The clonal relation of primary upper urinary tract urothelial carcinoma and paired urothelial carcinoma of the bladder.

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.

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.

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.

Prediction of Multiple Recurrent Events: A Comparison of Extended Cox Models in Bladder Cancer.

Recurrence of bladder cancer can occur repeatedly in the same patient after treatment of the primary tumor. Models predicting the risk of a next recurrence may inform individualized decision-making on surveillance frequency. We aimed to assess the usefulness of extensions of the Cox proportional hazards model for repeated events in this context. We analyzed 531 Dutch patients with bladder cancer (1990-2012) with information on 7 prespecified predictors at the time of diagnosis of the primary and recurrent tumors. We considered 3 aspects of model variants: how to model time to the repeated events (calendar time, gap time, elapsed time); the number of preceding events (predictor, stratum variable); and the within-subject correlation (ignored in a simple Cox model, robust standard errors in a variance-correction model, random effect in a frailty model). First to fourth recurrences of bladder cancer occurred in 313, 174, 103, and 66 patients, respectively, with median calendar follow-up times of 1.1, 2.5, 3.8, and 4.5 years, respectively. We focused on gap time in the detailed analyses, allowing for clinically meaningful predictions. Variance-correction models may be useful if predictor selection is part of the model development. Frailty models may be useful when within-subject correlation is strong.

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.

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.

Evaluation of an Epigenetic Profile for the Detection of Bladder Cancer in Patients with Hematuria.

PURPOSE: Many patients enter the care cycle with gross or microscopic hematuria and undergo cystoscopy to rule out bladder cancer. Sensitivity of this invasive examination is limited, leaving many patients at risk for undetected cancer. To improve current clinical practice more sensitive and noninvasive screening methods should be applied. MATERIALS AND METHODS: A total of 154 urine samples were collected from patients with hematuria, including 80 without and 74 with bladder cancer. DNA from cells in the urine was epigenetically profiled using 2 independent assays. Methylation specific polymerase chain reaction was performed on TWIST1. SNaPshot™ methylation analysis was done for different loci of OTX1 and ONECUT2. Additionally all samples were analyzed for mutation status of TERT (telomerase reverse transcriptase), PIK3CA, FGFR3 (fibroblast growth factor receptor 3), HRAS, KRAS and NRAS. RESULTS: The combination of TWIST1, ONECUT2 (2 loci) and OTX1 resulted in the best overall performing panel. Logistic regression analysis on these methylation markers, mutation status of FGFR3, TERT and HRAS, and patient age resulted in an accurate model with 97% sensitivity, 83% specificity and an AUC of 0.93 (95% CI 0.88-0.98). Internal validation led to an optimism corrected AUC of 0.92. With an estimated bladder cancer prevalence of 5% to 10% in a hematuria cohort the assay resulted in a 99.6% to 99.9% negative predictive value. CONCLUSIONS: Epigenetic profiling using TWIST1, ONECUT2 and OTX1 results in a high sensitivity and specificity. Accurate risk prediction might result in less extensive and invasive examination of patients at low risk, thereby reducing unnecessary patient burden and health care costs.

Stratification based on methylation of TBX2 and TBX3 into three molecular grades predicts progression in patients with pTa-bladder cancer.

The potential risk of recurrence and progression in patients with non-muscle-invasive bladder cancer necessitates followup by cystoscopy. The risk of progression to muscle-invasive bladder cancer is estimated based on the European Organisation of Research and Treatment of Cancer score, a combination of several clinicopathological variables. However, pathological assessment is not objective and reproducibility is insufficient. The use of molecular markers could contribute to the estimation of tumor aggressiveness. We recently demonstrated that methylation of GATA2, TBX2, TBX3, and ZIC4 genes could predict progression in Ta tumors. In this study, we aimed to validate the markers in a large patient set using DNA from formalin-fixed and paraffin-embedded tissue. PALGA: the Dutch Pathology Registry was used for patient selection. We included 192 patients with pTaG1/2 bladder cancer of whom 77 experienced progression. Methylation analysis was performed and log-rank analysis was used to calculate the predictive value of each methylation marker for developing progression over time. This analysis showed better progression-free survival in patients with low methylation rates compared with the patients with high methylation rates for all markers (P<0.001) during a followup of ten-years. The combined predictive effect of the methylation markers was analyzed with the Cox-regression method. In this analysis, TBX2, TBX3, and ZIC4 were independent predictors of progression. On the basis of methylation status of TBX2 and TBX3, patients were divided into three new molecular grade groups. Survival analysis showed that only 8% of patients in the low molecular grade group progressed within 5 years. This was 29 and 63% for the intermediate- and high-molecular grade groups. In conclusion, this new molecular-grade based on the combination of TBX2 and TBX3 methylation is an excellent marker for predicting progression to muscle-invasive bladder cancer in patients with primary pTaG1/2 bladder cancer.

