Ellen C. Zwarthoff

Molecular Grading of Urothelial Cell Carcinoma With Fibroblast Growth Factor Receptor 3 and MIB-1 is Superior to Pathologic Grade for the Prediction of Clinical Outcome

PURPOSE Fibroblast growth factor receptor 3 (FGFR3) mutations were recently found at a high frequency in well-differentiated urothelial cell carcinoma (UCC). We investigated the relationship between FGFR3 status and three molecular markers (MIB-1, P53, and P27kip1) associated with worse prognosis and determined the reproducibility of pathologic grade and molecular variables. PATIENTS AND METHODS In this multicenter study, we included 286 patients with primary (first diagnosis) UCC. The histologic slides were reviewed. FGFR3 status was examined by polymerase chain reaction-single strand conformation polymorphism and sequencing. Expression levels of MIB-1, P53, and P27kip1 were determined by immunohistochemistry. Mean follow-up was 5.5 years (range, 0.4 to 18.4 years). RESULTS FGFR3 mutations were detected in 172 (60%) of 286 UCCs. Grade 1 tumors had an FGFR3 mutation in 88% of patient samples and grade 3 tumors in 16% of patient samples. Conversely, aberrant expression patterns of MIB-1, P53, and P27kip1 were seen in 5%, 2%, and 3% of grade 1 tumors and in 85%, 60%, and 56% of grade 3 tumors, respectively. In multivariate analysis with recurrence rate, progression, and disease-specific survival as end points, the combination of FGFR3 and MIB-1 proved independently significant in all three cases. By using these two molecular markers, three molecular grades (mGs) could be identified: mG1 (mutation; normal expression), favorable prognosis; mG2 (two remaining combinations), intermediate prognosis; and mG3 (no mutation; high expression), poor prognosis. The molecular variables were more reproducible than pathologic grade (85% to 100% v 47% to 61%). CONCLUSION The FGFR3 mutation represents the favorable molecular pathway of UCC. Molecular grading provides a new, simple, and highly reproducible tool for clinical decision making in UCC patients.

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.

FGFR3 and P53 Characterize Alternative Genetic Pathways in the Pathogenesis of Urothelial Cell Carcinoma

Fibroblast growth factor receptor 3 (FGFR3) and P53 mutations are frequently observed in bladder cancer. We here describe the distribution of FGFR3 mutations and P53 overexpression in 260 primary urothelial cell carcinomas. FGFR3 mutations were observed in 59% and P53 overexpression in 25%. Interestingly, FGFR3 and P53 alterations were mutually exclusive, because they coincided in only 5.7% of tumors. Consequently, we propose that they characterize two alternative genetic pathways in urothelial cell carcinoma pathogenesis. The genetic alterations were reflected in the pathology and the clinical outcome, i.e., FGFR3 mutations were found in low-stage/-grade tumors and were associated with a favorable disease course, whereas P53 alterations were tied to adverse disease parameters.

Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi

Epidermal nevi are common congenital skin lesions with an incidence of 1 in 1,000 people; however, their genetic basis remains elusive. Germline mutations of the FGF receptor 3 (FGFR3) cause autosomal dominant skeletal disorders such as achondroplasia and thanatophoric dysplasia, which can be associated with acanthosis nigricans of the skin. Acanthosis nigricans and common epidermal nevi of the nonorganoid, nonepidermolytic type share some clinical and histological features. We used a SNaPshot multiplex assay to screen 39 epidermal nevi of this type of 33 patients for 11 activating FGFR3 point mutations. In addition, exon 19 of FGFR3 was directly sequenced. We identified activating FGFR3 mutations, almost exclusively at codon 248 (R248C), in 11 of 33 (33%) patients with nonorganoid, nonepidermolytic epidermal nevi. In 4 of these cases, samples from adjacent histologically normal skin could be analyzed, and FGFR3 mutations were found to be absent. Our results suggest that a large proportion of epidermal nevi are caused by a mosaicism of activating FGFR3 mutations in the human epidermis, secondary to a postzygotic mutation in early embryonic development. The R248C mutation appears to be a hot spot for FGFR3 mutations in epidermal nevi.

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.

Frequent FGFR3 mutations in urothelial papilloma.

