Zwarthoff, E.C.

FGFR3 Mutation Analysis in Voided Urine Samples to Decrease Cystoscopies and Cost in Nonmuscle Invasive Bladder Cancer Surveillance: A Comparison of 3 Strategies (Article)

2013-03-22T00:00:00Z

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.

Markers predicting response to bacillus Calmette-Guérin immunotherapy in high-risk bladder cancer patients: A systematic review (Article)

2012-01-01T00:00:00Z

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.

Spectrum of KIT/PDGFRA/BRAF mutations and Phosphatidylinositol-3-Kinase pathway gene alterations in gastrointestinal stromal tumors (GIST) (Article)

2011-12-15T00:00:00Z

Pathogenetic pathways of gastrointestinal stromal tumors (GIST) lacking mutations in KIT and PDGFRA (∼15%) are still poorly studied. Nearly nothing is known about PI3K alterations in GISTs and only a few GISTs with BRAF mutations have been reported. BRAF mutations (V600E) were found in 3/87 tumors (3.5%) concomitantly were wild type for KIT and PDGFRA. No mutations were detected in KRAS, NRAS, and FGFR3. For the first-time we demonstrated a PIK3CA mutation (H1047L) simultaneously occurring with a 15-bp deletion in KIT exon 11 in one tumor. We suggest that BRAF mutations are of pathogenetic significance in wild type GISTs. The PI3K pathway should be assessed in future studies.

Optimization of nonmuscle invasive bladder cancer recurrence detection using a urine based FGFR3 mutation assay (Article)

2011-08-01T00:00:00Z

Purpose: FGFR3 mutations occur in 70% of nonmuscle invasive bladder tumors. Although urine based FGFR3 mutation analysis can detect recurrence, its sensitivity may be limited if samples have few or no tumor cells. We determined whether test sensitivity depends on tumor size and the time point of urine collection, and how to increase sensitivity. Materials and Methods: A total of 440 urine samples from 18 patients with a suspicious bladder lesion at cystoscopy were collected during 6 days before surgery. Eight patients (300 samples) had an FGFR3 mutant tumor, including 4 each with a tumor greater than 3 and less than 1.5 cm. Polymerase chain reaction based FGFR3 analysis was done on all tumors and urine samples. Results: FGFR3 mutations were detected in 257 of the 300 urine samples (86%) from patients with an FGFR3 mutant tumor. Assay sensitivity was 100% for tumors greater than 3 cm and 75% for tumors less than 1.5 cm. It increased to 100% in patients with a less than 1.5 cm tumor when samples were pooled during 24 hours. Sensitivity was not influenced by the time of urine collection. All urine samples from patients with an FGFR3 wild-type tumor were negative for FGFR3 mutation. Conclusions: The sensitivity of tumor detection increased with tumor size. FGFR3 assay sensitivity depends on the number of shed tumor cells and improves by increasing urine volume. These findings suggest that there is an upper limit to the sensitivity of the FGFR3 assay when 1 urine sample is analyzed. This may also apply to other DNA or RNA based assays.

No evidence of FGFR3 mutations in prostate cancer (Article)

2011-05-01T00:00:00Z

BACKGROUND FGFR3 mutations are associated with a good clinical disease course in bladder tumors. Currently, prognostic markers to stratify prostate cancer (PCa) patients for conservative management are lacking. Conflicting results have been found on the presence of FGFR3 mutations in PCa. Our objective was to determine the prevalence of FGFR3 mutations in a subset of prostate tumors. Next, determine the prevalence of FGFR3 mutations in PCa patients with coexistent tumors in other tissues. METHODS Primary and locally advanced prostate tumors (n = 132) were collected at our medical center. From the 132 PCa patients, 28 (21%) were diagnosed with coexistent primary tumors (bladder, skin, pancreas, renal cell, gastric, colon, hepatic, and lung). Tumors were analyzed by FGFR3 mutation analysis on exon 7, 10, and 15, known to harbor the most frequent mutations. RESULTS The prevalence of FGFR3 mutations in patients with only PCa was 0%. Most PCa patients presented with coexistent bladder (n = 12) and bladder and skin tumors (n = 7). Other coexistent tumors in PCa patients included: bladder and pancreatic cancer (n = 1); bladder and renal cell carcinoma (n = 1); bladder and gastric carcinoma (n = 1); skin cancer (n = 1); colon cancer (n = 3); hepatic carcinoma (n = 1); and lung cancer (n = 1). FGFR3 mutations were detected in 9/15 (60%) analyzed bladder tumors. CONCLUSIONS FGFR3 mutations were absent in the investigated prostate tumors, suggesting a minor role of these mutations in tumorigenesis. Hence, FGFR3 mutation analysis is not suitable to select patients for conservative management. Interestingly, if a prostate tumor coincided with other tumors these were mostly bladder and skin.

