Presentation Abstract

Abstract Number: 1641
Presentation Title: Concordance between mutations identified by bar-coded multiplexed sequencing of BRAF, NRAS, and KIT in formalin fixed (FFPE) tissue from melanoma patients using the Ion Torrent Personal Genome Machine (PGM) and a real-time PCR assay
Presentation Time: Monday, Apr 02, 2012, 8:00 AM -12:00 PM
Location: McCormick Place West (Hall F), Poster Section 23
Poster Section: 23
Poster Board Number: 2
Author Block: George S. Watts, Marc M. Oshiro, Lee D. Cranmer. Univ. of Arizona Cancer Ctr., Tucson, AZ
Abstract Body: The standard chemotherapy for metastatic melanoma is dacarbazine, however, progression free survival with dacarbazine is a dismal two months. Efforts to improve the treatment of melanoma have focused on specific molecular defects following the discovery that the majority of patients have a mutation in one of three members of the BRAF-MAPK signaling pathway: KIT, BRAF, and RAS. Recent clinical trials have shown promise for drugs that target the molecular defects in melanoma, including the recently approved mutant BRAF inhibitor vemurafenib. As new drugs that target specific mutations move through clinical trials, the opportunity to perform precision medicine is becoming a reality. To deliver on the promise shown by the new generation of drugs it will be necessary to detect specific mutations in patients so they can be matched with the right drug. To this end, we have used an Ion Torrent Personal Genome Machine to sequence the six most commonly mutated codons in melanoma (BRAF 600, NRAS 12 and 13, NRAS 61, KIT 576, and KIT 642) from formalin fixed paraffin embedded tissue of late stage melanoma patients. All six codons of interest were sequenced in forty-six patients and mutations identified by comparison to normal controls. The A375 cell line, containing the known mutation V600E in BRAF was used as a positive control. Samples were sequenced in a multiplexed format using bar coding of the amplicons which allowed 14 patients and two controls to be sequenced simultaneously on each chip. Results were confirmed by real time RT-PCR assay specific for the BRAF V600E mutation. We demonstrate the feasibility of using rapid low cost next-generation sequencing to provide robust detection of mutations in fixed patient samples.