Human Pathology (2014) 45, 464–472
www.elsevier.com/locate/humpath
Original contribution
Immunohistochemical detection of BRAF V600E mutant protein using the VE1 antibody in colorectal carcinoma is highly concordant with molecular testing but requires rigorous antibody optimization☆ Shih-Fan Kuan, Sarah Navina, Kristi L. Cressman, Reetesh K. Pai ⁎ Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA Received 16 July 2013; revised 7 October 2013; accepted 24 October 2013
Keywords: Colorectal carcinoma; BRAF; V600E; VE1; Lynch syndrome; Immunohistochemistry
Summary The BRAF V600E mutation occurs in 15% of colorectal carcinomas (CRCs) and has important genetic, prognostic, and therapeutic implications. A monoclonal antibody (VE1) targeting the BRAF V600E mutant protein has become available with variable efficacy in literature reports. We investigated the utility of the VE1 antibody in detecting BRAF V600E mutant protein in two cohorts: (1) a retrospectively accrued series of 103 resected CRCs with (N = 57) and without (N = 46) known BRAF V600E mutation status by PCR and (2) a prospective series of 25 CRCs requiring BRAF analysis during routine screening for Lynch syndrome. All 74 cases with positive BRAF V600E mutation demonstrated cytoplasmic positivity with the VE1 antibody with most tumors (70/74, 95%) demonstrating moderate to strong staining. Of the 54 BRAF V600E–negative cases, 51/54 CRCs (94%) were negative with the VE1 antibody while 3 CRCs (6%) demonstrated weak cytoplasmic staining. The sensitivity and specificity of VE1 was 100% and 94%, respectively. Ten BRAF V600E–mutated CRCs had adjacent precursor lesions including 7 sessile serrated adenomas associated with CRCs with high-level microsatellite instability (MSI-H). All 10 precursor adenomas were positive for VE1 staining with the 7 sessile serrated adenomas maintaining preserved MLH1 expression. Our results indicate that VE1 immunohistochemistry is a useful surrogate for the detection of the BRAF V600E mutation in CRC, although weak staining must be evaluated by BRAF PCR analysis to exclude a false positive result. In addition, the BRAF V600E mutation appears to be an early event before the divergent development into MSS and MSI-H pathways. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Activating mutations of BRAF are found in 15% to 17% of colorectal carcinomas (CRCs) with most occurring in a ☆
The authors have no conflict of interest and have received no funding for this manuscript. ⁎ Corresponding author. Department of Pathology, University of Pittsburgh, Presbyterian Hospital, Pittsburgh, PA, USA. E-mail address: [email protected] (R. K. Pai). 0046-8177/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.10.026
hotspot of amino acid position 600 by a missense substitution of valine by glutamic acid, known as the BRAF V600E mutation [1]. Detection of the BRAF V600E mutation has important genetic, prognostic, and therapeutic implications for patients with CRC. First, most BRAF V600E–mutated CRCs exhibit sporadic high levels of microsatellite instability (MSI-H) and arise from a sessile serrated adenoma (SSA) [2]. Thus, BRAF mutation status helps to further stratify MSI-H CRCs into Lynch syndrome– associated CRCs which are negative for the BRAF V600E
BRAF VE1 immunohistochemistry in CRC mutation from sporadic MSI-H CRCs which are often positive for the BRAF V600E mutation. BRAF mutation analysis in MSI-H tumors has become a cost-effective assay in selecting patients who may benefit from further germline testing to evaluate for Lynch syndrome [3]. In addition, knowledge of BRAF mutation status has prognostic value in CRC. The presence of a BRAF V600E mutation is an unfavorable prognostic factor in microsatellite stable (MSS) CRCs as patients with BRAF–mutated MSS tumors typically follow an aggressive clinical course and often present with widespread metastatic disease [4]. Finally, the presence of a BRAF V600E mutation in CRC predicts resistance to anti-EGFR therapy in patients with a wild-type KRAS genotype [5]. BRAF and KRAS are downstream molecules of the Ras-Raf-MAPK signaling pathway lying downstream of EGFR. Current therapies for patients with CRC include anti-EGFR antibodies such as cetuximab and panitumumab as well as conventional chemoradiation. Several studies have shown that KRAS or BRAF mutations lead to constitutive activation of the MAPK pathway and result in the failure of anti-EGFR therapy [6]. Due to the high cost of anti-EGFR treatment, testing for the presence of KRAS and