FGFR3, HRAS, KRAS, NRAS AND PIK3CA mutations in bladder cancer and their potential as biomarkers for surveillance and therapy (Article)

2010-11-18T00:00:00Z

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.

Feasibility study of screening for bladder cancer with urinary molecular markers (the BLU-P project) (Article)

2010-11-01T00:00:00Z

Introduction: The prognosis of bladder cancer (BC) depends mainly on its histology, grade, and stage. Patients with superficial BC (70% of the urothelial carcinomas) have a relatively good prognosis, but patients diagnosed with invasive, high grade BC, and those who progress to invasive BC, have a poor prognosis and will not survive their disease in many cases due to their metastases, despite the currently available treatment options. Early detection can only be beneficial regarding mortality if the high risk cancers are recognized and treated at a localized stage. Materials and methods: Previous pilot studies on early detection consisted of home-based repeated hematuria testing and, in case of hematuria, a urologic evaluation with cytology and cystoscopy was carried out. This design resulted in too many cystoscopies. The recently initiated [Bladder Cancer Urine Marker Project (BLU-P) study www.blu-project.org] assesses the feasibility of a population-based screening for BC and at the same time evaluates a screening algorithm using next to hematuria testing, sensitive specific urine markers for BC (NMP22, FGFR3, MA analyses and MLPa) in an attempt to circumvent the high number of cystoscopies. Results: So far 1,611 men are included and 23.5% tested positive for hematuria (11.6% had one or more true positive test results). The additional molecular-based screening tests before referring to cystoscopy resulted in a decrease of the number of cystoscopies from 378 to 66 (82.5%). In those men referred for cystoscopy, so far only 1 BC case was detected. Conclusions: Further research is needed to evaluate whether this extremely low detection rate is caused by, e.g., a healthy screenee bias or that the additional selection step using the molecular urine tests is too strict and diagnoses are missed.

Fibroblast growth factor receptor 3 mutation analysis on voided urine for surveillance of patients with low-grade non-muscle - Invasive bladder cancer (Article)

2010-06-01T00:00:00Z

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.

Distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines (Article)

2010-05-01T00:00:00Z

Breast cancer has for long been recognized as a highly diverse tumor group, but the underlying genetic basis has been elusive. Here, we report an extensive molecular characterization of a collection of 41 human breast cancer cell lines. Protein and gene expression analyses indicated that the collection of breast cancer cell lines has retained most, if not all, molecular characteristics that are typical for clinical breast cancers. Gene mutation analyses identified 146 oncogenic mutations among 27 well-known cancer genes, amounting to an average of 3.6 mutations per cell line. Mutations in genes from the p53, RB and PI3K tumor suppressor pathways were widespread among all breast cancer cell lines. Most important, we have identified two gene mutation profiles that are specifically associated with luminal-type and basal-type breast cancer cell lines. The luminal mutation profile involved E-cadherin and MAP2K4 gene mutations and amplifications of Cyclin D1, ERBB2 and HDM2, whereas the basal mutation profile involved BRCA1, RB1, RAS and BRAF gene mutations and deletions of p16 and p14ARF. These subtype-specific gene mutation profiles constitute a genetic basis for the heterogeneity observed among human breast cancers, providing clues for their underlying biology and providing guidance for targeted pharmacogenetic intervention in breast cancer patients.