Activating point mutations in the FGFR3 gene occur frequently in low‐grade and low‐stage bladder carcinomas, whereas they are rare in high‐grade carcinomas. This study investigates the incidence of FGFR3 mutations in 12 urothelial papillomas and 79 pTaG1 tumours which were regraded according to the 1998 WHO/ISUP classification system, resulting in 62 papillary urothelial neoplasms of low malignant potential (PUNs‐LMP) and 17 low‐grade papillary urothelial carcinomas (LG‐PUCs). FGFR3 mutation analysis of 21 ovarian Brenner tumours was also performed. Seventy‐seven cases were detected with a mutation in the FGFR3 gene. The mutations were exclusively found in bladder neoplasms. In urothelial papilloma, generally considered a benign lesion, 9/12 (75%) mutations were found. This report is the first to describe a genetic defect in urothelial papilloma. A comparable percentage of mutations was found in PUNs‐LMP (85%) and LG‐PUCs (88%). No mutations were found in matched normal DNA from bladder tumour patients. The mean follow‐up was 5.78 years (range 0.21–17.60 years). Five patients developed high‐grade papillary urothelial carcinoma from 2.5 to 12 years after first diagnosis. Two patients died of bladder cancer. The mean number of recurrences (recurrence rate) per year was 0.03, 0.21, and 0.46, respectively, for papilloma, PUN‐LMP, and LG‐PUC. Urothelial papilloma is a rare lesion with a benign natural behaviour compared with PUN‐LMP and LG‐PUC of the bladder, but from a molecular perspective, papillomas should be classified together with all well‐differentiated urothelial neoplasms. Copyright

Molecular Grade (FGFR3/MIB-1) and EORTC Risk Scores Are Predictive in Primary Non–Muscle-Invasive Bladder Cancer

BACKGROUND The European Organization for Research and Treatment of Cancer (EORTC) risk scores are not validated in an independent patient population. Molecular grade (mG) based on fibroblast growth factor receptor 3 (FGFR3) gene mutation status and MIB-1 expression was proposed as an alternative to pathologic grade in bladder cancer (BCa) [1]. OBJECTIVE To validate the EORTC risk score and to determine its relation to mG in a series with long-term follow-up as well as to determine reproducibility of pathologic grade and mG. DESIGN, SETTING, AND PARTICIPANTS In this multicenter study, we included 230 patients with primary non-muscle-invasive BCa (NMIBC). MEASUREMENTS Four uropathologists reviewed the slides. FGFR3 mutation status was examined by two assays. MIB-1 was assessed by immunohistochemistry. The EORTC risk scores for recurrence and progression were determined. Multivariable analyses were used to find prognostic factors. RESULTS AND LIMITATIONS Median follow-up was 8.62 yr (interquartile range: 6.6-11.8). FGFR3 mutations were significantly related to favorable disease parameters, whereas altered MIB-1 was frequently seen with pT1, high grade, and high EORTC risk scores. EORTC risk scores were significant in multivariable analyses for recurrence and progression. In multivariable analyses for progression and disease-specific survival, the mG had independent significance. The addition of mG to the multivariable model for progression increased the predictive accuracy from 74.9% to 81.7% (p<0.001; Mantel-Haenszel test). The mG (89%) was more reproducible than the pathologic grade (41-74%). CONCLUSIONS We validated the EORTC risk scores for primary NMIBC in a clinical and biomarker setting. Next to EORTC risk score, mG proved highly reproducible and predictive. Our long-term results justify an independent prospective analysis of mG and EORTC risk scores.

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.

Novel fibroblast growth factor receptor 3 (FGFR3) mutations in bladder cancer previously identified in non-lethal skeletal disorders

Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several autosomal dominant craniosynostosis syndromes and chondrodysplasias i.e. hypochondroplasia, achondroplasia, SADDAN and thanatophoric dysplasia – a neonatal lethal dwarfism syndrome. Recently, activating FGFR3 mutations have also been found to be present in cancer, i.e. at high frequency in carcinoma of the bladder and rarely in multiple myeloma and carcinoma of the cervix. Almost all reported mutations in carcinomas corresponded to the mutations identified in thanatophoric dysplasia. We here screened a series of 297 bladder tumours and found three FGFR3 somatic mutations (G380/382R; K650/652M and K650/652T) that were not previously identified in carcinomas or thanatophoric dysplasia. Another novel finding was the occurrence of two simultaneous FGFR3 mutations in four tumours. Two of the three new mutations in bladder cancer, the G380/382R and the K650/652M mutations, were previously reported in achondroplasia and SADDAN, respectively. These syndromes entail a longer life span than thanatophoric dysplasia. The K650/652T mutation has not previously been detected in patients with skeletal disorders, but affects a codon that has been shown to be affected in some cases of thanatophoric dysplasia, SADDAN and hypochondroplasia. From a clinical perspective, the patients with FGFR3-related, non-lethal skeletal disorders might be at a higher risk for development of bladder tumours than the general population.

Markers Predicting Response to Bacillus Calmette-Guérin Immunotherapy in High-Risk Bladder Cancer Patients: A Systematic Review

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