Molecular and clinical support for a four-tiered grading system for bladder cancer based on the WHO 1973 and 2004 classifications.

Currently, the use of two classification systems for bladder cancer grade is advocated in clinical guidelines because the WHO2004 classification has not been sufficiently validated with biological markers and follow-up. The slides of 325 primary non-muscle invasive bladder cancers from three hospitals were reviewed by one uro-pathologist in two separate sessions for the WHO1973 (G1, G2 and G3) and 2004 (papillary urothelial neoplasm of low malignant potential (LMP), low-grade (LG) and high-grade (HG)) classifications. FGFR3 status was examined with PCR-SNaPshot analysis. Expression of Ki-67, P53 and P27 was analyzed by immuno-histochemistry. Clinical recurrence and progression were determined. We performed validation and cross-validation of the two systems for grade with molecular markers and clinical outcome. Multivariable analyses were done to predict prognosis and pT1 bladder cancer. Grade review resulted in 88 G1, 149 G2 and 88 G3 lesions (WHO1973) and 79 LMP, 101 LG and 145 HG lesions (WHO2004). Molecular validation of both grading systems showed that FGFR3 mutations were associated with lower grades whereas altered expression (Ki-67, P53 and P27) was found in higher grades. Clinical validation showed that the two classification systems were both significant predictors for progression but not for recurrence. Cross-validation of both WHO systems showed a significant stepwise increase in biological (molecular markers) and clinical (progression) potential along the line: G1-LG-G2-HG-G3. The LMP and G1 categories had a similar clinical and molecular profile. On the basis of molecular biology and multivariable clinical data, our results support a four-tiered grading system using the 1973 and 2004 WHO classifications with one low-grade (LMP/LG/G1) category that includes LMP, two intermediate grade (LG/G2 and HG/G2) categories and one high-grade (HG/G3) category.

Immunohistochemical and genetic profiles of endometrioid endometrial carcinoma arising from atrophic endometrium.

OBJECTIVE: Endometrial carcinomas are divided into type I endometrioid endometrial carcinomas (EECs), thought to arise from hyperplastic endometrium, and type II nonendometrioid endometrial carcinomas, thought to arise from atrophic endometrium. However, a minority (20%) of EECs have atrophic background endometrium, which was shown to be a marker of a worse prognosis. This study compares the immunohistochemical and genetic profiles of this possible third type to that of the known two types. METHODS: 43 patients with grade 1 EEC and hyperplastic background endometrium (type I), 43 patients with grade 1 EEC and atrophic background endometrium (type III) and 21 patients with serous carcinoma (type II) were included (n=107). Tissue microarrays of tumor samples were immunohistochemically stained for PTEN, L1CAM, ER, PR, p53, MLH1, PMS2, ß-catenin, E-cadherin and MIB1. The BRAF, KRAS, and PIK3CA genes were analyzed for mutations. RESULTS: A significantly higher expression of ER and PR, and a lower expression of L1CAM, p53 and MLH1 were found in type I and III compared to type II carcinomas. Expression of E-cadherin was significantly reduced in type III compared to type I carcinomas. Mutation analysis showed significantly less mutations of KRAS in type III compared to type I and II carcinomas (p<0.01). CONCLUSION: There appear to be slight immunohistochemical and genetic differences between EECs with hyperplastic and atrophic background endometrium. Carcinogenesis of EEC in atrophic endometrium seems to be characterized by loss of E-cadherin and a lack of KRAS mutations. As expected, endometrioid and serous carcinomas were immunohistochemically different.

TERT promoter mutations and BRAF mutations are rare in sporadic, and TERT promoter mutations are absent in NF1-related malignant peripheral nerve sheath tumors.

Hot spot mutations in the promoter region of telomerase reverse transcriptase (TERT promoter mutations) occur frequently in tumors of neuroectodermal origin such as melanoma and glioma. Many of these tumors are of neuroectodermal or ectomesenchymal origin which is suggestive of TERT promoter mutations playing a role in the development of malignant peripheral nerve sheath tumors (MPNSTs). In melanoma a correlation has been suggested between the occurrence of TERT promoter mutations and v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) mutations. We investigated TERT promoter and BRAF mutation frequency in respectively 94 and 86 consecutive MPNST cases from our institute. TERT promoter mutation analysis on DNA from formalin-fixed, paraffin-embedded specimens was performed by SNaPshot analysis. Sequence analysis of BRAF was performed by bidirectional DNA sequencing. We identified TERT C228T or C250T promoter mutations in 10 % (9/94) and BRAF V600E mutations in 3 % (3/86) of MPNSTs. All TERT promoter- and BRAF mutations occurred in NF1 unrelated tumors. One co-occurrence of a TERT promoter- and a BRAF mutation was observed. In comparison with other neuroectodermal derived malignant neoplasms, TERT promoter mutations occur at relatively low frequency in MPNSTs. The observation of TERT promotor and BRAF mutations in sporadic MPNSTs and the absence of TERT promotor and rarity of BRAF mutations in NF1 related tumors may imply an alternative genetic route of tumor progression in both patient groups.

