Background
BRAF, a member of the RAF family including ARAF, BRAF and RAF1, is a serine/threonine protein kinase encoded by
BRAF gene on chromosome 7q34 that activates the MAP kinase/ERK-signaling pathway mediating cellular responses to growth signals. It is the family member that is most easily activated by RAS, and the one with highest kinase activity [
1‐
3]. Frequent somatic mutational activation of
BRAF has been observed in human cancers, including melanomas, gliomas, colorectal cancers, lung cancers and others [
4]. Among primary central nervous system (CNS) neoplasms [
5‐
9], activation of the MAP kinase/ERK-signaling pathway appears to play an important role in the pathogenesis of a subset of glial/glioneuronal tumors, in particular, pilocytic astrocytoma (PA) [
8‐
13], PXA [
14], ganglioglioma (GG) [
9,
15], and dysembryoplastic neuroepithelial tumor (DNET) [
16].
BRAF activation in PA primarily results from tandem duplications at 7q34 with subsequent fusion between the 5’ end of a gene of unknown function,
KIAA1549, and the 3’ end of
BRAF; while in PXA, GG and DNET, constitutive
BRAF activation results from heterozygous missense mutation at codon 600 (V600E).
BRAF V600E mutation is characterized by exchange of T to A at base position c.1799 (c. 1799 T > A), which results in substitution of glutamic acid by valine at residue 600 (p. Val600Glut). Less frequent activating
BRAF mutations (e.g. V600K, V600D, V600M) have been observed in malignant melanoma [
17‐
20] and other non-CNS tumors [
4,
21] but only rarely have been identified in primary CNS tumors [
6].
The highest frequencies of
BRAF V600E mutation in primary CNS neoplasms have been reported in PXA (up to 60-65%) [
8,
9,
14,
22], a WHO grade II tumor [
23], with 30% recurrence and 80% overall survival rates at five years following primary resection. Histologically, PXA is characterized by marked cellular pleomorphism, nuclear atypia, and a variable number of bizarre, multinucleate giant cells (“classic PXA”), and occasionally shows increased mitotic activity and/or necrosis (“PXA with anaplastic features”) [
23,
24]. The main morphological differential diagnosis of PXA includes other pleomorphic and often more aggressive tumors such as glioblastoma (GBM)/giant cell or epithelioid GBM, a World Health Organization (WHO) grade IV tumor [
23]. Such critical clinical distinction with important prognostic and clinical implications may be morphologically challenging. Of note,
BRAF V600E mutation has been found in low frequency among GBM/giant cell GBM (approximately 5-10%) [
5,
7,
14], but in up to 54% among epithelioid GBM [
25]. Therefore,
BRAF V600E mutation assessment may be a potentially useful marker in the differential diagnosis of GBM/giant cell GBM
vs. “PXA with anaplastic features” and in identifying
BRAF V600E mutant astrocytic tumors suitable for targeted therapy.
Recently, a
BRAF V600E mutation-specific monoclonal antibody has been developed [
26] and validated as a reliable test among tumors that frequently harbor the
BRAF V600E mutation, including primary and metastatic melanoma [
18,
20,
27,
28], papillary thyroid carcinoma [
29,
30], hairy cell leukemia [
31], ovarian serous borderline tumors [
32], primary lung adenocarcinomas [
33], as well as in a large series of brain metastases and corresponding non-CNS primary tumors [
34].
Herein, we evaluated the IHC detection of BRAF V600E mutant protein in PXA by comparing to BRAF V600E mutation detection by molecular analysis, and investigated the interobserver variability of the IHC scoring. Detection of BRAF V600E mutation in PXA by immunohistochemistry was highly sensitive and specific, and showed a substantial/almost perfect interobserver agreement.
Discussion
In keeping with the majority of previous studies that also evaluated the immunohistochemical detection of the
BRAF V600E mutation in comparison to gold standard molecular testing in a variety of tumor types [
16,
18,
20,
26‐
34], we concluded that immunohistochemistry is an accurate detection method for the
BRAF V600E mutation in PXA. In our study, IHC results were comparable to the gold standard molecular testing for identification of
BRAF V600E mutation and resulted in high sensitivity and specificity. In contrast to the labor-intensive detection of
BRAF V600E mutation by molecular testing, identification of BRAF V600E mutant protein by immunohistochemistry is a rapid method that may be quickly implemented in diagnostic pathology practice since it is a widespread technique available in most academic centers as well as in non-academic pathology practices. However, validation of the BRAF V600E mutation IHC specificity by comparative molecular analysis should be performed for each tumor entity before routine diagnostic implementation, since it has been recently reported that in pituitary adenomas, the
BRAF V600E mutation-specific VE1 immunostaining is not associated with presence of
BRAF V600E mutation [
35].
