Clonal Relationship Between Lichen Sclerosus, Differentiated Vulvar Intra-epithelial Neoplasia and Non HPV-related Vulvar Squamous Cell Carcinoma.

BACKGROUND/AIM: Differentiated vulvar intraepithelial neoplasia (dVIN) and lichen sclerosus (LS) can give rise to vulvar squamous cell carcinoma (VSCC), but genetic evidence is currently still limited. We aimed to determine genetic abnormalities in VSCC and backtrack these abnormalities in the dVIN and LS lesions. MATERIALS AND METHODS: DNA from VSCC and patient-matched dVIN and LS samples of twelve patients was collected. High-resolution genome-wide copy number analysis was performed and subsequently, we sequenced TP53. RESULTS: Copy number alterations were identified in all VSCC samples. One dVIN lesion presented with three copy number alterations that were preserved in the paired VSCC sample. Targeted sequencing of TP53 identified mutations in five VSCCs. All five mutations were traced back in the dVIN (n=5) or the LS (n=1) with frequencies ranging from 3-19%. CONCLUSION: Our data provide genetic evidence for a clonal relationship between VSCC and dVIN or LS.

Immunohistochemical Profiles of Endometrioid Endometrial Carcinomas With and Without Metastatic Disease.

A minority of endometrial carcinomas present at an advanced stage with a poor prognosis, and should be identified to individualize treatment. Immunohistochemical markers have been studied, but most have not been directly linked to metastasis. This study analyzes the immunohistochemical profile of endometrioid endometrial carcinomas (EECs) with and without metastases, and corresponding metastases. Tissue microarray slides from stage I EECs, stage III-IV EECs, and corresponding metastases were stained and scored for expression of ß-catenin, E-cadherin, ER, PR, PTEN, p16, MLH1, PMS2, L1CAM, p53, p21, and MIB1. Scores were compared between primary stage I and III-IV EECs, stage III-IV EECs, and the corresponding metastases, and between intra-abdominal and distant metastases. Primary tumors with distant metastases had a significantly lower ER expression than those without metastases or with intra-abdominal metastases. Distant metastases had a significantly lower PR expression than the corresponding primary tumor and intra-abdominal metastases. In contrast, p16 and PTEN expression was significantly higher in intra-abdominal metastases compared with corresponding primary tumors. Immunohistochemistry predicts both presence and location of EEC metastases. Loss of ER and PR was related to distant spread, and increased expression of PTEN and p16 was related to intra-abdominal spread. Additional research should assess the use of these markers in the diagnostic workup as well as the possibility to target metastases through these markers.

Prognostic significance of highly sulfated chondroitin sulfates in ovarian cancer defined by the single chain antibody GD3A11.

OBJECTIVE: The extracellular matrix (ECM) of ovarian cancer may provide a number of potential biomarkers. Chondroitin sulfate (CS), a class of sulfated polysaccharides, is abundantly present in the ECM of ovarian cancer. Structural alterations of CS chains (i.e. sulfation pattern) have been demonstrated to play a role in cancer development and progression. In this study we investigate the potential of highly sulfated CS as a biomarker in ovarian cancer using the single chain antibody GD3A11 selected by the phage display technology. METHODS: The specificity of the antibody was determined by an indirect ELISA. GD3A11 epitope expression was assessed by immunohistochemistry in healthy organs, benign and malignant ovarian tumors (N=359) and correlated to clinical parameters. The CHST15 gene, responsible for the biosynthesis of highly sulfated CS was evaluated for mutation and methylation status. RESULTS: The GD3A11 epitope was minimally expressed in normal organs. Intense expression was observed in the ECM of different ovarian cancer subtypes, in contrast to benign ovarian tumors. Expression was independent of tumor grade, FIGO stage, and the use chemotherapy. For the aggressive ovarian cancer phenotype, intense expression was identified as an independent predictor for poor prognosis. CHST15 gene analysis showed no mutations nor an altered methylation status. CONCLUSION: Specific highly sulfated CS motifs expressed in the tumoral ECM hold biomarker potential in ovarian cancer patients. These matrix motifs constitute a novel class of biomarkers with prognostic significance and may be instrumental for innovative diagnostic and therapeutic applications (e.g. targeted therapy) in management of ovarian cancer.

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.

Mutational profiling of kinases in glioblastoma.

