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.

Genome-wide association study yields variants at 20p12.2 that associate with urinary bladder cancer.

Genome-wide association studies (GWAS) of urinary bladder cancer (UBC) have yielded common variants at 12 loci that associate with risk of the disease. We report here the results of a GWAS of UBC including 1670 UBC cases and 90 180 controls, followed by replication analysis in additional 5266 UBC cases and 10 456 controls. We tested a dataset containing 34.2 million variants, generated by imputation based on whole-genome sequencing of 2230 Icelanders. Several correlated variants at 20p12, represented by rs62185668, show genome-wide significant association with UBC after combining discovery and replication results (OR = 1.19, P = 1.5 × 10(-11) for rs62185668-A, minor allele frequency = 23.6%). The variants are located in a non-coding region approximately 300 kb upstream from the JAG1 gene, an important component of the Notch signaling pathways that may be oncogenic or tumor suppressive in several forms of cancer. Our results add to the growing number of UBC risk variants discovered through GWAS.

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.

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.

Hotspot mutations in PIK3CA associate with first-line treatment outcome for aromatase inhibitors but not for tamoxifen.

PIK3CA mutations occur frequently in breast cancer, predominantly in exons 9 and 20. The aim of this retrospective study is to evaluate the PIK3CA mutation status for its relationship with prognosis and first-line endocrine therapy outcome. PIK3CA exon 9 and 20 were evaluated for mutations in 1,352 primary breast cancer specimens by SnaPshot multiplex analyses. The mutation status was studied for their relationship with metastasis-free survival (MFS) in 342 untreated lymph node-negative (LNN) patients and to time to progression (TTP) in estrogen receptor (ER)-positive patients with metastatic disease treated with first-line tamoxifen (N = 447) or aromatase inhibitors (AIs; N = 84). We detected in 423 patients hotspot mutations for PIK3CA (31 %). Mutations in exon 20 were detected in 251 patients (59 %), with H1047L and H1047R mutations in 37 (15 %) and 214 (85 %) cases, respectively. Mutations in PIK3CA exon 9 were discovered in 173 patients (41 %), with E542K and E545K mutations in 57 (32 %) and 104 (60 %) cases as most prevalent ones. Evaluation of the untreated LNN patients for prognosis showed no relationship between MFS and PIK3CA mutations, neither for exon 9 [HR = 1.04 (95 % CI 0.57-1.89), P = 0.90] nor for exon 20 [HR = 0.98 (95 % CI 0.63-1.54); P = 0.94] when compared to wild-type. The PIK3CA mutation status was also not associated with treatment outcome after first-line tamoxifen. On the other hand, patients treated with first-line AIs showed a longer TTP when having a PIK3CA mutation in exon 9 [HR = 0.40 (95 % CI 0.17-0.95); P = 0.038] or exon 20 [HR = 0.50 (95 % CI 0.27-0.91); P = 0.024] compared to wild-types, both significant in uni- and multivariate analysis including traditional predictive factors. All results remained when only HER2-negative patients were evaluated for each cohort. PIK3CA mutations in ER-positive tumors were significantly associated with a favorable outcome after first-line AIs, which needs further confirmation in other datasets. Mutations were not associated with prognosis in untreated LNN patients nor predictive outcome after first-line tamoxifen therapy in advanced disease patients.

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.

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

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

A reported 20-gene expression signature to predict lymph node-positive disease at radical cystectomy for muscle-invasive bladder cancer is clinically not applicable.

BACKGROUND: Neoadjuvant chemotherapy (NAC) for muscle-invasive bladder cancer (MIBC) provides a small but significant survival benefit. Nevertheless, controversies on applying NAC remain because the limited benefit must be weight against chemotherapy-related toxicity and the delay of definitive local treatment. Therefore, there is a clear clinical need for tools to guide treatment decisions on NAC in MIBC. Here, we aimed to validate a previously reported 20-gene expression signature that predicted lymph node-positive disease at radical cystectomy in clinically node-negative MIBC patients, which would be a justification for upfront chemotherapy. METHODS: We studied diagnostic transurethral resection of bladder tumors (dTURBT) of 150 MIBC patients (urothelial carcinoma) who were subsequently treated by radical cystectomy and pelvic lymph node dissection. RNA was isolated and the expression level of the 20 genes was determined on a qRT-PCR platform. Normalized Ct values were used to calculate a risk score to predict the presence of node-positive disease. The Cancer Genome Atlas (TCGA) RNA expression data was analyzed to subsequently validate the results. RESULTS: In a univariate regression analysis, none of the 20 genes significantly correlated with node-positive disease. The area under the curve of the risk score calculated by the 20-gene expression signature was 0.54 (95% Confidence Interval: 0.44-0.65) versus 0.67 for the model published by Smith et al. Node-negative patients had a significantly lower tumor grade at TURBT (p = 0.03), a lower pT stage (p<0.01) and less frequent lymphovascular invasion (13% versus 38%, p<0.01) at radical cystectomy than node-positive patients. In addition, in the TCGA data, none of the 20 genes was differentially expressed in node-negative versus node-positive patients. CONCLUSIONS: We conclude that a 20-gene expression signature developed for nodal staging of MIBC at radical cystectomy could not be validated on a qRT-PCR platform in a large cohort of dTURBT specimens.

