Abstract 5531: Detection of co-occurring and potential resistance mutations in cell-free, circulating tumor DNA from patients with BRAF
mutant metastatic melanoma undergoing treatment with BRAF-targeted therapies
Authors: Corless Broderick , Chang Greg , Liu Weihua , Li Jin , Marziali Andre , Mai Laura , Wiggin Matthew , Wilson Melissa , Pavlick Anna , Osman Iman , Karlin-Neumann George , Spittle Cindy , Polsky David 
Published: August 2018

Abstract
Purpose Melanoma patients with BRAFmutant tumors often develop resistance to BRAF-inhibitor therapies. Co-occurring mutations, present at the time of treatment initiation, have been identified in patients with primary treatment resistance, and NRAS mutations have been associated with secondary resistance. We tested the ability of multiplex mutation detection assays to identify possible co-occurring and resistance mutations in cell-free, circulating tumor DNA (ctDNA) from patients undergoing treatment with BRAF-targeted therapies.
Methods Purified ctDNA samples remaining from a previous longitudinal study of metastatic melanoma patients, which measured BRAFmutant and NRASmutant ctDNA using droplet digital PCR (ddPCR) duplex assays, were used for this study. Twelve samples from 6 patients with BRAF V600E mutant (V600E) tumors, who were receiving BRAF-inhibitor therapy, were separately analyzed in different laboratories using the two different multiplex assays (Oncomine and OnTarget). Oncomine used approximately 20ng DNA; OnTarget used approximately 30ng DNA. Patient samples were chosen based on clinical response or disease progression at the time of blood draw.
Results Ten of 12 samples had V600E ctDNA detected by ddPCR. The On-Target detected V600E in 9/10 samples; the Oncomine detected V600E in 8/9 samples (1 sample was not analyzed due to limited sample availability). The 1 sample with a ddPCR detected mutation that was not detected by either OnTarget or Oncomine had a 0.01% fractional abundance, which is below the detection limit of these multiplex assays. Neither assay detected V600E DNA when it was not detected by ddPCR. One patient had 2 samples with a co-occurring p53 (R273H) mutation detected by OnTarget and Oncomine. This mutation was present at the time of partial response, and at disease progression with an associated increased fractional abundance as determined by both assays. The OnTarget detected NRASmtant ctDNA (Q61K x 2, Q61H x1) in 3 of the V600E samples; the Oncomine detected the Q61K in 1 of these 2 samples (the third sample was not analyzed as noted above). The NRAS mutations arose at times of disease progression. Oncomine also detected additional p53, GNAS, and FBXW7 mutations at generally low fractional abundances that were not detected by OnTarget.
Conclusion Co-occurring and potential resistance mutations are detectable in the plasma of metastatic melanoma patients using OnTarget or Oncomine assays. The assays demonstrate high sensitivity, as evidenced by their ability to identify V600E ctDNA. These findings suggest that studies analyzing patient ctDNA for resistance mutations while they are undergoing treatment are feasible. Results of such studies may eventually help inform treatment choices, such as switching therapies when resistance mutations emerge.
Citation Format: Broderick Corless, Greg Chang, Weihua Liu, Jin Li, Andre Marziali, Laura Mai, Matthew Wiggin, Melissa Wilson, Anna Pavlick, Iman Osman, George Karlin-Neumann, Cindy Spittle, David Polsky. Detection of co-occurring and potential resistance mutations in cell-free, circulating tumor DNA from patients with BRAFmutant metastatic melanoma undergoing treatment with BRAF-targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5531.