Heterogeneity of BRAF, NRAS, and TERT Promoter Mutational Status in Multiple Melanomas and Association with MC1R Genotype

Abstract

Data on somatic heterogeneity and germline-somatic interaction in multiple primary melanoma (MPM) patients are limited. We investigated the mutational status of BRAF, NRAS, and TERT promoter genes in 97 melanomas of 44 MPM patients and compared molecular and immunohistochemical findings. We further evaluated the association of somatic alterations with the germline MC1R genotype. Mutations in BRAF gene were identified in 41.2% (40/97) of melanomas, in NRAS in 2.1% (2/97), and in TERT promoter in 19.6% (19/97). Distribution of BRAF mutations did not differ across multiple melanomas (P = 0.85), whereas TERT promoter changes decreased from first to subsequent melanomas (P = 0.04). Intrapatient discrepancy of BRAF mutations among multiple tumors was detected in 14 of 44 MPM patients (32%) and of BRAF/NRAS/TERT promoter genes in 20 of 44 (45%). We observed a high rate of agreement between allele-specific TaqMan assay and immunohistochemistry in BRAF detection (κ = 0.83, P < 0.01) with 86 of 97 melanomas (88.7%) presenting similar BRAF status. Germline MC1R variants were identified in 81.4% (35/43) of MPM patients with no association of MC1R genotype with somatic mutations or with intrapatient concordance of somatic mutational profile. Our results support the genetic diversity of multiple melanomas and show that somatic heterogeneity is not influenced by inherited MC1R variants. Immunohistochemistry may be useful as an initial screening test.

Dermoscopic similarity is an independent predictor of BRAF mutational concordance in multiple melanomas

“…Intra‐patient concordance of the BRAF mutational status (either BRAF ‐mutated or BRAF wild‐type) between first and second melanoma was observed in 53.2% of our patients. A similar rate of BRAF mutational concordance ranging from 52.3% to 60% has been reported in previous studies . These data support the intra‐patient heterogeneity of BRAF mutational status in multiple melanomas of the same patient.…”

“…For competitive allele‐specific TaqMan TM PCR, the reaction containing 20 ng of DNA, 1X TaqMan TM Mutation Detection Assays (assay Hs00000111_mu for BRAF V600E ; Hs000000002_rm for BRAF V600K ), 1X TaqMan TM Genotyping Master Mix (Thermo‐Fisher) and water to reach the final volume of 20 µL was amplified using the standard TaqMan protocol on 7500 Fast Real‐Time PCR System (Thermo‐Fisher) . The ΔCt cut‐off was calculated using 5 DNA samples extracted from FFPE sections of melanomas positive for BRAF V600E or BRAF V600K and 3 extracted from FFPE sections of normal skin of unaffected individuals.…”

“…The most common oncogenic alteration is a hotspot mutation involving codon 600 of the BRAF gene, mainly the V600E change, occurring in 40%‐50% of cutaneous melanomas . A concordant BRAF mutational status (either V600‐mutated or wild‐type) between multiple melanomas within the same patient has been recently identified in 50% to 60% of patients, independently of location and time of melanoma occurrence. This supports the hypothesis of molecular heterogeneity of multiple melanomas in patients with MPM and has great impact in clinical practice when it is necessary to molecularly identify MPM subjects with discordant multiple melanomas for eligibility to target therapy with BRAF inhibitors.…”

Melanoma types by in vivo reflectance confocal microscopy correlated with protein and molecular genetic alterations: A pilot study

“…BRAF V600E and BRAF V600K mutations were analysed by competitive allele‐specific TaqMan PCR (castPCR Technology) while exon 3 of NRAS and exons 11 of c‐KIT were tested by Sanger sequencing. For competitive allele‐specific TaqMan assay, TaqMan Mutation Detection Assays (assays Hs00000111_mu for BRAF V600E and Hs00000769_mu for BRAF V600K ) were used to amplify the regions of interest according to the standard TaqMan protocol on a 7500 Fast Real‐Time PCR System (Thermo Fisher, Foster City, CA, USA) . Five DNA samples extracted from FFPE CM sections BRAF V600E positive and three from FFPE non‐CM sections were used to set a ΔCt cut‐off for mutation detection experiments.…”

“…Melanomas occurring on CSD regions have a different set of mutations in driver genes, such as NRAS and KIT . NRAS mutations mostly affect exon 2 at codon 61 and have been reported in approximately 20% of CM . About 1%‐3% of all melanoma show KIT alterations, including gene amplification and point mutations in exons 11 and 13 .…”