Can detection of Braf p.V600E mutation be improved? Comparison of allele specific multiplex sequencing to present tests

Objective: This is an investigative study to evaluate a new companion diagnostic platform, allele specific multiplex sequencing (ASMS). Detection of Braf p.V600E from solid tumors is used as the test model with the following objectives: 1) whether ASMS can detect Braf p.V600E/K mutations from a variety of solid tumors, 2) whether ASMS can detect all Braf p.V600E from samples that were positive for Braf V600E by SNaPshot or Ion Torrent, and 3) whether ASMS can detect Braf p.V600E among samples that were reported negative by SNaPshot or Ion Torrent. Methods: ASMS is a novel modification (US Patent 6197510) of traditional Sanger sequencing, with Lower Limit of Detection (LLOD) of 20 GE (Genome Equivalent) and 0.001% sensitivity. We compared ASMS to clinical samples previously tested either by SNaPshot or Ion Torrent methods. Results: We analyzed 83 DNA extracts from FFPE samples (41 tested by SNaPshot and 42 tested by Ion Torrent). There was a total of thirty-seven samples positive for Braf p.V600E (16 by Ion Torrent; 21 by SNaPshot), and all of these samples tested positive by ASMS for Braf p.V600E. Out of the 46 negatives for Braf p.V600E (20 by SNaPshot; 26 by Ion Torrent samples), ASMS detected Braf p.V600E positive results in 10 of the SNaPshot and in 18 of the Ion Torrent negative samples. ASMS could detect both Braf p.V600E and the wild-type Braf p.V600 simultaneously with 40 pg of FFPE DNA extracts. Conclusions: ASMS assay detected all Braf p.V600E positives from different types of solid tumors that previously tested positive by SNaPshot or Ion Torrent. Further, ASMS was able to detect Braf p.V600E among samples that were reported negative by SNaPshot or Ion Torrent.