BACKGROUND: Glioblastoma is a highly malignant brain tumor for which no cure is available. To identify new therapeutic targets, we performed a mutation analysis of kinase genes in glioblastoma. METHODS: Database mining and a literature search identified 76 kinases that have been found to be mutated at least twice in multiple cancer types before. Among those we selected 34 kinase genes for mutation analysis. We also included IDH1, IDH2, PTEN, TP53 and NRAS, genes that are known to be mutated at considerable frequencies in glioblastoma. In total, 174 exons of 39 genes in 113 glioblastoma samples from 109 patients and 16 high-grade glioma (HGG) cell lines were sequenced. RESULTS: Our mutation analysis led to the identification of 148 non-synonymous somatic mutations, of which 25 have not been reported before in glioblastoma. Somatic mutations were found in TP53, PTEN, IDH1, PIK3CA, EGFR, BRAF, EPHA3, NRAS, TGFBR2, FLT3 and RPS6KC1. Mapping the mutated genes into known signaling pathways revealed that the large majority of them plays a central role in the PI3K-AKT pathway. CONCLUSIONS: The knowledge that at least 50% of glioblastoma tumors display mutational activation of the PI3K-AKT pathway should offer new opportunities for the rational development of therapeutic approaches for glioblastomas. However, due to the development of resistance mechanisms, kinase inhibition studies targeting the PI3K-AKT pathway for relapsing glioblastoma have mostly failed thus far. Other therapies should be investigated, targeting early events in gliomagenesis that involve both kinases and non-kinases.

Novel single-chain antibody GD3A10 defines a chondroitin sulfate biomarker for ovarian cancer.

AIMS: Ovarian cancer has the highest case-to-fatality-index of all gynecological cancers. In this study, tumor-related alterations in the extracellular matrix, especially regarding chondroitin sulfate glycosaminoglycans, are proposed as a novel biomarker in ovarian cancer. MATERIALS & METHODS: Phage display technology was applied to select antibody GD3A10, which was obtained by biopanning using embryonic glycosaminoglycans as a source for carcinogenic antigens. GD3A10 antigen specificity was studied in situ using glycosaminoglycan degrading enzymes. A patient cohort (n = 159) was immunohistochemically stained. Scoring was correlated with clinical prognostic parameters and survival. Normal rat organs were used to study normal antigen distribution. RESULTS: GD3A10 is a specific anti-chondroitin sulfate antibody and the epitope was absent or very restricted in normal rat organs, normal ovaries and benign ovarian tumors. Strong stromal expression was observed in malignant ovarian tumors, and correlated with poor prognostic factors such as subtype, tumor grade and recurrence. CONCLUSION: tumor-associated glycosaminoglycans are an interesting source of biomarkers in ovarian cancer, as shown here using chondroitin sulfate antibody GD3A10.

Sulfated sugars in the extracellular matrix orchestrate ovarian cancer development: 'when sweet turns sour'.

Considering the high mortality of ovarian cancer, novel approaches for diagnostics and therapy are urgently needed. Cancer initiation, progression, and invasion occur in a complex and dynamic microenvironment which depends on the interplay between host cell responses and tumor activity. Chondroitin sulfate (CS), a special highly sulfated sugar, forms an important intermediate player in this respect. Depending on the (micro)structural diversity of chondroitin sulfate chains, various ligands interact with this special group of glycosaminoglycans, making it a key molecule for many physiological and pathological processes, including cancer development. This review focuses on the various functions of chondroitin sulfate in tumor growth, angiogenesis, dissemination and immunosilencing of ovarian cancer. We also shed light on possible future diagnostic and therapeutic modalities for ovarian cancer based on the large variety in chondroitin sulfate microstructure and function. It is concluded that the class of chondroitin sulfate represents an attractive target to interfere with the process of ovarian tumorigenesis.

The combination of IDH1 mutations and MGMT methylation status predicts survival in glioblastoma better than either IDH1 or MGMT alone.

