladder cancer and voided urine.

PURPOSE: Mutations in the fibroblast growth factor receptor 3 (FGFR3) occur in 50% of primary bladder tumors. An FGFR3 mutation is associated with good prognosis, illustrated by significantly lower percentage of patients with progression and disease-specific mortality. FGFR3 mutations are especially prevalent in low grade/stage tumors, with pTa tumors harboring mutations in 85% of the cases. These tumors recur in 70% of patients. Efficient FGFR3 mutation detection for prognostic purposes and for detection of recurrences in urine is an important clinical issue. In this paper, we describe a simple assay for the simultaneous detection of nine different FGFR3 mutations. EXPERIMENTAL DESIGN: The assay consists of one multiplex PCR, followed by extension of primers for each mutation with a labeled dideoxynucleotide. The extended primers are separated by capillary electrophoresis, and the identity of the incorporated nucleotide indicates the presence or absence of a mutation. RESULTS: The assay was found to be more sensitive than single-strand conformation polymorphism analysis. Mutations could still be detected with an input of only 1 ng of genomic DNA and in a 20-fold excess of wild-type DNA. Moreover, in urine samples from patients with a mutant tumor, the sensitivity of mutation detection was 62%. CONCLUSIONS: We have developed a fast, easy to use assay for the simultaneous detection of FGFR3 mutations, which can be of assistance in clinical decision-making and as an alternative for the follow-up of patients by invasive cystoscopy for the detection of recurrences in urine.

A new system for substaging pT1 papillary bladder cancer: a prognostic evaluation.

Superficially invasive (pT1) papillary urothelial cell carcinomas (UCCs) may run a variable course. Several attempts have been made for the substaging of UCC to identify the clinically aggressive tumors. We present a new substaging system, based on the extent of invasion. From a series of 53 primary pT1 UCC, 24 cases showed invasion of the subepithelial stroma by an invasive front extending more than a maximum length of 0.5 mm (pT1mic), and 29 showed extensively (>0.5 mm) infiltrating UCC (pT1ext). We tested diagnostic reproducibility between 2 pathologists and found 81% agreement. Furthermore, all cases were analyzed for mutations in the fibroblast growth factor receptor 3 (FGFR3) gene, which represents the favorable pathway of urothelial cell carcinogenesis. Mutant FGFR3 was commonly observed in pT1mic UCC (15/24, 63%), but rarely (2/29, 7%) in pT1ext UCC (chi2 test, P < .001). The presence of pT1ext at initial diagnosis proved to be the strongest predictor for progression, also when adjusted for FGFR3 mutation status in a Cox regression model. If confirmed on a larger series of pT1 UCC, this relatively simple and new substaging system for pT1 UCC may prove to be of prognostic value and supportive to clinical decision-making.