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

2010-03-30T00:00:00Z

Background. The 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 Presentation. We 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. Conclusions. Our findings would suggest that the BC disease course is determined by not only a patient's 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.

Cystoscopy Revisited as the Gold Standard for Detecting Bladder Cancer Recurrence: Diagnostic Review Bias in the Randomized, Prospective CEFUB Trial (Article)

2010-01-01T00:00:00Z

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.

Expression of the Endothelin Axis in Noninvasive and Superficially Invasive Bladder Cancer: Relation to Clinicopathologic and Molecular Prognostic Parameters (Article)

2009-11-01T00:00:00Z

Background: The endothelin (ET) axis plays a role in cancer biology and plays a potential role as a target for molecular therapy in urogenital tumours. Alterations of several proteins of the ET axis were detected in invasive bladder cancer. Objectives: To examine the potential role of the expression of ET axis proteins compared to other prognostic parameters (kinase inhibitor 67 [Ki-67], tumour protein 53 [TP53], and fibroblast growth factor receptor 3 gene [FGFR3] mutations) in noninvasive and invasive bladder cancer. Design, setting, and participants: Tissue microarrays from 154 consecutive patients with pTa-pT2 urothelial bladder cancer were immunohistochemically stained for endothelin 1 (ET-1), endothelin A and B receptors (ETAR, ETBR), TP53, and Ki-67. FGFR3 mutations were detected by SNaPshot analysis. Measurements: The results were correlated with clinicopathologic parameters and disease-specific survival, overall survival, and recurrence-free survival. Results and limitations: Proteins of the ET axis were frequently expressed in bladder cancer (ET-1 in 62% of tumours, ETAR in 93% of tumours, and ETBR in 84% of tumours). ET-1 expression was strongly correlated with tumour stage (p = 0.015), histologic grade (p = 0.008), and low proliferation status (p = 0.003). ETAR immunostaining was only associated with low proliferation status (p = 0.015). Kaplan-Meier survival analysis showed a significantly longer overall survival for patients with ET-1-expressing tumours (p = 0.007). A significantly longer disease-free survival was found in patients with ETAR-expressing tumours (p = 0.040), whereas ETBR expression was significantly correlated to a longer disease-free survival only in subgroups of patients with multifocal tumours (p = 0.031), low proliferation index (Ki-67 ≤10; p = 0.050), low TP53 expression (≤10; p = 0.018), and tumours with an FGFR3 mutation (p = 0.026). In the global model for recurrence-free survival, only high-grade (p = 0.048) and negative ETAR immunoreactivity (p = 0.048) were correlated with poor prognosis. Conclusions: In addition to other factors, particularly age at diagnosis and growth pattern, lack of ET-1 expression may be an independent negative prognostic factor for the overall-survival probability of bladder cancer patients. Lack of ETAR expression may be an independent negative marker for recurrence-free survival.

Clinical and pathological prognostic factors for recurrence, progression and mortality in non-muscle invasive bladder cancer: A meta-analysis (Article)

2009-11-01T00:00:00Z

Introduction: We aimed to quantify the importance of conventional clinical and pathological prognostic factors in the outcome of non-muscle invasive bladder cancer based on literature. Materials: We identified 14 publications that reported on the association between clinical and pathological prognostic factors for tumor recurrence, tumor progression and overall mortality of non-muscle invasive bladder cancer. Relative risks (RR) were estimated using meta-analytic techniques to combine results of separate studies. Results: In total, 2,536 of 5,021 patients had a tumor recurrence, 317 of 3,313 had tumor progression and 531 of 1,548 died. The strongest prognostic factor for tumor recurrence was the result of the first 3-monthly cystoscopy (RR 2.2, 95% confidence interval (CI) 1.8-2.7). The strongest prognostic factor for tumor progression was grade 3 compared to grade 1 (RR 6.7, 95% CI 4.7-9.5). The presence of carcinoma in situ had a strong effect on tumor progression (RR 4.4, 95% CI 3.4-5.5) and on the overall mortality (RR 4.0, 95% CI 2.4-6.5). Tumor shape, grade 3 and patient age also had strong effects on survival. Conclusion: Clinical and pathological prognostic factors could not predict tumor recurrence, but showed strong associations with tumor progression and overall mortality. To individualize surveillance strategies emphasis should be on the search for a panel of conventional and molecular prognostic factors that can reliably predict recurrence and progression.

