Abstract 1012: Detecting and intervening on rare pre-existing resistant subclones to BRAF/MEK inhibitors in metastatic melanoma
Authors: Nakazawa Michael , Cheng Lauren , Ahmed Mishal , Lazcano Rossana , Wani Khalida , Veiseh Omid , Kwong Lawrence 
Published: June 2023

Abstract
The combination of BRAF and MEK inhibitors (BRAFi/MEKi) in metastatic melanoma patients with BRAF activating mutations have resulted in dramatic responses in most patients, but unfortunately most patients progress after one year and limited subsequent treatment options are available. Although resistance mutations to BRAFi/MEKi mainly occur in the MAPK and PI3K pathways, after progression on BRAFi/MEKi drugs, targeting these mutations have been clinically unsuccessful. One possible cause of relapse is that a resistant subpopulation to BRAFi/MEKi exists prior to treatment, raising the possibility that earlier counter-resistance treatment could be administered and potentially be more successful. However, the prevalence and clinical significance of pre-existing resistance is currently unknown in melanoma. To address this, we used quantitative blocker displacement amplification (qBDA) to quantify the VAF (down to 0.01% resolution) of resistant subclones along the MAPK/PI3K pathways from FFPE biopsies on 149 metastatic melanoma patients. Due to possible intra-tumoral heterogeneity, multiple samples were taken when available per patient. Of 29 patients currently processed, 5/29 (17%) had detectable NRAS Q61L or Q61R, 5/29 (17%) had KRAS G12 or G13 mutations, and 3/29 (10.3%) had PIK3CA H1047R/Y. As expected, BRAF activating mutations were detectable in 29/29 patients, while VAF of the resistant subclones were found to be as low as 0.16%. We expect some of the putative resistance mutations to be artifacts, and all will be validated by ddPCR. To model the behavior of low-VAF resistance mutations, we conducted in vitro spike-in experiments using paired and labeled parental and BRAF/MEK inhibitor-resistant melanoma lines. Our results show that even down to 0.1%, the resistant lines can rapidly emerge as the dominant population while under drug treatment. We also tested the timing of applying a counter-resistance therapy: early vs late. Earlier intervention led to a significant delay in growth of the resistant population, as expected. However, we are currently preparing an experiment that will determine whether an earlier intervention might not simply delay resistance, but also alter its strength, at different % VAFs. We therefore anticipate mechanistic analyses of how %VAF and drug timing can optimize counter-resistance therapies. Overall, our interim data suggest a significant percentage of metastatic BRAF-mutated melanoma patients may harbor pre-existing resistant subclones, and that early treatment with counter-resistance therapy can potentially delay resistance to targeted therapy.
Citation Format: Michael Nakazawa, Lauren Cheng, Mishal Ahmed, Rossana Lazcano, Khalida Wani, Omid Veiseh, Lawrence Kwong. Detecting and intervening on rare pre-existing resistant subclones to BRAF/MEK inhibitors in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1012.