BRAF mutations before instituting therapy is recommended to predict therapeutic efficacy. The conventional polymerase chain reaction (PCR)– based methods for BRAF mutation detection are relatively time-consuming and expensive. Recently, an antibody (VE1) targeting the BRAF V600E mutant protein has become commercially available to detect its presence in formalinfixed, paraffin-embedded tissue [7–15]. This monoclonal antibody was raised against a synthetic peptide containing BRAF-mutated amino acid sequence from amino acid 596 to 606 (GLATEKSRWSG). A few studies using this antibody in various types of malignant tumors have demonstrated promising results [7–9]. However, the findings in CRC have been conflicting [10–14], likely due to the small number of cases included in the analyses and the lack of a standardized antibody optimization protocol. Herein, we investigated the immunohistochemical expression of BRAF V600E protein using the VE1 antibody in a series of resected CRCs with known BRAF V600E mutation status order to (1) determine the optimum assay conditions for BRAF V600E immunohistochemistry in formalin-fixed paraffin-embedded tissue from the resection specimens of CRCs, (2) assess the sensitivity and specificity of VE1 immunohistochemistry, and (3) analyze the presence of BRAF V600E in precursor lesions of CRC.
2. Materials and methods 2.1. Study group and molecular analysis We collected 2 cohorts of CRCs: (1) a retrospective series of 103 resected CRCs for the years 2009 through 2012 and (2) a prospectively accrued series of 25 resected CRCs analyzed for BRAF V600E during routine screening for
465 Lynch syndrome between June 2013 through September 2013. All tumors were analyzed for the presence of KRAS codon 12 and 13 mutations, BRAF V600E mutation, and MSI by either PCR or DNA mismatch repair (MMR) protein immunohistochemistry. This study was conducted under the guidelines of the University of Pittsburgh Institutional Review Board (IRB# PR012020335). For surgical resection specimens, manual microdissection was performed. Only specimens with a minimum of 50% tumor cellularity in a microdissection target were accepted for analysis. DNA was extracted from paraffin sections, using the DNeasy tissue kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. Detection of BRAF mutations was performed using real-time PCR and post-PCR fluorescence melting curve analysis on LightCycler (Roche Applied Science, Indianapolis, IN), as previously described [16]. Briefly, a pair of oligonucleotide primers flanking the mutation site was designed, together with 2 fluorescent probes, with the sensor probe spanning the nucleotide position 1799. Post-amplification FMCA was performed by gradual heating of samples at a rate of 0.2°C from 45°C to 95°C. All PCR products that deviated from the wild-type melting peak (placenta DNA) were sequenced to verify the presence of mutation. KRAS codon 12 and 13 mutations were assessed by direct sequencing in both directions (forward and reverse) as previously described [17]. Detection of MSI was performed using a National Cancer Institute (NCI)–recommended panel of microsatellite markers (BAT25, BAT26, D2S123, D5S346, and D17S250) as well as one novel quasi-monomorphic mononucleotide marker CAT25, as previously described [18].
2.2. Immunohistochemistry Immunohistochemical staining using VE1 mouse monoclonal antibody (Spring Biosciences, CA) was performed on 4-micron formalin-fixed paraffin-embedded tissue sections either by manual method or Ventana BenchMark Ultra automated stainer (Tucson, AZ). The manual staining method was used to evaluate the factors affecting the staining results including the pH of heat-induced antigen retrieval solutions and antibody concentrations. Briefly, 4-μm paraffin sections on negatively charged glass slides were deparaffinized in xylene solutions and rehydrated in graded alcohol solutions followed by washes in distilled water. Antigen retrieval was performed in a pressure cooker for 15 minutes in the following buffers: 10 mmol/L citric buffer (pH 6.0), 1 mmol/L EDTA (pH 8.0) or 20 mmol/L Tris-EDTA buffer (pH 9.0). The sections were allowed to cool to room temperature and then washed with PBS, 0.05% Tween 20, pH 7.0. The sections were incubated overnight in a humidified chamber at room temperature with various dilutions of VE1 antibody (1:50, 1:100, and 1:200). After washing with PBS, the sections were incubated with HRP-labeled polymer anti-mouse secondary antibody (DAKO Envision+ system, Carpinteria, CA) for 1