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.

Detection of multiple mutations in urinary exfoliated cells from male bladder cancer patients at diagnosis and during follow-up.

Most bladder cancer (BC) patients need life-long, invasive and expensive monitoring and treatment, making it a serious burden on the health system. Thus, there is a pressing need for an accurate test to assist diagnosis and surveillance of BC as an alternative to cystoscopy. Mutations in human TERT, FGFR3, PIK3CA, and RAS genes have been proposed as potential molecular markers in bladder tumor. Their concomitant presence in urine samples has not been fully explored.We investigated a panel of mutations in DNA from exfoliated urinary cells of 255 BC patients at diagnosis. Forty-one mutations in TERT, FGFR3, PIK3CA, and RAS were analyzed by SNaPshot assay in relation to clinical outcome. In 81 of these patients under surveillance, the same set of mutations was screened in additional 324 samples prospectively collected.The most common mutations detected in urine at diagnosis were in the TERT promoter. In non-invasive BC, these mutations were related to high risk and grade (p<0.0001) as well as progression to muscle-invasive disease (p=0.01), whereas FGFR3 mutations were observed in low-grade BC (p=0.02) and patients with recurrences (p=0.05). Stronger associations were observed for combined TERT and FGFR3 mutations and number of recurrences (OR: 4.54 95% CI: 1.23-16.79, p=0.02). Analyses of the area under the curve for combinations of mutations detected at diagnosis and follow-up showed an accuracy of prediction of recurrence of 0.80 (95% CI: 0.71-0.89).Mutations in urine of BC patients may represent reliable biomarkers. In particular, TERT and FGFR3 mutations have a good accuracy of recurrence prediction.

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

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.

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.

Defined morphological criteria allow reliable diagnosis of colorectal serrated polyps and predict polyp genetics.

Criteria for the diagnosis of serrated colorectal lesions (hyperplastic polyp, sessile serrated adenoma without or with dysplasia--which we called mixed polyp--and traditional serrated adenoma) for which consensus has been reached should be validated for applicability in daily practice in terms of inter-observer reproducibility and their association with clinical features and (epi)genetic events. A study set was created from a consecutive series of colorectal polyps (n = 1,926) by selecting all sessile serrated adenomas, traditional serrated adenomas and mixed polyps. We added consecutive series of hyperplastic polyps, classical adenomas and normal mucosa samples for a total of 200 specimens. With this series, we conducted an inter-observer study, encompassing ten pathologists with gastrointestinal pathology experience from five European countries, in three rounds in which all cases were microscopically evaluated. An assessment of single morphological criteria was included, and these were correlated with clinical parameters and the mutation status of KRAS, BRAF and PIK3CA and the methylation status of MLH1. Gender, age and localisation were significantly associated with certain types of lesions. Kappa statistics revealed moderate to good inter-observer agreement for polyp classification (κ = 0.56 to 0.63), but for single criteria, this varied considerably (κ = 0.06 to 0.82). BRAF mutations were frequently found in hyperplastic polyps (86 %, 62/72) and sessile serrated adenomas (80 %, 41/51). KRAS mutations occurred more frequently in traditional serrated adenomas (78 %, 7/9) and less so in classical adenomas (20 %, 10/51). Single morphological criteria for sessile serrated adenomas showed significant correlation with BRAF mutation (all p ≤ 0.001), and those for classical adenomas or traditional serrated adenoma correlated significantly with KRAS mutation (all p < 0.001). Therefore, single well-defined morphological criteria are predictive for genetic alterations in colorectal polyps.

Telomerase reverse transcriptase promoter mutations in bladder cancer: high frequency across stages, detection in urine, and lack of association with outcome.

BACKGROUND: Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. OBJECTIVES: To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). DESIGN, SETTING, AND PARTICIPANTS: A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and 21 mo, respectively). Voided urine from patients with suspicion of incident UBC (n=174), or under surveillance after diagnosis of non-muscle-invasive UBC (n=194), was tested using a SNaPshot assay. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival. RESULTS AND LIMITATIONS: In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p=0.0002). There was no association between TERT mutations and mRNA expression (p=0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature. CONCLUSIONS: Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences.

