Abstract 3394: Controls to detect limit of detection for BRAF V600E as an actionable mutation of interest by NextGen Sequencing and qPCR
Authors: Cristine L. Chisholm, Russell Garlick, Bharathi Anekella
Journal: Cancer Research (2015)

Abstract Background: Molecularly targeted therapy across tumor types has great potential to benefit cancer patients. In order to validate the assays being used to identify actionable mutations of interest, controls that establish an assay's limit of detection are needed. 40-60% of melanomas harbor a mutation in proto-oncogene BRAF, resulting in constitutive activation of the RAF/MEK/ERK signaling pathway; driving growth, differentiation and metastasis. Metastatic melanoma patients have a poor prognosis; 5 year survival after diagnosis is only 15%. BRAF mutations are also found in lymphomas, colorectal, thyroid and lung cancers. This study was undertaken to develop reference materials for establishment of the lowest limits of detection for identification of the BRAF V600E mutation with the cobas® 4800 BRAF V600 Mutation test, the THxIDTM BRAF kit, the Ion AmpliSeqTM Cancer Hotspot Panel. Reference materials with a limit of detection below 5% are useful for validation and identification of patient tumors with an extremely low percentage of the V600E mutation across multiple types of tumors. Methods: Melanoma cells containing the V600E allele, the V600D allele and the wild type allele were mixed at a range of allelic ratios from 50 to 1% in Histogel and embedded in FFPE. Genomic DNA was extracted from macro-dissected cells in 10μM FFPE sections and analyzed for quality and concentration using the KAPA Biosystems hgDNA Quantification and QC Kit. Quality scores (Q-ratios) generated with the kit may be used to predict the outcome of NGS library construction, with scores close to 1.0 being ideal for sequencing applications. Samples with Q scores close to 1.0, were subsequently tested on an ABI 7500 real time PCR system with the TrimGen BRAF V600 mutation kit. The extracted gDNA and unprocessed FFPE blocks and sections were then sent to diagnostic testing laboratories for analysis on the the cobas® 4800 BRAF V600 Mutation test, the THxIDTM BRAF kit, and the Ion AmpliSeqTM Cancer Hotspot Panel. Results: We were able to isolate high quality and quantity (&amp;gt;700ng per 2 10μM curls, Quality scores ∼1.0) gDNA suitable in fragment size and concentration for use in V600 companion diagnostic and NGS analysis from FFPE at a range of allelic ratios. Our 50% and 10% V600E mutant samples were detected by the TrimGen BRAF V600 mutation kit. Testing of mutations below 10% is currently ongoing on companion diagnostic and NGS platforms in clinical diagnostic laboratories. Conclusions: Prototype FFPE and extracted genomic DNA process controls were generated for use in NGS and companion diagnostics for BRAF V600. The advantage of this material is that it contains the mutation of clinical significance at well defined, consistent low levels, at and below the current minimum detectable mutant allele ratio of 10%. These reference controls may allow laboratories to verify assay detection limits, establish run to run reproducibility, and monitor testing quality over time. Citation Format: Cristine L. Chisholm, Russell Garlick, Bharathi Anekella. Controls to detect limit of detection for BRAF V600E as an actionable mutation of interest by NextGen Sequencing and qPCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3394. doi:10.1158/1538-7445.AM2015-3394