BACKGROUND: Genetic and epigenetic profiling of glioblastomas has provided a comprehensive list of altered cancer genes of which only O(6)-methylguanine-methyltransferase (MGMT) methylation is used thus far as a predictive marker in a clinical setting. We investigated the prognostic significance of genetic and epigenetic alterations in glioblastoma patients. METHODS: We screened 98 human glioblastoma samples for genetic and epigenetic alterations in 10 genes and chromosomal loci by PCR and multiplex ligation-dependent probe amplification (MLPA). We tested the association between these genetic and epigenetic alterations and glioblastoma patient survival. Subsequently, we developed a 2-gene survival predictor. RESULTS: Multivariate analyses revealed that mutations in isocitrate dehydrogenase 1 (IDH1), promoter methylation of MGMT, irradiation dosage, and Karnofsky Performance Status (KFS) were independent prognostic factors. A 2-gene predictor for glioblastoma survival was generated. Based on the genetic and epigenetic status of IDH1 and MGMT, glioblastoma patients were stratified into 3 clinically different genotypes: glioblastoma patients with IDH1mt/MGMTmet had the longest survival, followed by patients with IDH1mt/MGMTunmet or IDH1wt/MGMTmet, and patients with IDH1wt/MGMTunmet had the shortest survival. This 2-gene predictor was an independent prognostic factor and performed significantly better in predicting survival than either IDH1 mutations or MGMT methylation alone. The predictor was validated in 3 external datasets. DISCUSSION: The combination of IDH1 mutations and MGMT methylation outperforms either IDH1 mutations or MGMT methylation alone in predicting survival of glioblastoma patients. This information will help to increase our understanding of glioblastoma biology, and it may be helpful for baseline comparisons in future clinical trials.

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.

Müllerian precursor lesions in serous ovarian cancer patients: using the SEE-Fim and SEE-End protocol.

Serous ovarian cancer is suggested to develop from epithelium embryologically derived from the Müllerian ducts. The aim of the current study is to thoroughly, analyze the epithelium derived from the Müllerian ducts (cervix, endometrium and fallopian tubes) in serous ovarian cancer patients. Sixty women diagnosed with serous ovarian carcinoma were included in this multicentre, observational study. Tissues were embedded completely for histological assessment, in accordance with the SEE-Fim and SEE-End protocol (Sectioning and Extensively Examining of the Fimbriated end; and-Endometrium), and prevalence of cervical, as well as endometrial and tubal pathology was analyzed. In 31 (52%) cases, a pathologic lesion was identified, and in 16 (27%) of these cases coexistence of pathologic lesions. In 1 case, severe dysplasia was found in the cervix, in 9 (15%) cases endometrial intraepithelial carcinoma, in 19 (32%) cases atypical hyperplasia, and in 23 (43%) cases serous tubal intraepithelial carcinoma. Serous tubal intraepithelial carcinoma was seen significantly more often concurrent with endometrial atypical hyperplasia or endometrial intraepithelial carcinoma than with benign endometrium (64 vs 28%; P=0.01). To conclude, histological assessment of epithelium derived from Müllerian ducts of serous ovarian cancer patients resulted in the identification of endometrial intraepithelial carcinoma, serous tubal intraepithelial carcinoma and/or endometrial atypical hyperplasia in more than half of cases. Coexistence of these pathologic lesions was common, and might represent an effect of field carcinogenesis or tumor implantation of migrating cells.

Histopathologic assessment of the entire endometrium in asymptomatic women.

Knowledge on the nature of the endometrium in women without symptoms of endometrial disease is poor. Therefore, the aim of this prospective study was to describe the endometrium of a cohort of asymptomatic women. The entire endometrium of premenopausal and postmenopausal women was embedded for histologic examination. All included patients underwent a hysterectomy on indication of uterovaginal prolapse, from July 2011 to October 2012, in 3 hospitals in the South of the Netherlands. Exclusion criteria were symptoms of postmenopausal vaginal blood loss or premenopausal disordered vaginal bleeding. As a result, 68 women were included in the study, 48 women were postmenopausal and 20 were premenopausal. In the endometrium of 10 women, simple hyperplasia was found (15%); 1, complex hyperplasia (2%); 2, simple atypical hyperplasia (3%); 2, complex atypical hyperplasia (3%); and 2, a small focus of intramucosal endometrioid endometrial carcinoma (3%). In general, the endometrium was heterogeneous, and most lesions were not present in the entire endometrium. In conclusion, after examining the entire endometrium, a remarkable high prevalence of endometrial pathology was found in asymptomatic women. The clinical meaning of these lesions is not yet clear, but endometrial pathology may frequently exist without symptoms.