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

2009-07-31T00:00:00Z

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 (Euro 4104 vs Euro 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 >/=61%, had a specificity of 73%, and decreased the costs of the MA test per follow-up sample from Euro 158 to

Sexual function of patients under surveillance for bladder cancer (Article)

2009-07-01T00:00:00Z

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.

Detection of urothelial bladder cancer cells in voided urine can be improved by a combination of cytology and standardized microsatellite analysis (Article)

2009-06-01T00:00:00Z

Purpose: To evaluate molecular and immunohistochemical markers to develop a molecular grading of urothelial bladder cancer and to test these markers in voided urine samples. Experimental Design: 255 consecutive biopsies from primary bladder cancer patients were evaluated on a tissue microarray. The clinical parameters gender, age, adjacent carcinoma in situ, and multifocality were collected. Uro-Vysion fluorescence in situ hybridization (FISH) was done. Expression of cytokeratin 20, MIB1, and TP53 was analyzed by immunohistochemistry. Fibroblast growth factor receptor 3 (FGFR3) status was studied by SNaPshot mutation detection. Results were correlated with clinical outcome by Cox regression analysis. To assess the predictive power of different predictor subsets to detect high grade and tumor invasion, logistic regression models were learned. Additionally, voided urine samples of 119 patients were investigated. After cytologic examination, urine samples were matched with their biopsies and analyzed for loss of heterozygosity (LOH), FGFR3 mutation, polysomy, and p16 deletion using Uro-Vysion FISH. Receiver operator characteristic curves for various predictor subsets were plotted. Results: In biopsies, high grade and solid growth pattern were independent prognostic factors for overall survival. A model consisting of Uro-Vysion FISH and FGFR3 status (FISH + FGFR3) predicted high grade significantly better compared with a recently proposed molecular grade (MIB1 + FGFR3). In voided urine, the combination of cytology with LOH analysis (CYTO + LOH) reached the highest diagnostic accuracy for the detection of bladder cancer cells and performed better than cytology alone (sensitivity of 88.2% and specificity of 97.1%). Conclusions: The combination of cytology with LOH analysis could reduce unpleasant cystoscopies for bladder cancer patients. Copyright

The development of multiple bladder tumour recurrences in relation to the FGFR3 mutation status of the primary tumour (Article)

2009-05-01T00:00:00Z

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.Inthe 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.

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]) (Article)

2009-03-01T00:00:00Z

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.

Bladder cancer biomarkers and their role in surveillance and screening (Article)

2009-01-01T00:00:00Z

Early detection of bladder cancer and its recurrences is essential for improved prognosis and long-term survival. The detection and follow-up of these patients is currently based on cystoscopy, which is expensive and invasive, and, in most cases, cytology, which is non-invasive but not very sensitive. During recent years, many urine-based tests have been developed and tested in different patient populations. In this review we discuss new developments for biomarkers in bladder cancer that have potential use in surveillance and screening. In almost all publications authors compare sensitivity of the test with a concomitantly executed cystoscopy, for example, determine cross-sectional sensitivity. However, it has also been shown that false positive test results may be followed by a positive cystoscopy in the near future, showing that cystoscopy itself does not provide 100% sensitivity. This suggests that for a proper evaluation of urine-based tests, longitudinal studies should be carried out and the results communicated to the urologist.

