Assessment of prognostic implication of a panel of oncogenes in bladder cancer and identification of a 3-gene signature associated with recurrence and progression risk in non-muscle-invasive bladder cancer.

This study evaluated the prognostic value of a panel of 29 oncogenes derived from the analysis of The Cancer Genome Atlas (TCGA data) or from the recent literature on bladder tumors on a well-characterized series of muscle-invasive bladder cancer (MIBC) and non-MIBC (NMIBC) samples and tried to identify molecular prognostic markers. Mutations of HRAS, FGFR3, PIK3CA and TERT were found in 2.9%, 27.2%, 14.9% and 76.7% of tumor samples, respectively. Concerning NMIBC, on multivariate analysis, RXRA and FGFR3 levels were associated with recurrence-free survival (RFS) (p = 0.0022 and p = 0.0069) and RXRA level was associated with progression to muscle-invasive disease (p = 0.0068). We identified a 3-gene molecular signature associated with NMIBC prognosis. FGFR3 overexpression was associated with reduced response to Bacillus Calmette-Guerin treatment (p = 0.037). As regards MIBC, on multivariate analysis, ERCC2 overexpression was associated with RFS (p = 0.0011) and E2F3 and EGFR overexpression were associated with overall survival (p = 0.014 and p = 0.035). RT-PCR findings were confirmed by IHC for FGFR3. Genomic alterations in MIBC revealed in TCGA data also concern NMIBC and seem to be associated with prognosis in terms of recurrence and progression. Correcting these alterations by targeted therapies seems a promising pharmacological approach.

Baseline Computed Tomography Radiomic and Genomic Assessment of Head and Neck Squamous Cell Carcinoma.

PURPOSE: To determine the relationship between computed tomography (CT) radiomic features and gene expression levels in head and neck squamous cell carcinoma (HNSCC). METHODS: This retrospective study included 66 patients with HNSCC primary lesions (36 oropharyngeal, 6 hypopharyngeal, 10 laryngeal, 14 oral cavity). Gene expression information for 6 targetable genes (fibroblast growth factor receptor [FGFR]1, epidermal growth factor receptor [EGFR], FGFR2, FGFR3, EPHA2, PIK3CA) was obtained via Agilent microarrays from samples collected between 1997 and 2010. Pretreatment contrast-enhanced soft tissue neck CT scans were reviewed, and 142 radiomics features were derived. R was used to calculate Pearson correlation coefficients were calculated between gene expression levels and each radiomic feature. P values were adjusted using the false discovery rate (FDR) method. RESULTS: There were significant correlations between FGFR1 and 5 gray level cooccurrence matrix (GLCM) features with FDR-adjusted P values less than 0.05: inertia (r = 0.366, FDR-adjusted P = 0.006), absolute value (r = 0.31, FDR-adjusted P = 0.024), contrast (r = 0.366, FDR-adjusted P = 0.006), difference average (r = 0.31, FDR-adjusted P = 0.024), and difference variance (r = 0.37, FDR-adjusted P = 0.005). There was 1 correlated feature for FGFR2 with an FDR-adjusted P value less than 0.05: fractal dimension box-coarse (r = 0.33, FDR-adjusted P = 0.018). There was 1 correlated feature for EPHA2 with an FDR-adjusted P value less than 0.05: GLCM entropy (r = -0.28, FDR-adjusted P = 0.049). Six of the 7 features that showed significant correlation belonged to the GLCM class of features. CONCLUSIONS: The CT radiomic features demonstrate correlations with FGFR1 status in HNSCC and should be further investigated for their potential to predict FGFR1 status.

Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation.

Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: 'microgenomics'. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.

TP53 and FGFR3 Gene Mutation Assessment in Urine: Pilot Study for Bladder Cancer Diagnosis.

AIM: To assess, in a prospective clinical research study, a new non-invasive and reliable test to accurately detect tumor protein 53 (TP53) and fibroblast growth factor receptor-3 (FGFR3) mutations in cells in urine. MATERIALS AND METHODS: TP53 mutations were analyzed using the functional analysis of separated allele in yeast (FASAY) method, which allows functional analysis of the P53 protein, and FGFR3 mutations were assessed with the SNaPshot system, detecting the eight most frequent point-mutations of this gene. Chi-square test or Fisher's exact test were used to compare TP53 and FGFR3 mutations in the tumors according to tumor stage and grade. RESULTS: TP53 and FGFR3 mutations in bladder tumors increased and decreased respectively with increasing tumor stage and cellular grade (p<0.05 and p<0.001, respectively). A total of 103 tumor/urinary sediment couples were analyzed. TP53 or FGFR3 mutations were observed in 76 tumors. The sensitivity for the detection of this type of mutation in urine was 46%, the specificity was 81%, the positive predictive value was 94% and the negative predictive value was 37%. CONCLUSION: Our original data confirmed the feasibility of TP53 and FGFR3 mutation detection in urine sediment. These measurements, together with urine cytology, may increase tumor detection. The sensitivity of the TP53/FGFR3 phenotype test in the urine was less than 50% and was not able to replace standard cystoscopy in the diagnosis of bladder tumors.

Role of dimerization efficiency of transmembrane domains in activation of fibroblast growth factor receptor 3.

Mutations in transmembrane (TM) domains of receptor tyrosine kinases are shown to cause a number of inherited diseases and cancer development. Here, we use a combined molecular modeling approach to understand molecular mechanism of effect of G380R and A391E mutations on dimerization of TM domains of human fibroblast growth factor receptor 3 (FGFR3). According to results of Monte Carlo conformational search in the implicit membrane and further molecular dynamics simulations, TM dimer of this receptor is able to form a number of various conformations, which differ significantly by the free energy of association in a full-atom model bilayer. The aforementioned mutations affect dimerization efficiency of TM segments and lead to repopulation of conformational ensemble for the dimer. Particularly, both mutations do not change the dimerization free energy of the predominant (putative "non-active") symmetric conformation of TM dimer, while affect dimerization efficiency of its asymmetric ("intermediate") and alternative symmetric (putative "active") models. Results of our simulations provide novel atomistic prospective of the role of G380 and A391E mutations in dimerization of TM domains of FGFR3 and their consecutive contributions to the activation pathway of the receptor.

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.

Direct assessment of the effect of the Gly380Arg achondroplasia mutation on FGFR3 dimerization using quantitative imaging FRET.

The Gly380Arg mutation in FGFR3 is the genetic cause for achondroplasia (ACH), the most common form of human dwarfism. The mutation has been proposed to increase FGFR3 dimerization, but the dimerization propensities of wild-type and mutant FGFR3 have not been compared. Here we use quantitative imaging FRET to characterize the dimerization of wild-type FGFR3 and the ACH mutant in plasma membrane-derived vesicles from HEK293T cells. We demonstrate a small, but statistically significant increase in FGFR3 dimerization due to the ACH mutation. The data are consistent with the idea that the ACH mutation causes a structural change which affects both the stability and the activity of FGFR3 dimers in the absence of ligand.