466
S. -F. Kuan et al.
Fig. 1 Effects of pH and antibody concentrations on the staining of BRAF V600E VE1 antibody in colorectal carcinoma. A-C, Heat-induced antigen retrieval was performed in a pressure cooker for 15 minutes in the following buffers: 10 mmol/L citric buffer pH 6.0 (A), 10 mmol/L EDTA buffer, pH 8.0 (B) and 20 mmol/L Tris-EDTA buffer, pH 9.0 (C). The antibody dilution was 1:200 in A, B, and C. D-F, Antigen retrieval was performed using 10 mmol/L EDTA buffer, pH 8.0, for 15 minutes, then incubated with VE1 antibody in following dilutions: 1:200 (D), 1:100 (E), and 1:50 (F). All images are at 100× magnification.
hour at room temperature. Color visualization was performed with liquid DAB chromogen in imidazole-HCl buffer (pH 7.5) containing hydrogen peroxide until the brown color fully developed. The sections were counterstained with hematoxylin and coverslipped with permanent mounting media. After optimization of staining conditions by the manual method, all cases were immunostained in a Ventana BenchMark Ultra stainer to avoid variation by manual processing. The automated program for immunohistochemistry included deparaffinization at 72°C, pre-primary oxidase inhibition for 4 minutes, cell condition (CC1, pH 8.0) for 56 minutes at 100°C; incubation with 1:200 diluted VE1 antibody at 37°C for 20 minutes, incubation with OptiView HRP Linker for 8 minutes, incubation with OptiView HRP multimer for 8 minutes, and incubation with hematoxylin and bluing reagent for 4 minutes each.
For all cases, VE1 immunohistochemistry was independently read as positive and negative blinded to the BRAF mutation status by PCR. For the retrospective series, each case was reviewed by 3 gastrointestinal pathologists (S.F.K., S.N., and R.K.P.). For both the retrospective and prospectively accrued cases, a semiquantitative assessment of intensity was given by one pathologist (S.F.K.) using the following scoring criteria: 0, negative with no cytoplasmic staining visualized at any magnification; 1+, weak, difficult to recognize brown color on the slide requiring a 10× or greater objective for confirmation; 2+, moderate, easy to recognize staining seen with a 2× or 4× objective; and 3+, strong color on glass slides by naked eyes with confirmation of strong staining using 2× microscopic objective. MMR protein immunohistochemistry for MLH1, PMS2, MSH2, and MSH6 was performed as previously described [19]. Normal expression was defined as nuclear staining within
BRAF VE1 immunohistochemistry in CRC tumor cells, using infiltrating lymphocytes as positive internal control. Negative protein expression was defined as complete absence of nuclear staining within tumor cells with concurrent positive labeling in internal non-neoplastic tissues.
3. Results 3.1. Optimization of staining conditions by manual method We investigated the optimal staining conditions including pH of retrieval solutions and antibody concentrations for the VE1 antibody using 10 resection cases of CRC that had been confirmed to be BRAF V600E–mutated by PCR. When heatinduced antigen retrieval was performed in citric acid buffer, (pH 6.0), BRAF-mutated CRC showed heterogeneous and weak cytoplasmic staining in some cases (Fig. 1A) and complete absence of staining in some cases. EDTA buffer (pH 8.0) pretreatment provided satisfactory intensity and homogeneity of VE1 staining in all cases (Fig. 1B). TrisEDTA buffer (pH 9.0) further increased the intensity but also created nonspecific staining of nuclei (Fig. 1C). We then tested the effects of various dilutions of VE1 antibody after antigen retrieval in EDTA (pH 8.0) solution. Although all 3 dilutions (1:50, 1:100, and 1:200) of the antibody generated recognizable brown color in tumor cytoplasm (Fig. 1D-F), we identified a nonspecific nuclear staining in some areas of normal colonic mucosa at 1:50 or 1:100 dilutions. A 1:200 dilution of antibody reduced the chance of nonspecific nuclear staining. During the experimentation, we also noticed that the automated stainer produced more consistent and satisfactory staining quality than the manual method. We therefore decided to conduct our study on the Ventana automated stainer because (1) the automated stainer has been adapted as the sole instrumentation by many clinical laboratories including our institution and (2) it was the only staining method recommended by the vendor.
