title: High-throughput detection of fusion genes in cancer using the Sequenom MassARRAY platform.
abstract: Fusion genes have pivotal roles in the development and progression of human cancer and offer potential for rational drug design. Massively parallel sequencing has identified a panoply of in-frame expressed fusion genes, but early reports suggest that the majority of these are present at very low prevalence or are private events. Conventional methods for the identification of recurrent expressed fusion genes in large cohorts of cancers (eg fluorescence in situ hybridization (FISH) and reverse transcriptase PCR (RT-PCR)) are time consuming and prone to artifacts. Here, we describe a novel high-throughput strategy for the detection of recurrent fusion genes in cancer based on the Sequenom MassARRAY platform. Fusion genes were initially identified by massively parallel sequencing of breast cancer cell lines. For each fusion gene, two Sequenom probes were designed. Primary human breast cancers and cancer cell lines were interrogated for 10 fusion genes. Sensitivity, specificity, and predictive values of the MassARRAY method were then determined using FISH and qRT-PCR as the 'gold standard.' By combining two probes per fusion gene, the negative and positive predictive values were 100 and 71.4%, respectively. All fusion genes identified by massively parallel sequencing were accurately detected. No recurrent fusion genes were found. The MassARRAY-based approach described here may, therefore, be employed as a high-throughput screening tool for known fusion genes in human cancer. In keeping with other highly sensitive assays, further refinement of this technique is necessary to reduce the number of false-positive results.
authors.affiliation: Molecular Pathology Team, The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK.
authors.forename: Maryou B K
authors.initials: MB
authors.lastname: Lambros
authors.forename: Paul M
authors.initials: PM
authors.lastname: Wilkerson
authors.forename: Rachael
authors.initials: R
authors.lastname: Natrajan
authors.forename: Neill
authors.initials: N
authors.lastname: Patani
authors.forename: Vidya
authors.initials: V
authors.lastname: Pawar
authors.forename: Radost
authors.initials: R
authors.lastname: Vatcheva
authors.forename: Marthe
authors.initials: M
authors.lastname: Mansour
authors.forename: Mirja
authors.initials: M
authors.lastname: Laschet
authors.forename: Beatrice
authors.initials: B
authors.lastname: Oelze
authors.forename: Nicholas
authors.initials: N
authors.lastname: Orr
authors.forename: Susanne
authors.initials: S
authors.lastname: Muller
authors.forename: Jorge S
authors.initials: JS
authors.lastname: Reis-Filho
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D005260:Female; D005820:Genetic Testing / Q000592:standards; D006367:HeLa Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D046228:Microarray Analysis / Q000379:methods* / Q000592:standards; D015514:Oncogene Proteins, Fusion; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D015514:Oncogene Proteins, Fusion