Massive Parallel Sequencing To Assess the Mutational Landscape of Fine Needle Aspirate Samples: A Pilot Study
Jose L Costa, Rene Gerhard, Esther Diana Rossi, Luis Cirnes, Ana Justino, Jose C Machado, Fernando Schmitt. IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal; Catholic University of Sacred Heart, Rome, Italy; Medical Faculty of the University of Porto, Porto, Portugal
Background: Nowadays, massive parallel sequencing(MPS)is shaping research in the field of life sciences.This fast evolving technology is starting to move into the clinical diagnostic arena.However,only with the recent introduction of the benchtop sequencers it can be adopted by the majority of molecular pathology laboratories.The possibility of performing genomic studies with small amounts of material obtained,for example,by fine-needle aspiration(FNA),can minimize invasive procedures and allow the monitoring of cancer,including therapeutic response,with repeated testing.
Design: In this pilot study we aim to address the possibility of using this technology for assessing the mutational landscape of FNA samples.Four samples collected from Breast cancer patients were used to isolate genomic DNA.A single tube multiplex PCR amplification designed to detect 739 mutations from 46 oncogenes and tumor suppressor genes was performed and the resulting amplicons sequenced using the Ion Torrent PGM sequencer.
Results: Genomic DNA of good quality was obtained from all samples.This enabled the amplification and sequencing of all targeted regions.High coverage was obtained for all amplicons.The use of the variant caller plug-in from the Torrent Suite v2.2 resulted in the determination of the mutational landscape of the FNA samples.Importantly, mutations in genes that can be therapeutically intervened were identified using this strategy,namely PIK3CA, SMAD4 and KDR.This resulted in findings that could be used for a better management of the patients,and that would not be obtained using the standard routine practice.
Conclusions: In this study we present a workflow that provides a comprehensive genetic screening tool for FNA samples, in a fast and cost-efficient manner.This may provide a valuable tool for the management of cancer patients that can be implemented in most molecular pathology laboratories.
Tuesday, March 5, 2013 1:00 PM
Poster Session IV # 71, Tuesday Afternoon