Dynamic and unpredictable changes in mutant allele fractions of BRAF and NRAS during visceral progression of cutaneous malignant melanoma

Data indicate that primary cutaneous melanomas are characterized by clonal heterogeneity associated with oncogenic drivers. Less data are available on the clonal changes occurring during melanoma progression. We therefore wished to analyse these changes in skin melanomas in common sites of visceral metastases as compared to the primary tumor.

An autopsy cohort of 50 patients with BRAF- and NRAS-mutant cutaneous metastatic melanomas including 139 visceral metastases was analysed for mutant allele fractions (MAF), determined by pyrosequencing and corrected for tumor/normal ratio. MAF levels were also classified as high (> 40%), medium (15–40%) or low (< 15%).

Contrary to NRAS mutant cases, in BRAF-mutant melanomas MAFs were found to be significantly increased in visceral metastases compared to the primary due to the significantly higher levels in lung-, adrenal gland-, intestinal- and kidney metastases. The incidence of the three MAF variants in BRAF-mutant primaries was similar, whereas the high MAF cases were found to be increased in metastases. On the other hand, medium MAF levels were more common in case of NRAS-mutant tumors. Only 31.3% of BRAF mutant- and 50% of NRAS mutant cases maintained the MAF profile of the primary in metastasis. In the majority of multiple metastatic tumors, (BRAF:71.8%, NRAS:75%) metastases were relatively homogeneous regarding MAF. However, in 6/32(18.7%) of BRAF mutant cases low MAF primaries switched to high MAF in metastases. In heterogeneous BRAF mutant metastatic cases low to high or high to low MAF conversions occurred in a further 4/32(12.5%) cases in individual metastases as compared to the primary tumors. At lower frequency, in NRAS mutant tumor such changes also observed (2/12,16.7%).

We provided evidence for the selection of BRAF-mutant melanoma cells during metastatic progression to the lung, intestine, adrenal gland and kidney. Our findings suggest that in visceral metastases of malignant melanoma BRAF- or NRAS-MAFs are rather heterogeneous and cannot be predicted from data of the primary tumor. These data may have clinical significance when using targeted therapies.