3.2. Automated BRAF VE1 immunohistochemistry in colorectal carcinoma study groups The retrospective study group included 103 cases of surgically resected CRCs with known BRAF and KRAS Table 1
467 mutation by PCR (Table 1). Among these cases, 57 were BRAF V600E–mutated, 21 displayed KRAS codon 12 or 13 mutations, and the remaining 25 cases were BRAF/KRAS wild type. All 57 cases with a known BRAF V600E mutation were positive by VE1 antibody. The intensity was scored strong (3+) in 35 cases, moderate (2+) in 19 cases, and weak (1+) in 3 cases (Fig. 2A-C). All 46 cases of BRAF wild-type CRC, including 21 KRAS mutants, were negative for VE1 staining (Fig. 2D). The independent evaluation (positive vs negative) by 3 GI pathologists showed 100% concordance of the reading and scoring. It is noteworthy that a diffuse and homogeneous cytoplasmic staining pattern was observed in all ranges of tumor differentiation and histological subtypes such as mucinous (Fig. 2E) and large cell neuroendocrine carcinomas (Fig. 2F). Nine cases of CRCs with known history of Lynch syndrome were stained negative. The 25 CRCs included in the prospective study group of CRCs were identified during routine screening of CRC for DNA MMR protein expression at our institution over a 3month period. All 25 CRCs demonstrated loss of immunohistochemical expression for MLH1 and PMS2 requiring evaluation for BRAF V600E in an attempt to distinguish sporadic MSI-H CRC from possible Lynch syndrome– associated CRC. BRAF VE1 immunohistochemistry was independently evaluated using the 0-3+ scoring scheme and without knowledge of the BRAF PCR results. All 16 CRCs with 2+ or 3+ VE1 staining were positive for BRAF V600E by PCR (Table 2). All 5 CRCs with negative (0) staining were negative for BRAF V600E by PCR. Of the four cases of CRCs demonstrating weak (1+) staining, three cases were negative for BRAF V600E by PCR while one case was positive for BRAF V600E by PCR.
3.3. BRAF V600E protein expression in precursor lesions in the study group Ten separate BRAF V600E–mutated CRC cases also contained precursor lesions adjacent to the main tumor (Table 3). Seven cases were MSI-H, and 3 were MSS. All 7 precursors adjacent to those 7 MSI-H CRC cases were classified as sessile serrated adenomas and stained moderately to strongly positive by VE1 antibody (Fig. 3A and B). Of the three precursor lesions adjacent to MSS CRCs, one was a traditional serrated adenoma (Fig. 3C) and 2 were conventional tubular adenomas. All 3 also demonstrated positive staining with VE1 antibody.
Correlation between VE1 immunophenotype and BRAF/KRAS genotype in the retrospective study group
BRAF V600E genotype
KRAS genotype
No. with positive VE1 staining
Staining intensity
Mutant
Wild type
57/57
Wild type Wild type
Mutant Wild type
0/21 0/25
3+ (N = 35); 2+ (N = 19); 1+ (N = 3) All negative All negative
468
S. -F. Kuan et al.
Fig. 2 Immunohistochemistry of BRAF V600E VE1 antibody in colorectal carcinoma. A-D, The intensity of staining was scored as weak (1+, A), moderate (2+, B), strong (3+, C) and negative (0, D). E and F, Diffuse and homogenous staining were seen in different histological subtypes such as mucinous (E) and large cell neuroendocrine (F) colorectal carcinomas. All images are at 100× magnification.