MN1 affects expression of genes involved in hematopoiesis and can enhance as well as inhibit RAR/RXR-induced gene expression (Article)

2008-10-13T00:00:00Z

The oncoprotein meningioma 1 (MN1) is overexpressed in several subtypes of acute myeloid leukemia (AML) and overexpression was associated with a poor response to chemotherapy. MN1 is a cofactor of retinoic acid receptor/retinoic x receptor (RAR/RXR)-mediated transcription and this study identified genes in the promonocytic cell line U937 that were regulated by MN1. We found that MN1 can both stimulate and inhibit transcription. Combining MN1 expression with all-trans retinoic acid (ATRA), the ligand of the RAR/RXR dimer, showed that MN1 could both enhance and repress ATRA effects. Many of the identified genes are key players in hematopoiesis and leukemogenesis (e.g. MEIS1 and BMI1). Another interesting target is DHRS9. DHRS9 is involved in the synthesis of ATRA from vitamin A. MN1 inhibited DHRS9 expression and completely abolished its induction by ATRA. MN1 is also the target of a rare AML-causing translocation encoding the MN1-TEL protein. MN1-TEL induces expression of only a few genes and its most pronounced effect is inhibition of a large group of ATRA-induced genes including DHRS9. In conclusion, both MN1 and MN1-TEL interfere with the ATRA pathway and this might explain the differentiation block in leukemias in which these genes are involved.

DNA: stabiel en veranderlijk (Inaugural Lecture)

2008-10-10T00:00:00Z

DNA en kanker zijn onlosmakelijk met elkaar verbonden. Kanker is een ziekte van het DNA. Het zijn veranderingen in het DNA die van een gewone cel een kankercel maken. In de Verenigde Staten van Amerika is kanker inmiddels de belangrijkste doodsoorzaak|1. De sterfte aan hart en vaatziekten is de laatste decennia hard omlaag gegaan. Bij kanker gaat dat helaas beduidend minder snel. Waarom is dat het geval en wat maakt kanker zo’n lastig probleem? Ik zal proberen u in grote lijnen een beeld te schetsen van de huidige stand van de wetenschap met betrekking tot de moleculaire achtergronden van tumorontwikkeling en de problemen die er zijn in het onderzoek aan kanker. Rede, in verkorte vorm uitgesproken ter gelegenheid van het aanvaarden van het ambt van bijzonder hoogleraar met als leeropdracht Moleculaire Pathologie van Tumorontwikkeling aan het Erasmus MC, faculteit van de Erasmus Universiteit Rotterdam op 10 oktober 2008.

Detection of tumours of the urinary tract in voided urine (Article)

2008-10-06T00:00:00Z

Patients with non-muscle-invasive bladder cancer are treated by transurethral resection. About 60-70% of these patients will develop recurrences and in 11% of these cases progression to a muscle-invasive tumour occurs. Surveillance of patients by cystoscopy is therefore carried out every 3-4 months in the first 2years and yearly thereafter. Several biomarkers have been developed that potentially can detect recurrent bladder cancer in voided urine samples and may present an alternative for the invasive cystoscopy procedure. Recently, van Rhijn reviewed the performance of several of these biomarkers regarding detection of recurrent disease in patients under surveillance. In general, sensitivities were much lower when only patients under surveillance were taken into account than when the patient cohorts included patients with primary disease or patients with high-grade tumours. In this article recent new data on those markers that displayed a sensitivity and specificity of at least 70% as mentioned in the review by van Rhijn are reviewed. The literature selected was limited to those papers in which the performance of makers was assayed only on urine samples of patients under surveillance. The markers with sensitivity and specificity over 70% that were selected from the previous study are Lewis X, NMP22, microsatellite analysis (MA), CYFRA 21.1, cytokeratin 20 and the UroVysion fluorescence in situ hybridization (FISH) test. Recent new developments such as the use of FGFR3 mutation analysis and methylation detection are also discussed. In conclusion, tests such as the UroVysion FISH test and MA are able to detect most concomitant recurrences and to predict recurrent disease. In general, lesions that are missed are pTa and low grade. With MA several upper tract recurrences were identified that were missed by cystoscopy. The value of the most promising urine tests needs to be established in longitudinal studies and exclusively on patients under surveillance for recurrent disease. A longitudinal setting allows subsequent urine samples to be tested and this increases sensitivity because a negative test outcome sometimes occurs between positive ones. Stratification of patients according to the genetic status of their primary tumours and smoking habits should be investigated. Decision models should be developed that recommend at which points in time cystoscopy or urine testing should be performed.

