Mutational Analysis and Single Cell Sequencing of Melanoma Brain Metastases

Abstract

<h3>Purpose/Objective(s)</h3> Melanoma brain metastases are a significant source of morbidity and mortality. While radiosurgery and (CNS) penetrant therapies have improved clinical outcomes, understanding the molecular landscape and microenvironment of brain metastases is critical to devise improved treatments for patients. Here, we perform bulk and single cell genomic analysis of melanoma brain metastases to identify molecular mechanisms underlying therapeutic response. <h3>Materials/Methods</h3> 50 consecutive patients who underwent surgical resection with histo-pathologically confirmed melanoma brain metastases and known BRAF^V600E status treated at a single institution were retrospectively identified. In 25 patients (50%) with sufficient brain metastasis tissue for targeted next generation sequencing, DNA mutations were assessed with a CLIA certified sequencing assay spanning over 500 cancer genes. Single cell RNA sequencing was performed on n=5 samples with sufficient frozen tissue. Overall survival (OS) and CNS progression free survival (CNS PFS) were estimated using the Kaplan-Meier method from time of brain metastasis diagnosis. Single cell analysis was performed using the 10x platform and Seurat R package. <h3>Results</h3> The median patient age was 64 years old (27 – 78 years), and the median clinical follow up was 17 months. 21 patients (42%) had BRAF^V600E melanoma brain metastases. BRAF^V600E mutation was associated with worse CNS PFS (p=0.04, log-rank test) and OS (p=0.03, log-rank test). Multivariate analysis incorporating age, Karnofsky performance score, and presence of extracranial disease supports <i>BRAF</i> status as an independent prognostic factor for OS (p<0.05). In patients undergoing targeted next generation sequencing, the most common pathogenic variant was <i>TERT</i> promoter mutation (n=19; 76%) followed by <i>TP53</i> mutation (n=9; 36%). The four TCGA molecular melanoma subgroups were equally represented: <i>NRAS</i> mutant (n=8; 32%), <i>NF1</i> mutant (n=6; 24%), <i>BRAF</i> mutant (n=6; 24%) or triple wildtype (n=5; 20%) Evaluation of clinical outcomes in the context of next generation sequencing results revealed no differences by <i>TERT</i> status but demonstrated worse overall survival in <i>BRAF</i> mutant tumors (median OS: 11 months; p=0.01, log-rank test). Single cell sequencing of 31,219 nuclei across 5 samples revealed distinct subpopulations between <i>BRAF</i> mutant and <i>BRAF</i> wildtype tumors in both the tumor and the brain microenvironment, with increased immune infiltration in <i>BRAF</i> wildtype tumors and greater neuronal subpopulations in <i>BRAF</i> mutant lesions. <h3>Conclusion</h3> Our data suggest <i>BRAF</i> may be useful as a prognostic biomarker and <i>TERT</i> promoter mutations as a recurrent intracranial molecular alteration in melanoma. Single cell analysis shows brain metastases exhibit distinct neuronal and immune populations. Future work will require integration of molecular status with treatment to help guide therapy and interrogation of larger, prospective, multi-institutional cohorts to validate these observations.