To further investigate the sequence of BRAF V600E mutation and loss of MLH1 expression, we performed immunostaining of VE1 and anti-MLH1 antibodies on the serial sections of 7 pairs of SSA-associated MSI-H tumors. In all 7 cases, the areas of SSA without cytological dysplasia expressed both MLH1 and BRAF V600E mutant protein, while the areas of invasive carcinoma expressed BRAF mutant protein but lost MLH1 expression (Fig. 3E and F). These findings support the notion that BRAF V600E
Table 2
mutation occurs before the loss of MLH1 mismatch repair protein in the SSA-MSI-H pathway for CRC.
4. Discussion Our results indicate that VE1 immunohistochemistry is highly sensitive and specific for detecting colorectal carcinomas harboring a BRAF V600E mutation. However,
Prospective comparison of BRAF VE1 immunohistochemistry with BRAF V600E PCR
BRAF VE1 immunohistochemistry
Negative Weak staining (1+) Moderate to strong staining (2+ or 3+)
No. of cases
BRAF PCR V600E positive (%)
V600E negative (%)
5 4 16
0 1 (25) 16 (100)
5 (100) 3 (75) 0
BRAF VE1 immunohistochemistry in CRC Table 3
469
Analysis of VE1 immunohistochemistry in colorectal carcinomas with adjacent precursor lesions
Case
Age
Sex
Cancer histology
Carcinoma MSI status
Precursor histology
Cancer VE1
Precursor VE1
1 2 3 4 5 6 7 8 9 10
81 85 75 83 80 62 77 66 83 67
F F F F F F F F F M
Medullary carcinoma PD adenocarcinoma MD adenocarcinoma MD adenocarcinoma MD adenocarcinoma Mucinous/SRC MD adenocarcinoma LC-NEC MD adenocarcinoma LC-NEC
MSI-H MSI-H MSI-H MSI-H MSI-H MSI-H MSI-H MSS MSS MSS
SSA SSA SSA SSA SSA SSA SSA TSA TA TA
3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+
2+ 3+ 3+ 2+ 2+ 2+ 3+ 2+ 3+ 2+
Abbreviations: MD, moderately differentiated; PD, poorly differentiated; SRC, signet-ring cell; LC-NEC, large cell neuroendocrine carcinoma; TSA, traditional serrated adenoma; TA, tubular adenoma.
our study highlights several important points concerning the implementation of BRAF V600E VE1 immunohistochemistry. First, optimization of the staining protocol is essential
before VE1 can be reliably applied in a clinical or research setting. Second, nonspecific nuclear staining is not uncommon, especially in high antibody concentrations and high pH
Fig. 3 CRC and adjacent precursors using the BRAF V600E VE1 antibody. A and B, Two colorectal carcinomas with MSI-H and adjacent SSA were strongly stained with the VE1 antibody. C, A microsatellite stable colorectal carcinoma and its adjacent traditional serrated adenoma (TSA) were positively stained by VE1. D-F, Serial sections of a MSI-H colorectal carcinoma (left lower) arising in a sessile serrated adenoma (right upper) which were stained with hematoxylin and eosin (D), BRAF V600E VE1 antibody (E) and MLH1 (F). All images are at 40× magnification.