Prediction of Progression of Non-Muscle-Invasive Bladder Cancer by WHO 1973 and 2004 Grading and by FGFR3 Mutation Status: A Prospective Study (Article)

2008-10-01T00:00:00Z

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 (Article)

2008-05-01T00:00:00Z

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.

FGFR3 Mutations and a Normal CK20 Staining Pattern Define Low-Grade Noninvasive Urothelial Bladder Tumours (Article)

2007-09-01T00:00:00Z

Objectives: Molecular markers superior to conventional clinicopathologic parameters are needed to predict disease courses in bladder cancer patients. In this study, we investigated four markers (Ki-67, TP53, CK20, FGFR3) in primary urothelial bladder tumours and compared them with traditional pathologic features. Methods: Tissue microarrays were used to analyse CK20, TP53, and Ki-67 expression immunohistochemically in 255 unselected patients. FGFR3 mutations were detected by SNaPshot analysis. Results: Abnormal CK20 expression was strongly associated with higher tumour grades and stages (p < 0.001); however, 65% of pTa tumours revealed an abnormal CK20 pattern. In the group of pTaG1 tumours, 59% presented with an abnormal CK20 pattern, whereas 82% carried the FGFR3 mutation. In the group of bladder tumours with normal CK20 pattern, the FGFR3 gene was mutated in 89%, whereas a mutated FGFR3 gene was found in only 37% of cases with abnormal CK20 expression (p < 0.001). All markers proved to be strong predictors of disease-specific survival in univariate studies. However, in multivariate analyses they were not independent from classical pathologic parameters. None of the molecular markers was significantly associated with tumour recurrence. Conclusions: Dysregulation of CK20 expression is an early event in the carcinogenesis of papillary noninvasive bladder cancer, but occurs later than FGFR3 mutations. The group of low-grade noninvasive papillary tumours is defined by the presence of an FGFR3 mutation and a normal CK20 expression pattern.

Low frequency of molecular changes and tumor recurrence in inverted papillomas of the urinary tract (Article)

2007-06-01T00:00:00Z

AIM: Inverted papilloma (IP) of the urinary tract can be difficult to distinguish from noninvasive urothelial carcinoma with prominent inverted growth pattern (invNIUC). Ancillary markers may help to resolve such cases and clarify the reported malignant potential of some IPs. METHODS: Eighty-nine urothelial lesions initially diagnosed as IP were reviewed by 4 experienced urologic pathologists and studied immunohistochemically (Ki67, p53, CK20, MSH2, MLH1, and MSH6). Mutations of the FGFR3 gene, deletions (loss of heterozygosity) of 9p, 9q, and 17p, microsatellite instability, and elevated microsatellite instability at selected tetranucleotides were also analyzed. RESULTS: Considerable interobserver variability in histopathologic diagnoses was noticed. Only 62 (69.7%) initial diagnoses were confirmed by the review pathologists whereas 23 tumors (25.8%) were redefined as invNIUC. Molecular analyses revealed infrequent alterations in IPs, including microsatellite instability (1.8%), elevated microsatellite instability at selected tetranucleotides (13.2%), FGFR3 mutations (9.8%), 9p deletions (3.9%), 9q deletions (13.2%), 17p deletions (5.1%), nuclear p53 accumulation (18.9%), and aberrant immunostaining for MSH2 (5.8%), MLH1 (11.8%), and MSH6 (3.8%). IP and invNIUC differed in FGFR3 mutations and Ki-67 labeling index (P<0.001 each), and 9q loss of heterozygosity (P=0.03). There were fewer recurrences in IP (5.4%) compared with invNIUC (40.9%; P<0.0001). CONCLUSIONS: IP is a benign lesion that lacks specific genetic alterations found in exophytic noninvasive papillary urothelial tumors. These lesions could be reactive in nature, perhaps secondary to chronic inflammation or a neoplastic process that lack specific genetic alterations. Nevertheless given the clinical and molecular data of this study a conservative clinical approach is appropriate.

