Effect of BRAF V600E Heterogeneity on Melanoma Pathogenesis

To determine if BRAF V600E is conserved during melanoma disease progression and the effect of heterogeneity on patient outcomes, clinical data was collected from 17 patients with cobas molecularly determined BRAF status. HRM analysis followed by Sanger sequencing was performed on tumor-rich regions micro-dissected from all available matched FFPE tissue samples from each patient to elucidate spatial and temporal differences in BRAF status. Notably, 4/7 patients (57%) were found to be misdiagnosed by the cobas test. Intra- and inter-tumor heterogeneity with respect to BRAF V600E was revealed in 6/13 patients (46%). Three patients had discordant results between primary and metastatic tumors, suggesting that BRAF is not necessary to initiate metastasis. In this cohort, patients harboring heterogeneous melanoma were found to have an increased likelihood of progressing or recurring during a given time period (HR 4.74, 95% CI 0.410-54.745, p = 0.1953) and were 3.566 times more likely to experience a new metastasis (95% CI 0.741-17.162, p = 0.0951) than patients with non-heterogeneous BRAF V600E melanoma. Our findings suggest a complex picture of melanoma pathogenesis whereby tumors are comprised of at least two distinct types of malignant clones that differ in BRAF mutation status and the presence of both negatively influences patient outcomes. Further investigation of melanoma heterogeneity is crucial to facilitate personalized treatment strategies for this aggressive malignancy