Original contributionUtility of BRAF V600E Immunohistochemistry Expression Pattern as a Surrogate of BRAF Mutation Status in 154 Patients with Advanced Melanoma☆,☆☆
Introduction
Investigations into the genomic landscape of cutaneous melanoma have identified mutations in the BRAF oncogene in 50% to 60% of patients as the most common oncogenic driver mutation [1], [2]. Among the activating BRAF mutations in melanomas, the majority (>99%) involves exon 15. The most frequent BRAF mutation type encountered (~75%-90%) is BRAF V600E, which involves a threonine-to-alanine (T to A) single-DNA base substitution [1], [2], [3]. Compared with the kinase activity of the BRAFWT protein, the BRAF V600E protein exhibits 480 times higher kinase activity; furthermore, patients with BRAF V600E mutant melanomas treated with selective small-molecule BRAF inhibitors (eg, vemurafenib) had better clinical response, progression-free survival, and overall survival rates than did patients treated with standard chemotherapy [4], [5].
Various molecular testing platforms are available for determining BRAF mutations, including the Cobas 4800 BRAF V600 mutation test (Roche Molecular Diagnostics, Branchburg, NJ) as well as next-generation sequencing (NGS) platforms [6].
BRAF V600E can also be detected at the level of protein expression using immunohistochemical (IHC) methods with monoclonal anti–BRAF V600E. This antibody provides highly sensitive (97%-100%) and specific (97%-100%) insight into the BRAF V600E mutation status of patients with melanoma [7], [8], [9], [10], [11], [12]. Homogeneous BRAF V600E IHC expression in melanoma highly correlates with the presence of BRAF V600E mutation when compared with sequencing methods as the gold standard. However, we and others have observed a subset of tumors exhibiting intratumoral or intertumoral heterogeneity for BRAF V600E IHC expression, and these tumors appear to harbor the BRAF V600E mutation at variable frequencies by molecular testing methods [9], [13]. Of note, however, Wilmott et al [14] showed that the pattern of BRAF V600E protein expression did not predict response in patients receiving BRAF inhibitor therapy. Nevertheless, knowledge of the clinical relevance of heterogeneous tumor expression with anti–BRAF V600E immunostaining and correlation with BRAF mutation status is limited. Here, we compared the expression patterns from BRAF V600E IHC test patients' mutation status, determined by concomitant NGS in 154 patients with metastatic melanoma. Our results confirmed the high sensitivity and specificity of the BRAF V600E IHC assay and further underscored the importance of recognizing and reporting heterogeneous labeling patterns.
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Patient selection and data collection
With the approval of the institutional review board at The University of Texas MD Anderson Cancer Center, we retrospectively reviewed the results of clinical BRAF mutation testing performed over a 2-year period (January 1, 2011–January 31, 2013) in patients with melanoma who were treated at MD Anderson, and their tumor samples were analyzed by the Clinical Laboratory Improvement Amendments (CLIA)–certified Molecular Diagnostics Laboratory of the Division of Pathology and Laboratory Medicine.
Clinical tumor characteristics and association with BRAF V600E IHC expression
A total of 154 patients (male-to-female ratio, 100:54; median age, 61 years; range, 26-87 years) met the study criteria: 131 (85%) patients had a known anatomic site of their primary melanoma, and 23 (15%) had melanoma of unknown primary. Table 1 shows the clinical tumor characteristics used for IHC and NGS testing.
There was no significant association with BRAF V600E IHC expression or intensity of staining with the anatomic site of primary melanomas or between the methods of tissue procurement.
Discussion
Here, we report our experience with the largest single collection of melanomas assayed by both BRAF V600E IHC and NGS platform in a CLIA-certified environment. We showed that cumulative homogeneous and heterogeneous BRAF V600E IHC expression had a sensitivity of 98% and a specificity of 96%. Our findings are in agreement with prior studies showing high sensitivity (overall 97%, from 534/550 samples; range, 97%-100%) and specificity (overall 98%, from 723/738 samples; range, 97%-100%) for this
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Competing interest: The authors declare no conflicts of interest.
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Funding/Support: Portion of this work was supported by National Cancer Institute Grant P30CA016672. R. Broaddus is funded by the National Institutes of Health SPORE in Uterine Cancer (NIH 2P50 CA098258-08).
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Both authors contributed equally to the manuscript.