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

2005-11-01T00:00:00Z

Purpose:Mutations in the fibroblast growth factor receptor 3 (FGFR3) occur in 50% of primary bladder tumors.An FGFR3 mutationis associatedwith goodprognosis, 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 detectionof recurrences inurine is animportant clinical issue. Inthis 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 amutation. 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 1ng 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 detectionwas 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.

Targeted disruption of the Mn1 oncogene results in severe defects in development of membranous bones of the cranial skeleton. (Article)

2005-05-01T00:00:00Z

Fusion of the MN1 gene to TEL (ETV6) results in myeloid leukemia. The fusion protein combines the transcription activating domain of MN1 and the DNA binding domain of TEL and is thought to act as a deranged transcription factor. In addition, disruption of the large first exon of the MN1 gene is thought to inactivate MN1 function in a meningioma. To further investigate the role of MN1 in cancer, we generated Mn1 knockout mice. Mn1(+/-) animals were followed for 30 months, but they had no higher incidence of tumor formation than wild-type littermates. Mn1 null mice, however, were found to die at birth or shortly thereafter as the result of a cleft palate. Investigation of newborn or embryonic day 15.5 (E15.5) to E17.5 null mice revealed that the development of several bones in the skull was abnormal. The affected bones are almost exclusively formed by intramembranous ossification. They are either completely agenic at birth (alisphenoid and squamosal bones and vomer), hypoplastic, deformed (basisphenoid, pterygoid, and presphenoid), or substantially thinner (frontal, parietal, and interparietal bones). In heterozygous mice hypoplastic membranous bones and incomplete penetrance of the cleft palate were observed. We conclude that Mn1 is an important factor in development of membranous bones.

FGFR3 and P53 characterize alternative genetic pathways in the pathogenesis of urothelial cell carcinoma (Article)

2004-01-01T00:00:00Z

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.

Combined microsatellite and FGFR3 mutation analysis enables a highly sensitive detection of urothelial cell carcinoma in voided urine (Article)

2003-01-01T00:00:00Z

PURPOSE: Fibroblast growth factor receptor 3 (FGFR3) mutations were reported recently at a high frequency in low-grade urothelial cell carcinoma (UCC). We investigated the feasibility of combining microsatellite analysis (MA) and the FGFR3 status for the detection of UCC in voided urine. EXPERIMENTAL DESIGN: In a prospective setting, 59 UCC tissues and matched urine samples were obtained, and subjected to MA (23 markers) and FGFR3 mutation analysis (exons 7, 10, and 15). In each case, a clinical record with tumor and urine features was provided. Fifteen patients with a negative cystoscopy during follow-up served as controls. RESULTS: A mutation in the FGFR3 gene was found in 26 (44%) UCCs of which 22 concerned solitary pTaG1/2 lesions. These mutations were absent in the 15 G3 tumors. For the 6 cases with leukocyturia, 46 microsatellite alterations were found in the tumor. Only 1 of these was also detected in the urine. This was 125 of 357 for the 53 cases without leukocyte contamination. The sensitivity of MA on voided urine was lower for FGFR3-positive UCC (15 of 21; 71%) as compared with FGFR3 wild-type UCC (29 of 32; 91%). By including the FGFR3 mutation, the sensitivity of molecular cytology increased to 89% and was superior to the sensitivity of morphological cytology (25%) for every clinical subdivision. The specificity was 14 of 15 (93%) for the two (molecular and morphological) cytological approaches. CONCLUSIONS: Molecular urine cytology by MA and FGFR3 mutation analysis enables a highly sensitive and specific detection of UCC. The similarity of molecular profiles in tumor and urine corroborate their clonal relation.

