Massarray Technology: High Throughput Method for Unlocking the Presence of Recurrent Fusion Gene in Cancer.
Maryou B Lambros, Rachael Natrajan, Radost Vatcheva, Henning Gohlke, Malcolm Plant, Susanne Muller, Jorge S Reis-Filho. ICR, London, United Kingdom; Sequenom GmbH, Hamburg, Germany
Background: Fusion genes result from chromosomal structural rearrangements and play pivotal roles in the biology of many solid tumours. We and others have recently demonstrated that breast cancers harbour fusion genes, however most of the fusion genes identified in breast cancer appear not to be recurrent with the exception of ETV6-NTRK3 in secretory carcinomas and MYB-NFIB in adenoid cystic carcinomas. Conventional approaches to determine whether a fusion gene is recurrent (e.g. fluorescence in situ hybridisation (FISH) and reverse transcriptase PCR (RT-PCR)) are either labour intensive or prone to false positive results. The aim of our study was to develop a high throughput method to investigate multiple samples for the presence of novel fusion genes based on the MassArray technology (iPLEX® assay).
Design: Nine breast cancer cell lines previously subjected to massively parallel sequencing and known to harbour 10 specific fusion genes previously validated by Sanger sequening and FISH were used as the study group. RNA was extracted from 126 formalin fixed paraffin embedded (FFPE) breast cancers, 9 breast cancer cell lines and HeLa cells, and converted to cDNA. Small amplicons (70-150bp) were designed based on the breakpoint positions within the cDNA sequence of a given fusion gene, followed by a single primer extension from one partner of the fusion gene to the other. Two pairs of extension primers were used to extend from one partner of a given fusion gene to the other and vice versa. A multiplex of 13 PCR amplicons, were used to identify 13 fusion genes discovered recently in breast cancer cell lines. Positive results were validated by RT-PCR.
Results: MassArray displayed a sensitivity of 98.9%, a specificity of 95.2%, a positive predictive value of 38.6% and a negative predictive value of 99.9% to detect all fusion genes tested. All positive results were validated by RT-PCR and sequencing. Fusion genes were accurately detected in cDNA dilutions of up to 1:5.000. Although no recurrent fusion genes were detected and validated in the small cohort of primary tumours analysed in this study, all fusion genes were accurately detected in the panel of cell lines used.
Conclusions: The high level of sensitivity of the MassArray assay suggests that such high throughput assay can be used as a screening tool to investigate the presence of known fusion genes. Further work is required to minimise the number of false positive results. The small PCR amplicons (70-150 bp) used for the MassArray assay facilitates the usage of FFPE tissue samples.
Monday, February 28, 2011 8:30 AM
Platform Session: Section G 1, Monday Morning