In surgical neuropathology,
BRAF V600E mutation-specific immunohistochemistry has a potential clinically relevant role. IHC detection of BRAF V600E mutant protein is an accurate and reliable alternative method that may be diagnostically useful when dealing with morphologically challenging pleomorphic astrocytic tumors in which the differential diagnoses include PXA and GBM/giant cell GBM. In addition,
BRAF V600E mutation has also been identified to a lesser extent in epithelioid GBM[
25], ganglioglioma and pilocytic astrocytoma, predominantly in extra-cerebellar location [
9], expanding the diagnostic repertoire use of BRAF V600E mutation-specific IHC testing among primary CNS tumors and potentially the identification of tumors suitable for
BRAF V600E mutation-targeted therapy.
Of note, low tumor cell content may prevent identification of the
BRAF V600E mutation by molecular analysis and result in false-negative results, but this limitation has been shown to be overcome by the use of BRAF V600E monoclonal antibody [
18,
29,
30,
32,
34]. Thus, small biopsies that would be deemed unsuitable for molecular testing may be successfully evaluated by immunohistochemistry. This is particularly important in surgical neuropathology, in which biopsy samples of small size and/or with low tumor cell content are not infrequent. In addition, BRAF V600E IHC analysis allows identification of tumor cells at the single cell level, which would not only aid in the diagnosis of biopsies with very low tumor cell content but would also allow localization of the mutation at a cellular type level, as recently highlighted by the demonstration of neuronal tumor cells as the predominant tumor cell population harboring the
BRAF V600E mutation in gangliogliomas [
15].
In our study, BRAF V600E IHC interpretation was usually straightforward with high (at least substantial) interobserver agreement. The presence of rare cases with nonspecific staining is a potential pitfall, which could lead to rare false positive results. Similarly, others have also reported negative cases with nonspecific background staining [
15,
32,
34]. In ambiguous cases, sequential molecular confirmatory testing is advocated [
20,
27]. We recommend that pathologists not only be aware of the possibility of nonspecific staining but also train during validation of the IHC testing to correctly identify such cases and avoid misinterpretation of nonspecific staining as positive staining.
Methods
Case selection
All studies were conducted according to Mayo Institutional Review Board–approved protocols. This study was approved by the Mayo IRB as a minimal risk study with waiver of consent.
According to the Minnesota law, the Minnesota research authorization status was reviewed and only patients whose Research Status was "yes" were included in the study.
Fifty pleomorphic xanthoastrocytoma (PXA) cases upon first surgical resection were selected from the files from Mayo Clinic (n = 32) and Johns Hopkins University (n = 18) from 1965 to 2011. All existing diagnostic slides were retrieved and reviewed by at least two of the authors (C.G. and C.M.I.), and the diagnosis of PXA was confirmed according to previously described criteria [
23]. Tumors showed a relatively solid growth pattern and were composed of a combination of spindle-shaped, xanthic and pleomorphic, multinucleated giant astrocytes, associated with both pale and bright eosinophilic granular bodies. They included both classic PXA (≤5 mitotic figures per 10 high power fields) and PXA with anaplastic features (including >5 mitotic figures per 10 high power fields and/or necrosis). All 50 cases were classified as either classic PXA (n = 34) or PXA with anaplastic features (n = 16) and were included in the study. Of these, 46 cases (92%) had available tissue for IHC and/or molecular analysis.