KRAS and BRAF mutation analysis in routine molecular diagnostics: comparison of three testing methods on formalin-fixed, paraffin-embedded tumor-derived DNA.

Accurate mutation detection assays are strongly needed for use in routine molecular pathology analyses to aid in the selection of patients with cancer for targeted therapy. The high-resolution melting (HRM) assay is an ideal prescreening tool, and SNaPshot analysis offers a straightforward genotyping system. Our present study was determined to compare these mutation testing methods on formalin-fixed, paraffin-embedded (FFPE) tumor-derived DNA. We compared the performance of HRM, followed by cycle sequencing (HRM-sequencing); multiplex PCR assay, followed by SNaPshot analysis (multiplex mutation assay); and a successor assay using HRM, followed by SNaPshot (HRM-SNaPshot) for mutation analysis of both KRAS (codon 12/13/61) and BRAF (codon 600/601). In a series of 195 FFPE-derived DNA specimens, a high genotypic concordance between HRM-sequencing and multiplex mutation assay was found (κ, 0.98; 95% CI, 0.94 to 1), underlining the potential of a combined HRM-SNaPshot approach. In reconstruction experiments, the analytical sensitivity of HRM-SNaPshot was twofold to fourfold higher than HRM-sequencing and multiplex mutation assay, respectively. In addition, HRM-SNaPshot had a good performance rate (99%) on FFPE tumor-derived DNA, and mutation detection was highly concordant with the predecessor assays (κ for both, 0.98). The occurrence of BRAF and KRAS mutations is mutually exclusive. HRM-SNaPshot is an attractive method for mutation analysis in pathology, given its good performance rate on FFPE-derived DNA, high analytical sensitivity, and prescreening approach.

Selection of microsatellite markers for bladder cancer diagnosis without the need for corresponding blood.

Microsatellite markers are used for loss-of-heterozygosity, allelic imbalance and clonality analyses in cancers. Usually, tumor DNA is compared to corresponding normal DNA. However, normal DNA is not always available and can display aberrant allele ratios due to copy number variations in the genome. Moreover, stutter peaks may complicate the analysis. To use microsatellite markers for diagnosis of recurrent bladder cancer, we aimed to select markers without stutter peaks and a constant ratio between alleles, thereby avoiding the need for a control DNA sample. We investigated 49 microsatellite markers with tri- and tetranucleotide repeats in regions commonly lost in bladder cancer. Based on analysis of 50 blood DNAs the 12 best performing markers were selected with few stutter peaks and a constant ratio between peaks heights. Per marker upper and lower cut off values for allele ratios were determined. LOH of the markers was observed in 59/104 tumor DNAs. We then determined the sensitivity of the marker panel for detection of recurrent bladder cancer by assaying 102 urine samples of these patients. Sensitivity was 63% when patients were stratified for LOH in their primary tumors. We demonstrate that up-front selection of microsatellite markers obliterates the need for a corresponding blood sample. For diagnosis of bladder cancer recurrences in urine this significantly reduces costs. Moreover, this approach facilitates retrospective analysis of archival tumor samples for allelic imbalance.

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

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.

Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.

Recently a number of randomized trials have shown that patients with advanced colorectal cancer do not benefit from therapies targeting the epidermal growth factor receptor when their tumors harbor mutations in the KRAS, BRAF and PIK3CA genes. We developed two multiplex assays that simultaneously screen 22 nucleotides in the KRAS, NRAS, BRAF and PIK3CA genes for mutations. The assays were validated on 294 tumor DNA samples from patients with advanced colorectal cancer. In these samples 119 KRAS codon 12 and 13 mutations had been identified by sequence analysis, 126 tumors were wild-type for KRAS and the analysis failed in 49 of the 294 samples due to poor DNA quality. The two mutation assays detected 130 KRAS mutations, among which were 3 codon 61 mutations, and in addition 32 PIK3CA, 13 BRAF and 6 NRAS mutations. In 19 tumors a KRAS mutation was found together with a mutation in the PIK3CA gene. One tumor was mutant for both PIK3CA and BRAF. In summary, the mutations assays identified 161 tumors with a mutation, 120 were wild-type and the analysis failed in 13. The material cost of the 2 mutation assays was calculated to be 8-fold lower than the cost of sequencing required to obtain the same data. In addition, the mutation assays are less labor intensive. We conclude that the performance of the two multiplex mutation assays was superior to direct sequencing. In addition, these assays are cheaper and easier to interpret. The assays may also be of use for selection of patients with other tumor types.

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.