Elsevier

Human Pathology

Volume 44, Issue 11, November 2013, Pages 2563-2570
Human Pathology

Original contribution
Comparison of 2 monoclonal antibodies for immunohistochemical detection of BRAF V600E mutation in malignant melanoma, pulmonary carcinoma, gastrointestinal carcinoma, thyroid carcinoma, and gliomas

https://doi.org/10.1016/j.humpath.2013.06.018Get rights and content

Summary

BRAF mutation is seen in a variety of human neoplasms including cutaneous malignant melanoma, papillary thyroid carcinoma, colorectal carcinoma, non–small cell lung carcinoma, pleomorphic xanthoastrocytoma, and others. Currently, there are 2 commercially available monoclonal antibodies for the detection of BRAF V600E mutation; however, a full and practical comparison of their performance in various tumor types on an automated staining platform has not been done. We investigated their sensitivity and specificity in detecting the BRAF V600E mutation in a series of 152 tumors including 31 malignant melanomas, 25 lung carcinomas, 32 gastrointestinal carcinomas, 23 thyroid carcinomas, 35 gliomas, and 6 other malignancies. In this series, the concordance rate between immunohistochemistry (IHC) and mutational analyses was 97% (148/152) for VE1 and 88% (131/149) for anti–B-Raf. The sensitivity and specificity were 98% (60/61) and 97% (88/91) for monoclonal VE1 and 95% (58/61) and 83% (73/88) for anti–B-Raf, respectively. There were 4 cases with discordant IHC and mutational results for monoclonal VE1 in contrast to 18 cases for anti–B-Raf. Our studies showed that IHC with monoclonal VE1 has a better performance compared with anti–B-Raf in an automated staining platform and confirmed that clone VE1 provides excellent sensitivity and specificity for detecting the BRAF V600E mutation in a variety of tumor types in a clinical setting.

Introduction

In approximately 100% of hairy cell leukemia [1], 66% of pleomorphic xanthoastrocytomas [2], 40% to 60% of malignant melanomas [3], [4], 45% papillary thyroid carcinomas [5], 13% of ovarian borderline tumors [6], 20% of biliary tract carcinomas [7], 10% of colorectal carcinomas [8], and 4.6% of lung adenocarcinomas [9], BRAF has been found to be mutated. An early clinical trial demonstrated a partial or complete response to the BRAF kinase inhibitor vemurafenib for most patients with metastatic melanoma harboring BRAF V600E mutation, whereas a later trial showed improved 6-month survival versus cytotoxic chemotherapy [10], [11]. Although resistance to BRAF inhibitor therapy and progression of disease eventually occur for most patients receiving this therapy for metastatic melanoma, promising new targeted therapies for other members of the MAP kinase pathway and combined targeted therapies are under investigation [12], [13], [14].

Detection of BRAF mutations may also guide therapies for several nonmelanoma malignancies. The detection of BRAF V600E mutations in papillary thyroid carcinoma offers diagnostic confirmation and may predict a more aggressive disease course [5]. Metastatic colorectal carcinomas with BRAF V600E mutations have demonstrated a worse prognosis and resistance to anti-epidermal growth factor receptor (EGFR) therapy [15]. Preclinical experiments have demonstrated the potential for BRAF inhibitor therapy for malignant astrocytomas with BRAF V600E mutations [16]. Responses to BRAF inhibitor therapy have been reported in single patients with hairy cell leukemia and lung adenocarcinoma with BRAF mutations [17], [18].

A number of methods are available for identifying the BRAF V600E mutations, including Sanger sequencing, pyrosequencing, high-resolution melting analysis, and allele-specific polymerase chain reaction (PCR)–based methods [19]. Most popular methods use PCR assays for BRAF and subsequent nucleic acid sequencing [20]. Although specific mutations can be identified reliably, molecular methods for identifying BRAF mutations are expensive and may require weeks from the time of tissue collection, especially when paraffin-embedded tissue must be sent to a reference laboratory. Furthermore, because the tumor cells must make up at least 20% of the tissue sample in some laboratories, minute foci of tumor may be difficult to isolate and assess for molecular changes. Technical difficulty with molecular assays can be encountered in some instances in which the melanomas possess marked melanin pigmentation [21].

Recently, the development of an immunohistochemical stain for the BRAF V600E mutation has raised the possibility of a more rapid screening test. As clinical interest in BRAF mutations expands, the application of immunohistochemistry (IHC) for detecting BRAF V600E needs to be verified across a spectrum of tumor types. Although the VE1 monoclonal antibody has been shown to be a highly sensitive and specific antibody for the immunohistochemical detection of BRAF V600E mutation in comparison with sequencing methods in studies of malignant melanoma, thyroid, lung, ovary, and brain tumors [19], [22], [23], [24], [25], [26], [27], [28] and anti–B-Raf for the detection of melanoma [29], a full and practical comparison of their performance in a clinical setting has not been done. The primary goal of this study was to compare the sensitivity and specificity of these 2 monoclonal antibodies using an automated platform and the same staining protocol for various tumor types as compared with sequencing methods.

Section snippets

Materials and methods

Archival, formalin-fixed, paraffin-embedded materials of tumors submitted for SNaPshot (Applied Biosystems, Grand Island, NY) genotyping assay at the Molecular Diagnostic Laboratory, Massachusetts General Hospital, Boston, MA, between July 7, 2009, and May 10, 2012, were retrieved from the pathology files. Whenever possible, the same tumor block on which molecular testing was performed was selected for immunohistochemical studies.

Results

The study included a total of 152 specimens (Table 1). They include 31 cases of malignant melanoma (19 primary cutaneous and 12 metastatatic melanomas), 25 cases of pulmonary carcinomas (19 primary and 6 metastatic carcinomas), 32 cases of gastrointestinal carcinomas (23 primary and 9 metastatic carcinomas), 23 cases of thyroid carcinomas (19 primary and 4 metastatic carcinomas), 35 gliomas (16 ganglioglioma, 7 pleomorphic xanthoastrocytoma, 7 glioblastoma, 3 astrocytoma, 1 anaplastic

Discussion

Although VE1 has previously been shown to be a highly sensitive and specific antibody in the immunohistochemical detection for BRAF V600E in various tumor types included in this study, with the exception of gastrointestinal carcinomas [21], [22], [23], [24], [25], [26], [27], [28], none has shown that the same protocol can be reliably used for a spectrum of tumors in an automated laboratory setting. In addition, we also compared the performance of 2 commerically available monoclonal antibodies.

References (36)

  • H. Davies et al.

    Mutations of the BRAF gene in human cancer

    Nature

    (2002)
  • G.V. Long et al.

    Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma

    J Clin Oncol

    (2011)
  • R.P. Tufano et al.

    BRAF mutation in papillary thyroid cancer and its value in tailoring initial treatment: a systematic review and meta-analysis

    Medicine

    (2012)
  • M. Preusser et al.

    Expression of BRAF V600E mutant protein in epithelial ovarian tumors

    Appl Immunohistochem Mol Morphol

    (2013)
  • A. Tannapfel et al.

    Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma

    Gut

    (2003)
  • W.S. Samowitz et al.

    Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers

    Cancer Res

    (2005)
  • A. Marchetti et al.

    Clinical features and outcome of patients with non–small-cell lung cancer harboring BRAF mutations

    J Clin Oncol

    (2011)
  • P.B. Chapman et al.

    Improved survival with vemurafenib in melanoma with BRAF V600E mutation

    N Engl J Med

    (2011)
  • Cited by (100)

    View all citing articles on Scopus

    The authors have no conflicts of interest to disclose.

    View full text