BRAF V600E immunohistochemical analysis
Four-micron freshly cut sections (<2 weeks) of formalin-fixed, paraffin-embedded (FFPE) tissue of 46 (of 50) cases were dried and melting at 62°C oven for 20 minutes. Subsequently, they were stained with mouse monoclonal BRAF V600E antibody (1/100 titer; clone VE1) and raised against a synthetic peptide corresponding to amino acids 596–606 (GLATEKSRWSG) of mutant BRAF (Spring Bioscience) with slight modifications to the manufacturer’s protocol. Briefly, staining was performed on the Ventana BenchMark XT (Ventana Medical Systems Inc.). The staining protocol included online deparaffinization, HIER (Heat Induced Epitope Retrieval) with Ventana Cell Conditioning 1 for 32 minutes and primary antibody incubation for 32 minutes at 37°C. Antigen-antibody reactions were visualized using Ventana OptiViewTM Amplification kit, followed by Ventana OptiViewTM Universal DAB Detection Kit (Optiview HQ Linker 8 min, Optiview HRP Multimer 8 min, Optiview Amplifier H2O2/Amplifier 4 min, Optiview Amplifier Multimer 4 min, Optiview H2O2/DAB 8 min, Optiview Copper 4 min). Counterstaining was obtained online using Ventana Hematoxylin II for 8 minutes followed by bluing reagent for 4 minutes. Finally, all slides are removed from the stainer, dehydrated, and coverslipped for microscopic examination. Positive control included a known BRAF V600E mutant skin malignant melanoma. Cases were scored as positive (“IHC-positive”), negative (“IHC-negative”), and “negative-nonspecific” (“IHC negative/nonspecific”). Only tumor cells showing non-ambiguous cytoplasmic staining for BRAF V600E immunostain were scored as positive (“IHC positive”). Faint, weak granular stain was noted in a few cases and considered nonspecific. These cases were scored as “IHC negative/nonspecific” for tracking purposes. IHC slides were scored first by a primary reviewer and later by three additional independent reviewers, all four blind to the molecular results. The primary reviewer (CG) was the one most intimately involved with the development of the immunostain, who had gained most experience through the process, and was most familiar with variations in stain intensity and distribution not only among PXA cases but also with other positive and negative controls. Because of the level of expertise with reading the BRAF V600E immunostain and of the sequence of events, the primary reviewer was used as the “gold standard” to which the results of the BRAF V600E mutation molecular analysis were compared (see below). The four reviewers were compared to each other in regards to the scoring of BRAF V600E immunohistochemistry.
BRAF V600E mutation status by allele-specific PCR with fragment analysis
After review of hematoxylin and eosin-stained slides, of all the 50 cases, 37 (74%) cases had sufficient (≥20%) viable tumor for BRAF V600E mutation molecular analysis. DNA was extracted from 5-micron sections of FFPE tissue (four to eight slides per case) using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer's instructions with few modifications (samples were lysed overnight; 10 uL of additional proteinase K was used on the following day; the tissue was allowed to complete lysis for additional one to two hours). DNA was quantified using a Qubit fluorometer and Qubit Quant-iT dsDNA BR Assay Kit (Invitrogen). Testing for the BRAF V600E mutation was performed following clinically validated protocols. BRAF allele-specific fluorescent PCR was performed with primers specifically designed to detect the base mutant and wild type base at position c.1799, and fragment analysis was completed on the ABI 3730 DNA Analyzer (Applied Biosystems). The primers were differentially labeled and had a size variance in addition to the different fluorophore. Wild-type peak was approximately 155.2 base pairs, and the mutant peak was approximately 158 base pairs (predetermined bins were set at +/− 1.2 base pairs from the expected size, i.e. 155.2 +/−1.2 bp and 158 +/−1.2 bp).
Statistical analysis
Sensitivity and specificity along with 95% confidence intervals (score method) were calculated for BRAF V600E IHC analysis based on the reading of the primary reviewer compared to the BRAF V600E mutation molecular analysis result considered the “gold standard”.
To evaluate the degree of agreement for IHC interpretation among the four observers, overall kappa for >2 reviewers were calculated. Kappa values may vary from 0 to 1.0. Values of 0.4 to 0.6 are considered evidence of “moderate” agreement, >0.6-0.8 of “substantial,” and >0.8-1 of “almost perfect” agreement according to Landis and Koch [
36]. Kappa was calculated based on the IHC scoring results in two scenarios: when cases were scored as “positive/negative” and when negative/nonspecific staining was also taken into account [
36‐
39]. All analyses were performed using SAS version 9 (Cary, NC).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Cristiane M. Ida, MD, planned project, reviewed cases, wrote manuscript. Julie A. Vrana performed BRAF V600E immunostaining, reviewed manuscript. Fausto J. Rodriguez, MD, provided and reviewed part of the cases, reviewed manuscript. Mark E. Jentoft, MD, reviewed immunohistochemical stains, reviewed manuscript. Alissa A. Caron performed DNA extraction for BRAF V600E mutation molecular analysis, reviewed manuscript. Sarah M. Jenkins performed statistical analysis, reviewed manuscript. Caterina Giannini, MD, planned project, reviewed cases, prepared figures, wrote manuscript. All authors read and approved the final manuscript.