Usefulness of immunocytochemistry for the detection of the BRAF(V600E) mutation in circulating tumor cells from metastatic melanoma patients.

TO THE EDITOR Metastatic melanoma patients harboring a BRAF gene mutation on codon 600 can be treated with targeted therapies (Flaherty, 2012). Depending on the content of tumor cells and on the analytical sensitivity, BRAF mutations are found in 50–70% of metastatic melanoma patients (Davies et al., 2002). Around 80% display a valineto-glutamic acid substitution (V600E) and B16% harbor a valine-tolysine substitution (V600K) causing constitutive kinase activation (Wan et al., 2004; Rubinstein et al., 2010). BRAF mutation analysis is currently performed in daily clinical practice on tissue samples using various molecular biology technologies. Moreover, the detection of the BRAF mutation in blood samples from melanoma patients in the context of translational research and clinical trials has been described (Board et al., 2009; Sakaizawa et al., 2012). Metastatic dissemination correlates with the presence of circulating tumor cells (CTCs) detected in blood samples (Paterlini-Brechot et al., 2011; Alix-Panabières et al., 2012). The detection of circulating melanoma tumor cells (CMCs) can be performed using different technologies, in particular by the isolation by size of epithelial tumor cells (ISET) method, a direct method that allows cytopathological analysis of CMCs (De Giorgi et al, 2010). Moreover, ancillary methods for CTC characterization can be performed on cells isolated by ISET (De Giorgi et al., 2010; Ilie et al., 2012). Recent studies highlighted the value of immunohistochemistry (IHC) using the VE1 antibody for the detection of the BRAF mutation in melanoma (Capper et al., 2012). The aim of this work was to combine ISET and immunocytochemistry (ICC) using the VE1 antibody to investigate the presence of BRAF in CMCs from metastatic melanoma patients. Therefore, 98 metastatic melanoma patients were screened for BRAF both by pyrosequencing and by IHC anti-VE1. Concomitantly and blindly, ICC for the BRAF mutation was performed on CMCs isolated by ISET (See Supplementary Data). Population data are shown in Supplementary Table S1. Of 98 patients, 53 (54%) had a BRAF mutation detected by pyrosequencing in tissue samples. Among these patients, 51/53 (96%) showed strong immunostaining with the VE1 antibody in tissue sections (Supplementary Table S2). Homogenous intracytoplasmic staining without associated nuclear staining was demonstrated in melanoma cells only (Figure 1). Among the tumors negative for the BRAF mutation by pyrosequencing, none had positive VE1 immunostaining (Supplementary Table S2; Figure 1). An excellent concordance was found between these two methods (Supplementary Table S2). The IHC antiVE1 demonstrated 96% sensitivity and 100% specificity when compared with the sequencing results. CMCs were isolated in 87/98 (89%) patients. Of 87 patients, 54 (62%) demonstrated positive immunostaining on ISET filters as detected by VE1 ICC (Table 1; Figure 1). Forty-six out of fifty-four (85%) patients with CMCs positively stained by ICC had a BRAF mutation detected in tissue specimen by pyrosequencing (Table 1; Figure 1). Eight out of fifty-four (15%) patients with positive VE1-immunostained CMCs lacked BRAF in tumor tissues, analyzed both by pyrosequencing and IHC (Table 1; Figure 1). The ICC VE1 CMC-based assay LETTER TO THE EDITOR