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.

Hypermethylation of the polycomb group target gene PCDH7 in bladder tumors from patients of all ages.

PURPOSE: Bladder tumors in patients younger than 20 years show a low incidence of the genetic and epigenetic aberrations typically found in older patients. One of the most common epigenetic aberrations in human malignancies is DNA hypermethylation. Polycomb group complexes have an important role during lineage choices in embryogenesis and their target genes are 12 times more likely to be methylated than nonpolycomb group target genes. We hypothesized that methylation of polycomb group target genes is an early event in urothelial carcinogenesis and thus might be observed in young patients. MATERIALS AND METHODS: We stratified 167 patients by age into 4 groups, including age less than 20 years in 14, 20 to 40 in 48, 40 to 60 in 47 and greater than 60 in 58. Five previously identified polycomb group target genes (MEIS1, ONECUT2, OTX1, PCDH7 and SOX21) were selected for methylation analysis. Methylation ratios were calculated by using the unmethylated and methylated signal. The outcome represented the fraction of methylated cells within one tumor. Genes with similar methylation ratios in all age groups were considered as potential bladder cancer initiating candidates. RESULTS: Three genes showed higher methylation ratios in tumors from older patients, including ONECUT2, SOX21 and OTX1 (each p <0.001). MEIS1 showed a similar methylation ratio in all groups but the median methylation ratio was low. PCDH7 showed a similar median methylation percent in all age categories, ie 54% at less than 20, 59% at 20 to 40, 59% at 40 to 60 and 67% at greater than 60 years (p = 0.1). CONCLUSIONS: Tumors from young patients showed less methylation for most markers. PCDH7 showed high methylation ratios in all age categories. Therefore, it could have an important role in early urothelial carcinogenesis.

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.

FGFR3 mutation analysis in voided urine samples to decrease cystoscopies and cost in nonmuscle invasive bladder cancer surveillance: a comparison of 3 strategies.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Keratinocytic epidermal nevi are associated with mosaic RAS mutations.

BACKGROUND: Activating RAS mutations in the germline cause rare developmental disorders such as Costello syndrome. Somatic RAS mutations are found in approximately 30% of human cancers. Keratinocytic epidermal nevi (KEN) represent benign congenital skin lesions arranged along Blaschko's lines. A subgroup of KEN is caused by hotspot oncogenic FGFR3 and PIK3CA mutations in mosaicism, but the majority lack these mutations. METHODS: This study screened 72 KEN for activating mutations in RAS genes and other oncogenes. RESULTS: Activating RAS mutations were identified in 28/72 (39%) of KEN. HRAS was the most commonly affected oncogene (86%), with the HRAS p.G13R substitution representing a new hotspot mutation. CONCLUSION: These results indicate that activating RAS somatic mutations leading to mosaicism result in benign KEN of the skin. Given the prevalence of KEN, mosaic HRAS mutations appear to be more common in patients than germline ones. These findings identify KEN as a mosaic RASopathy and lend further support to the notion that genetic mosaicism is an important contributor to disease.

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.

The FGFR3 mutation is related to favorable pT1 bladder cancer.

PURPOSE: Stage pT1 bladder cancer comprises a heterogeneous group of tumors for which different management options are advocated. FGFR3 mutations are linked to favorable (low grade/stage) pTa bladder cancer while altered P53 is common in cases of high grade, muscle invasive (pT2 or greater) bladder cancer. We determined the frequency of FGFR3 mutations and P53 alterations in patients with pT1 bladder cancer and correlated these data to histopathological variables and clinical outcomes. MATERIALS AND METHODS: We included 132 patients with primary pT1 bladder cancer from a total of 2 academic centers. A uropathologist reviewed the slides for grade and confirmed the pT1 diagnosis. FGFR3 mutation status was examined by SNaPshot® analysis and P53 expression was determined by standard immunohistochemistry. Kaplan-Meier and multivariate analyses were used to assess progression. RESULTS: FGFR3 mutations were detected in 37 of 132 pT1 bladder cancer cases (28%) and altered P53 was seen in 71 (54%). Only 8% of patients had the 2 molecular alterations (p = 0.001). FGFR3 mutation correlated with lower grade and altered P53 correlated with high grade pT1 bladder cancer. Median followup was 6.5 years. FGFR3 mutation status and carcinoma in situ were significant for predicting progression on univariate and multivariate analyses but P53 status was not. CONCLUSIONS: FGFR3 mutations selectively identify patients with pT1 bladder cancer who have favorable disease characteristics. Further study may confirm that FGFR3 identifies those who would benefit from a conservative approach to the disease.

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

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 patients with 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.