Immunohistochemistry is highly sensitive and specific for detection of BRAF V600E mutation in pleomorphic xanthoastrocytoma

Background High frequencies of the BRAF V600E mutation have been reported in pleomorphic xanthoastrocytoma (PXA). Recently, a BRAF V600E mutation-specific antibody has been developed and validated. We evaluated the immunohistochemical (IHC) detection of BRAF V600E mutation in PXA by comparing to gold standard molecular analysis and investigating the interobserver variability of the IHC scoring. We performed BRAF V600E IHC in 46 cases, of which 37 (80%) cases had sufficient tumor tissue for molecular analysis. IHC detection was performed using monoclonal mouse antibody VE1 (Spring Bioscience). IHC slides were scored independently by four reviewers blind to molecular data, including a primary (gold standard) and three additional reviewers. BRAF V600E mutation status was assessed by allele-specific polymerase chain reaction (PCR) with fragment analysis. Results All 46 cases showed interpretable BRAF V600E IHC results: 27 (59%) were positive (strong cytoplasmic staining), 19 (41%) were negative (6 of these cases with focal/diffuse weak cytoplasmic staining, interpreted as nonspecific by the primary reviewer). By molecular analysis, all 37 cases that could be tested had evaluable results: 22 (59%) cases were positive for BRAF V600E mutation and were scored as “IHC-positive”, and 15 (41%) were negative (including 11 cases scored as “IHC-negative” and 4 cases scored as negative with minimal nonspecific staining). IHC detection of BRAF V600E mutant protein was congruent in all 37 cases that were successfully evaluated by molecular testing (sensitivity and specificity of 100%). Agreement for IHC scoring among the 4 reviewers was almost perfect (kappa 0.92) when cases were scored as “positive/negative” and substantial (kappa 0.78) when minimal nonspecific staining was taken into account. Conclusions We conclude that detection of BRAF V600E mutation by immunohistochemistry is highly sensitive and specific. BRAF V600E IHC interpretation is usually straightforward, but awareness of possible nonspecific staining is necessary and training is recommended. It is a practical rapid method that may avoid the need of labor-intensive molecular testing and may be most valuable in small biopsies unsuitable for molecular analysis.

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.
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.

BRAF V600E mutation immunohistochemical and molecular analyses
All 46 (100%) cases with available tissue were evaluable by immunohistochemistry for BRAF V600E mutant protein.
Cases were scored as positive if non-ambiguous tumor cell cytoplasmic staining was identified ("IHC-positive"). BRAF V600E immunoreactivity in most cases was characterized by intense cytoplasmic stain, with a somewhat granular appearance, likely due to the visualization/amplification set used and with variable distribution. In some cases, all tumor cells stained; while in others, the stain was patchy in the tissue sections. Isolated tumor cells infiltrating parenchyma were also visualized by the immunostain. According to the primary reviewer, 27 (59%) cases were "IHC-positive," and 19 (41%) cases were "IHC-negative," including 6 cases "IHC-negative/nonspecific." Examples of immunostaining pattern are shown in Figures 1 and 2. Immunostaining varied in intensity from strong ( Figure 1A) to weak ( Figure 1B), as well as in distribution, with cases showing isolated infiltrating tumor cells ( Figure 1C). Among cases considered negative ("IHC-negative"), although the majority of cases did not have any degree of immunostaining ( Figure 2A), a few showed very focal and/or weak tumor cell cytoplasmic staining as shown in Figures 2B and 2C. This focal/weak immunostaining was interpreted as nonspecific ("IHC-negative/nonspecific").

Interobserver variability of BRAF V600E immunohistochemical analysis
Most (26) cases scored as "IHC-positive" by the primary reviewer were also interpreted as "IHC-positive" by the other three reviewers ( Figure 3: Table 2 ), with the exception of one "IHC negative/nonspecific" interpretation by a single reviewer. However, reviewers three and four also scored as positive one additional case among those scored as "IHC-negative/nonspecific" by the primary reviewer ( Figure 3: Table 2 ). All 13 cases scored as "IHC-negative" by the primary reviewer were also scored as "IHC-negative" by reviewers two and three, while reviewer four scored two of them as "IHC-negative/ nonspecific." The results of the review of the remaining six cases interpreted as "IHC-negative/nonspecific" by the primary reviewer as well as the results of the BRAF V600E mutation molecular analysis are detailed in Figure 3: Table 2. In summary: two cases were scored as "IHC-negative" by the other three observers; two cases were also scored as "IHC-negative/nonspecific"  by one observer and as "IHC-negative" by the other two observers; one case was scored as "IHC-negative" by two observers and as "IHC-positive" by one observer; one case was also scored as "IHC-negative/nonspecific" by one observer and as "IHC-positive" by the other two observers.

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][27][28][29][30][31][32][33][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 mutationspecific 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 mutationspecific 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.

Conclusions
Detection of BRAF V600E mutation in PXA by immunohistochemistry is highly sensitive and specific with substantial/almost perfect interobserver agreement. BRAF V600E IHC interpretation is usually straightforward, but awareness of possible nonspecific staining is necessary, and training on the IHC stain interpretation is recommended. It is a practical rapid method that may avoid the need of labor-intensive molecular testing and may be extremely valuable in biopsies unsuitable for molecular analysis due to either small size or low tumor cell content.

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 formalinfixed, 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. 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][37][38][39]. All analyses were performed using SAS version 9 (Cary, NC).

Additional files
Additional file 1: Figure S1. Fragment analysis electropherograms of a BRAF V600E mutant case (case 14): mutant BRAF V600E peak in addition to the wild-type BRAF peak, consistent with presence of BRAF V600E mutation (A); positive control-mutant BRAF V600E peak in addition to the wild-type BRAF peak, consistent with presence of BRAF V600E mutation (B); negative control-BRAF wild-type peak without mutant BRAF V600E peak, consistent absence of BRAF V600E mutation (C); Blank/No DNA-Absence of distinct peaks, indicating no sample contamination (D).
Additional file 2: Figure S2. Fragment analysis electropherograms of a BRAF V600E non-mutant case (case 20): BRAF wild-type peak without mutant BRAF V600E peak, consistent with absence of BRAF V600E mutation (A); positive control-mutant BRAF V600E peak in addition to the wild-type BRAF peak, consistent with presence of BRAF V600E mutation (B); negative control-BRAF wild-type peak without mutant BRAF V600E peak, consistent absence of BRAF V600E mutation (C); Blank/No DNA-Absence of distinct peaks, indicating no sample contamination (D).