A Rare Complex BRAF Mutation Involving Codon V600 and K601 in Primary Cutaneous Melanoma: Case Report

Abstract

BRAF is one of the most common mutated kinases detected in human cancer, particularly in cases of primary cutaneous melanomas (PCM). Mutations of the BRAF proto-oncogene, at the p.V600 codon, has been detected in more than 50% of primary and metastatic melanoma cells in clinical samples. In addition to the most frequent BRAF p.V600E mutation, corresponding to the single base pair substitution c.1799T>A, rarer mutations, within and outside the V600 codon, have been described. Expectedly, BRAF and MEK inhibitors (or their combination) have been poorly explored as potential therapeutic strategies in metastatic melanomas harboring this rare mutation. By using a set of sequencing techniques and immunohistochemistry, this work reports the genomic and clinical features of two melanoma patients showing a rare complex mutation affecting codon V600 and K601 of the BRAF gene, leading to a V600E2; K601I change. Specifically, these two patients show a distinct clinical behavior and significantly differ in their responses to BRAF and MEK inhibitors. Indeed, although this treatment has proven to be effective and safe in both cases, the observed variability between the two patients resulted as a direct consequence of the baseline extent of brain involvement, intracranial treatment failure as well as on the PTEN status.

Keywords: BRAF; BRAF inhibitors; MEK inhibitors; NGS; metastatic melanoma; mutation.

Figure 1

Melanoma histology in Patient 1 (PT#1) and Patient 2 (PT#2) at different sites. Sections of PT#1 and PT#2 melanomas are from different melanoma sites as indicated and stained for haematoxilin and eosin. Magnification: 200x; sk, skin; ov, ovary; br, brain.

Figure 2

BRAF status in PT#1 and PT#2. Sections are from PT#1 and PT#2 and stained for anti-VE1 as labeled. Primary (PT#1 and PT#2^sk) and metastatic (PT#2°v and PT#2^br) melanomas are illustrated. A granular cytoplasmic stain for anti-BRAFV600E was detected in melanoma cells from all samples; no reactivity is observed in endothelial cells an immune cells. Sections were counterstained with hematoxylin. Magnification: 400x. Sequencing data illustrate the BRAF substitutions (Igv screenshot of amplicon that cover codon 600 and 601 of BRAF) at codon V600 and K601 in PT#1 and PT#2 by using Ion S5 system (PT#1) and Illumina MiSeq (PT#2^sk, PT#2^ov, and PT#2^br).

Figure 3

PTEN status in PT#1 and PT#2. Sections are from PT#1 and PT#2 and stained for anti-PTEN as labeled. Primary (PT#1 and PT#2^sk) and metastatic (PT#2°v and PT#2^br) melanomas are illustrated. Loss of PTEN reactivity was observed in melanoma cells from all samples except PT#2^sk; internal positive control are represented by vessel. Sections were counterstained with hematoxylin and imaged at 100x magnification. Sequencing data illustrate the PTEN sequencing analysis (Igv screenshot of amplicon that cover exon 2 of PTEN) by Illumina MiSeq in P#T1 and PT#2 samples. A PTEN substitution is observed in samples PT#2^ov and PT#2^br. sk, skin; ov, ovary; br, brain.