470
S. -F. Kuan et al.
antigen retrieval solutions. Finally, a scoring system needs to be established for identifying borderline cases which require additional testing. Heat-induced antigen retrieval (HIAR) has been widely used in many laboratories, although the mechanism is not yet fully understood [20,21]. Citric buffer, pH 6.0 is the most popular solution for HIAR. However, pre-treatment of sections in citric buffer (pH 6.0) only revealed weak, heterogeneous and inconsistent staining in the cytoplasm with the VE1 antibody. Adackapara and colleagues analyzed a series of 52 colorectal carcinomas with known BRAF mutation status by PCR and found that VE1 immunohistochemistry had a low sensitivity (71%) and specificity (74%) for detecting BRAF V600E mutation (Table 4) [10]. They argued that VE1 immunohistochemistry is not a useful surrogate for detecting BRAF mutation in colorectal carcinoma. However, in their analysis, manual staining with citrate buffer pre-treatment was employed which, in our experience, results in weak heterogeneous immunoreactivity that is difficult to interpret. EDTA buffer (pH 8.0) proved to be a retrieval agent that produced robust and homogenous cytoplasmic staining by VE1 antibody. The remaining literature reports analyzing the utility of VE1 immunohistochemistry in detecting BRAF mutation all employed EDTApretreatment and automated staining and identified a sensitivity and specificity of VE1 immunohistochemistry approaching 100% [11–14], similar to our series (Table 4). Tris-EDTA buffer (pH 9.0) further increased the positive signals, but it also significantly increased the chance of nonspecific nuclear staining. A recent study by Emoto and colleagues demonstrated that heat treatment cleaves the intraand intermolecular methylene bridge induced by formaldehyde [21]. Once the antigen molecule is denatured by heat, the polypeptide extends to expose the hydrophilic and hydrophobic portions. In the setting of solution around neutral pH (4.5-7.5), the antigenic determinants are conTable 4
cealed after the solutions cool because the peptides are ready to self-associate. In contrast, in more basic pH solutions, the extended peptides are charged negatively which prevents the refolding of peptide. This hypothesis may explain our observation that optimal staining can only be achieved by EDTA (pH 8.0) or higher pH solutions but not by near neutral solutions (pH 6.0). The presence of nonspecific nuclear staining is a troublesome but intrinsic property of the VE1 antibody. We speculate that there is a low-affinity nuclear protein that may cross-react with the 11-amino-acid peptide that was used to produce VE1 antibody. First, higher antibody concentration increased the chance of nuclear staining because more free antibody was available in the solution to bind this low-affinity protein. Second, retrieval solutions with more basic pHs also enhanced nuclear staining, suggesting that this nuclear protein required more basic retrieval solutions to better expose its antigen determinants, as discussed previously. Finally, in order to correctly interpret the results, users of VE1 antibody need to be aware of this and other nonspecific staining. These include the occasional background staining in smooth muscle and in degraded tissue, either due to tumor necrosis or drying artifacts during the staining procedure. When an immunohistochemical assay is utilized to guide clinical management, it is crucial to develop a standard scoring system to define the positivity. Various scoring criteria have been employed in early literature reports using the VE1 antibody. Two reports used 80% and 75% positive tumor cells respectively as the cut-off for positivity, although both admitted their staining results were diffuse and homogeneous [11,14]. Two other groups used a 4-tiered system (negative, weak, moderate, strong) to assess the intensity [10,12]. Our findings indicate that a scoring system relying on a cut-off percentage of positive tumor cells may not apply to BRAF mutant protein. We believe that a scoring
Comparison of literature reports of VE1 BRAF V600E immunohistochemistry in colorectal carcinoma
Authors
Adackapara et al
Affolter et al
Sinicrope et al
Capper et al
Toon et al
Kuan et al
Reference No. of cases Sensitivity (%) Specificity (%) Tissue type
[10] 52 71 74 Whole sections
[13] 31 100 100 Whole sections
[12] 75 100 100 Whole sections
[11] 91 100 98.8 Whole sections
Current Study 128 100 94 Whole sections
HIAR solution/ instrument VE1 concentration VE1 incubation/ method
Citrate pH 6.0 Pressure cooker 1:50
EDTA pH 9.0 Pressure cooker 1:600
CC1 pH 8.0 Ventana Stainer 1:45
CC1 pH 8.0 Ventana Stainer 1:5
[14] 201 98 a 100 a TMA and Whole sections Leica ER2 pH 9.0 Leica Bond III 1:80
Overnight, 4°C Manual
1 hour, 35°C Ventana Stainer
16 min, 37°C Ventana Stainer
32 min, 37°C Ventana Stainer
Strong, Mod, Weak, Neg
Not done
3+; 2+; 1+; 0
Positive (N80% cancer cells +)
Scoring a
30 min, room temperature Leica Stainer Positive (N75% cancer cells +)
Using the reported combined BRAF V600E multiplex PCR assay and real-time BRAF PCR assay results [14].