A 3-plex methylation assay combined with the FGFR3 mutation assay sensitively detects recurrent bladder cancer in voided urine.

PURPOSE: DNA methylation is associated with bladder cancer and these modifications could serve as useful biomarkers. FGFR3 mutations are present in 60% to 70% of non-muscle invasive bladder cancer (NMIBC). Low-grade bladder cancer recurs in more than 50% of patients. The aim of this study is to determine the sensitivity and specificity of a urine assay for the diagnosis of recurrences in patients with a previous primary NMIBC G1/G2 by using cystoscopy as the reference standard. EXPERIMENTAL DESIGN: We selected eight CpG islands (CGI) methylated in bladder cancer from our earlier genome-wide study. Sensitivity of the CGIs for recurrences detection was investigated on a test set of 101 preTUR urines. Specificity was determined on 70 urines from healthy males aged more than 50 years. A 3-plex assay for the best combination was developed and validated on an independent set of 95 preTUR, recurrence free, and nonmalignant urines (n=130). RESULTS: The 3-plex assay identified recurrent bladder cancer in voided urine with a sensitivity of 74% in the validation set. In combination with the FGFR3 mutation assay, a sensitivity of 79% was reached (specificity of 77%). Sensitivity of FGFR3 and cytology was 52% and 57%, respectively. CONCLUSION: The combination of methylation and FGFR3 assays efficiently detects recurrent bladder cancer without the need for stratification of patients regarding methylation/mutation status of the primary tumor. We conclude that the sensitivity of this combination is in the same range as cystoscopy and paves the way for a subsequent study that investigates a modified surveillance protocol consisting of the urine test followed by cystoscopy only when the urine test is positive.

DNA methylation-based biomarkers in bladder cancer.

Urinary bladder cancer is the fifth most common cancer in the Western world. Increasing evidence has shown that DNA methylation in bladder cancer is expansive and is implicated in pathogenesis. Furthermore, distinct methylation patterns have been identified between non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC), as well as between FGFR3-mutant and wild-type tumours. Given these distinctions in expression, methylated genes have been proposed as diagnostic and prognostic biomarkers for patients with bladder cancer. Indeed, several studies have revealed that methylated genes--including CDH1, FHIT, LAMC2, RASSF1A, TIMP3, SFRP1, SOX9, PMF1 and RUNX3--are associated with poor survival in patients with MIBC. Further validation of these markers for prognostication as well as surveillance (of patients with NMIBC) is required. Validated markers for progression, diagnosis, survival and BCG response will contribute to clinical decision-making and individualized treatment.

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.

Genome-wide analysis of CpG island methylation in bladder cancer identified TBX2, TBX3, GATA2, and ZIC4 as pTa-specific prognostic markers.

BACKGROUND: DNA methylation markers could serve as useful biomarkers, both as markers for progression and for urine-based diagnostic assays. OBJECTIVE: Identify bladder cancer (BCa)-specific methylated DNA sequences for predicting pTa-specific progression and detecting BCa in voided urine. DESIGN, SETTING, AND PARTICIPANTS: Genome-wide methylation analysis was performed on 44 bladder tumours using the Agilent 244K Human CpG Island Microarray (Agilent Technologies, Santa Clara, CA, USA). Validation was done using a custom Illumina 384-plex assay (Illumina, San Diego, CA, USA) in a retrospective group of 77 independent tumours. Markers for progression were identified in pTa (n = 24) tumours and validated retrospectively in an independent series of 41 pTa tumours by the SNaPshot method (Applied Biosystems, Foster City, CA, USA). MEASUREMENTS: The percentage of methylation in tumour and urine samples was used to identify markers for detection and related to the end point of progression to muscle-invasive disease with Kaplan-Meier models and multivariate analysis. RESULTS AND LIMITATIONS: In the validation set, methylation of the T-box 2 (TBX2), T-box 3 (TBX3), GATA binding protein 2 (GATA2), and Zic family member 4 (ZIC4) genes was associated with progression to muscle-invasive disease in pTa tumours (p = 0.003). Methylation of TBX2 alone showed a sensitivity of 100%, a specificity of 80%, a positive predictive value of 78%, and a negative predictive value of 100%, with an area under the curve of 0.96 (p<0.0001) for predicting progression. Multivariate analysis showed that methylation of TBX3 and GATA2 are independent predictors of progression when compared to clinicopathologic variables (p = 0.04 and p = 0.03, respectively). The predictive accuracy improved by 23% by adding methylation of TBX2, TBX3, and GATA2 to the European Organisation for Research and Treatment of Cancer risk scores. We further identified and validated 110 CpG islands (CGIs) that are differentially methylated between tumour cells and control urine. The limitation of this study is the small number of patients analysed for testing and validating the prognostic markers. CONCLUSIONS: We have identified four methylation markers that predict progression in pTa tumours, thereby allowing stratification of patients for personalised follow-up. In addition, we identified CGIs that will enable detection of bladder tumours in voided urine.