Molecular evolution of multiple recurrent cancers of the bladder (Article)

2000-01-01T00:00:00Z

We describe the reconstruction of bladder tumor development in individual patients spanning periods of up to 17 years. Genomic alterations detected in the tumors were used for hierarchical cluster analysis of tumor subclones. The cluster analysis highlights the clonal relationship between tumors from each patient. Based on the cluster data we were able to reconstruct the evolution of tumors in a genetic tree, where tumors with few aberrations precede those with many genetic insults. The sequential order of the tumors in these pedigrees differs from the chronological order in which the tumors appear. Thus, a tumor with few alterations can be occult for years following removal of a more deranged derivative. Extensive genetic damage is seen to accumulate during the evolution of the tumors. To explain the type and extent of genetic damage in combination with the low stage and grade of these tumors, we hypothesize that in bladder cancer pathogenesis an increased rate of mitotic recombination is acquired early in the tumorigenic process.

Generation of antisera to mouse insulin-like growth factor binding proteins (IGFBP)-1 to -6: comparison of IGFBP protein and messenger ribonucleic acid localization in the mouse embryo (Article)

1999-01-01T00:00:00Z

The insulin-like growth factor (IGF) system is an important regulator of fetal growth and differentiation. IGF bioavailability is modulated by IGF binding proteins (IGFBPs). We have generated six different antisera, directed to synthetic peptide fragments of mouse IGFBP-1 through -6. The specificity of the produced antisera was demonstrated by enzyme-linked immunosorbent assay, Western blotting, and by immunohistochemistry on sections of mouse embryos of 13.5 days post coitum. Specificity for the IGFBP-2 through -6 antisera also was confirmed immunohistochemically in liver and lung of corresponding gene deletion (knock-out) mutant mice and wild-type litter mates. Immunohistochemistry and messenger RNA (mRNA) in situ hybridization on sections of mouse embryos of 13.5 days post coitum revealed tissue-specific expression patterns for the six IGFBPs. The only site of IGFBP-1 protein and mRNA production was the liver. IGFBP-2, -4, and -5 protein and mRNA were detected in various organs and tissues. IGFBP-3 and -6 protein and mRNA levels were low. In several tissues, such as lung, liver, kidney, and tongue, more than one IGFBP (protein and mRNA) could be detected. Differences between mRNA and protein localization were extensive for IGFBP-3, -5, and -6, suggesting that these IGFBPs are secreted and transported. These results confirm the different spatial localization of the IGFBPs, on the mRNA and protein level. The overlapping mRNA and protein localization for IGFBP-2 and -4, on the other hand, may indicate that these IGFBPs also function in an auto- or paracrine manner.

A G-->A transition creates a branch point sequence and activation of a cryptic exon, resulting in the hereditary disorder neurofibromatosis 2 (Article)

1998-01-01T00:00:00Z

We describe a G-->A transition within intron 5 of the NF2 gene. This mutation creates a consensus splice branch point sequence. To our knowledge this is the first report of a mutation that creates a functional branch point sequence in a human hereditary disorder. The new branch point sequence is located 18 bp upstream of a consensus splice acceptor site. A consensus splice donor site is found 106 bp 3' of the acceptor site. Asa consequence the G-->A transition results in an alternatively spliced mRNA containing an additional exon 5a of 106 bp derived from intron sequences. We cloned the mutant cDNA and show that due to an in-frame stop codon the cDNA codes for a truncated NF2 protein. The mutation was observed in three affected members of an NF2 family. In a tumour of one of the family members both alternatively spliced and wild-type mRNA were found, although the wild-type allele of the gene is absent due to an interstitial deletion on chromosome 22. We also show that immunoprecipitations reveal the presence of full-length wild-type NF2 protein in the tumour lysate. These data support the hypothesis that some degree of normal splicing of the mutant precursor RNA is taking place. It is therefore likely that this residual activity of the mutant allele explains the relatively mild phenotype in the family. These data also indicate that complete inactivation of the gene is not required for tumour formation.