Abstract 1682: Non-V600 BRAF mutants in melanoma: Multiple MAPK mutations and distinctive RNA expression
Authors: Kiernan Elizabeth , Bastian Paul , Dorfman Liza , Anastopoulos Ioannis N. , Wubbenhorst Brad , Krepler Clemens , Rebecca Vito , Herlyn Meenhard , Nathanson Katherine 
Published: August 2019

Abstract
Large discovery sequencing analyses conducted by our group and others enabled classification of melanomas into major and minor subtypes, helping to elucidate the role and diversity of activating mutations within the MAPK pathway. We sequenced 482 melanoma PDX, cell lines and patient biopsies, of which 20 (4.1%) carry non-V600 BRAF mutations. Of the 20 mutations, 14 are pathogenic, 5 are likely pathogenic and 1 is a variant of unknown significance. Interestingly, 78.5% of tumors with pathogenic non-V600 mutants and all likely pathogenic or VUS non-V600 mutants carry additional mutations within the MAPK pathway. To explore this pattern, we queried publicly available melanoma sequencing data from six previously published sequencing studies of melanoma. Consistent with our findings, nonV600-BRAF mutations were identified in 0 - 9% of melanoma sequencing studies. Of the tumors carrying non-V600 BRAF mutants, 26 of 42 (62%) carry additional mutations in the MAPK pathway. Given the co-occurrence of MAPK mutations with non-V600 mutants, we considered whether these mutations are found together within the same cell or if mutants are encoded in distinct cell populations and reflect admixture of a heterogenous tumor. We compared the variant allele frequency (VAF) of co-occurring MAPK mutants with their non-V600 BRAF counterparts in 41 available samples. The VAF between mutants was discordant (&gt;0.10 change in VAF between mutants) in 27 of 41 (68%) of the samples. To more precisely assess variants at single-cell resolution, we established a PCR based method for variant calling using single-cell-derived cDNA libraries. Pilot studies supported distinct expression patterns and we are working to generate robust and reproducible sequencing data. We also compared RNA expression data between BRAFV600 melanomas and non-V600 melanomas. This analysis revealed distinct transcriptomes that were easily distinguished based on previously established quaternary structure and kinase activity of various BRAF mutants. Interestingly, MAPK-pathway genes did not account for most of the differential expression, and an AXL-high/MITF-low expression program was highly enriched in non-V600 BRAF tumors relative to their V600 BRAF counterpart (FDR &lt;0.01). Taken together, non-V600 BRAF mutant melanomas represent a subtype characterized by a tendency for multiple MAPK mutations and an AXL-high/MITF-low enriched RNA expression pattern differentiated based on BRAF structure and function. Further studies of this rare sub-type of melanoma are important to demonstrate the diversity and interplay of MAPK dysregulation in cancer and may reveal mechanisms of tumorigenesis.
Citation Format: Elizabeth Kiernan, Paul Bastian, Liza Dorfman, Ioannis N. Anastopoulos, Brad Wubbenhorst, Clemens Krepler, Vito Rebecca, Meenhard Herlyn, Katherine Nathanson. Non-V600 BRAF mutants in melanoma: Multiple MAPK mutations and distinctive RNA expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1682.