In silico analysis of kinase expression identifies WEE1 as a gatekeeper against mitotic catastrophe in glioblastoma.

Kinases execute pivotal cellular functions and are therefore widely investigated as potential targets in anticancer treatment. Here we analyze the kinase gene expression profiles of various tumor types and reveal the wee1 kinase to be overexpressed in glioblastomas. We demonstrate that WEE1 is a major regulator of the G(2) checkpoint in glioblastoma cells. Inhibition of WEE1 by siRNA or small molecular compound in cells exposed to DNA damaging agents results in abrogation of the G(2) arrest, premature termination of DNA repair, and cell death. Importantly, we show that the small-molecule inhibitor of WEE1 sensitizes glioblastoma to ionizing radiation in vivo. Our results suggest that inhibition of WEE1 kinase holds potential as a therapeutic approach in treatment of glioblastoma.

Bladder cancer: novel molecular characteristics, diagnostic, and therapeutic implications.

Bladder cancer (BC) comes in two flavors: as non-muscle invasive (NMI) and as muscle invasive (MI) disease. These two subtypes differ in their genetic aberrations. In NMI-BC mutations in the FGFR3 oncogene are found with a frequency of 75%, whereas mutations in the TP53 tumor suppressor gene prevail in MI-BC. Mutations in the RAS genes occur in 15% of BC of all stages and are mutually exclusive with FGFR3 mutations. Mutations in the PIK3CA gene are found in about 13% and these almost exclusively co-occur with FGFR3 mutations. NMI-BC with FGFR3 mutations are genetically stable, but FGFR3 wild type NMI-BC and MI tumors are genetically unstable. In this paper, we discuss the use of these genetic aberrations in relation to recurrence, progression, surveillance, and therapeutic options. As of yet, there is no biomarker that can predict recurrences or the rate of recurrences when they occur. We show that FGFR3 mutations are associated with a decreased risk of progression, and a better survival both in BC and in upper urinary tract cancer. Microsatellite analysis (MA) in order to detect loss-of-heterozygosity can be used to detect recurrences in urinary cells of patients under surveillance. The results of a Dutch randomized trial show that consecutive positive MA results are a strong predictor for future recurrences. Using FGFR3 mutation analysis for those patients who have an FGFR3 mutant tumor will enhance performance of urine-based surveillance. Although FGFR3 mutations occur in only 20% of MI tumors, these tumors often have a high expression of the FGFR3 protein. This suggests that this receptor could present a target for adjuvant therapy in MI-BC. However, whether the FGFR3 pathway is active in these tumors remains to be established.

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.

Absence of AKT1 mutations in glioblastoma.

BACKGROUND: Oncogenic activation of the PI3K signalling pathway plays a pivotal role in the development of glioblastoma multiforme (GBM). A central node in PI3K downstream signalling is controlled by the serine-threonine kinase AKT1. A somatic mutation affecting residue E17 of the AKT1 gene has recently been identified in breast and colon cancer. The E17K change results in constitutive AKT1 activation, induces leukaemia in mice, and accordingly, may be therapeutically exploited to target the PI3K pathway. Assessing whether AKT1 is activated by somatic mutations in GBM is relevant to establish its role in this aggressive disease. METHODOLOGY/PRINCIPAL FINDINGS: We performed a systematic mutational analysis of the complete coding sequence of the AKT1 gene in a panel of 109 tumor GBM samples and nine high grade astrocytoma cell lines. However, no somatic mutations were detected in the coding region of AKT1. CONCLUSIONS/SIGNIFICANCE: Our data indicate that in GBM oncogenic deregulation of the PI3K pathway does not involve somatic mutations in the coding region of AKT1.

Bladder cancer biomarkers and their role in surveillance and screening.

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.