Whole exome paraffin sequencing (WEPS) of same-patient (pt) primary, extracranial, and melanoma brain metastases (PEB) to identify new driver and clonal evolution genetic events.

e20033 Background: To date, little is known about how primary melanomas clonally evolve to distant metastases, including brain, and how driver mutations are selected during melanoma progression. To identify new driver and clonal evolution genetic events beyond the known mutations in BRAF/NRAS, we performed WEPS of same-pt PEB. Methods: DNA from tumor (T) and adjacent normal (AN) was extracted from macrodissected tissue slides. DNA libraries were captured using Agilent SureSelect Human All Exon v4 and sequenced at 2x48 bp. Variant calling was performed on T vs. matched AN using VARSCAN (default settings). Single nucleotide variations (SNV) were filtered using prior knowledge (COSMIC). We report only genes having probably or possibly damaging non-synonymous SNV (PolyPhen2 score > 0.45) by ANNOVAR analysis and nonsense mutations. Focused analysis for gene copy number alterations previously described in melanoma was performed using our previously published NGScopy package (Bioconductor). We considered analysis for gene copy number alterations if median coverage depth > 20X. We computed T/AN copy number ratio at the gene against the chromosome level. Results: To date, 4 pts with all four tissues (PEB-AN) have been analyzed. Mean coverage depth was 68X and read quality was 37. 85% of reads were uniquely mapped. The 10 most frequently mutated genes across all 12 T samples were APC, ARID1A, CDKN2A, EGFR, FBXW7, KIT, KMT2C, NF1, PTEN, and TET2 (FDR < 0.0001). NF1 mutations were present in all T from 4 pts. All T from 3 out of 4 pts had mutations in BRAF (2 with confirmed V600E), PTEN, IDH2, TET2, CCDC6, PCSK7, and members of the ARID and KMT2 gene family. Both metastatic T from 2 pts had mutations in TRIM27, NCOA1, BCL6, and BCL10 that were not present in primary. All T from 2 pts and all T from one pt had > 30% amplification of the KIT and KDR genes, respectively. Conclusions: Aggressive melanoma bears known mutations in signaling pathways that coexist with mutations in genes associated with chromatin modification, histone/nucleotide methylation, and metabolism. Identifying such mutations in primaries may predict future prognosis.