CC1 pH 8.0 Ventana Stainer 1:200 20 min, 37°C Ventana Stainer 3+; 2+; 1+; 0
BRAF VE1 immunohistochemistry in CRC assessment based on staining intensity such as that was established in EGFR mutation-specific antibodies is probably more appropriate for the current BRAF V600E VE1 immunoassay [22]. In our retrospective analysis, we identified three cases with weak (1+) but diffuse staining that was clearly distinguishable from the surrounding nonneoplastic tissue, and confirmed by PCR to harbor the BRAF V600E mutation. However, in our prospective analysis, we identified three cases of colorectal carcinoma with weak (1+) staining that lacked BRAF V600E by PCR. Our results indicate that weak (1+) staining with VE1 is not diagnostic of BRAF V600E protein expression and requires additional testing by PCR analysis. In our practice, we routinely perform BRAF PCR analysis on any tumor demonstrating weak (1+) staining with VE1. During our prospective evaluation, we also encountered variability in staining with the VE1 antibody, particularly between new antibody lots purchased from the vendor or when new reagents are used in the assay. This indicates that rigorous antibody optimization must be performed if any new reagents are to be used. The staining variability with different conditions and reagents is a major limitation of the VE1 antibody as it requires considerable time to fully validate the antibody. Until such variability in staining quality is reduced by the vendor, use of the VE1 antibody for clinical and diagnostic purposes may not be entirely practical. Another interesting issue is whether immunohistochemistry or PCR-based method is more sensitive in detecting BRAF V600E mutation. Currently, PCR-based assays are the gold standard to detect BRAF mutation. However, PCR-based methods require the presence of an adequate number of cancer cells to detect the mutation. Capper and colleagues recently reported a case which demonstrated strong staining for VE1, but no BRAF mutation could be detected by repeated Sanger sequencing analysis, which they attributed to the rare cancer cells within abundant stroma in that specimen [11]. Similarly, Toon and colleagues found that BRAF VE1 immunohistochemistry outperformed the PCR MassArray assay used in their analysis for detecting BRAF V600E mutation in colorectal carcinoma using real-time PCR as a gold standard [14]. In addition, 15 cases failed to amplify by BRAF PCR indicating that PCR-based methods may not always provide a result when assessing for BRAF mutation status. Since proteins could sometimes be more abundant or better preserved than DNA molecules in certain conditions of tissue processing, it is not surprising that immunohistochemistry may be more sensitive than PCR-based assay in detecting BRAF mutation in small specimens such as needle aspiration. However, the application of VE1 antibody in small biopsies or aspiration samples should be more fully validated. The 2 major pathways for colorectal carcinogenesis are chromosomal instability and microsatellite instability (MSI) [23]. In the chromosomal instability pathway, CRCs often follow the classic adenoma-carcinoma sequence of tumorigenesis. According to Kinzler and Vogelstein, mutation of the APC tumor suppressor gene is frequently an early event
471 while the activating mutation of the KRAS oncogene occurs at the intermediate stage of adenoma progression [24]. In contrast, CRCs within the MSI pathway commonly arise in serrated adenomas (so-called “serrated neoplastic pathway”) [25,26]. MSI-H CRCs often involve BRAF mutation and hypermethylation of CpG islands (CIMP) for several genes, such as MLH1, CDKN2A, and CACNA1G [27]. The coexpression of BRAF mutation and CIMP phenotypes has an odds ratio of 203 [28]. However, the time sequence or cause-effect relationship between BRAF mutation and CIMP is not well defined. In the past, the sequence of various gene mutations in the carcinogenesis pathway could only be analyzed by microdissection and PCR-based technology. With the development of mutation-specific immunohistochemistry, it becomes possible to dissect the sequence of mutation events on a single slide for a particular tumor. Indeed, in our series, we identified 7 colorectal carcinomas arising in SSAs. All 7 cases demonstrated BRAF V600E–mutated protein in the precursor SSA as well as cancerous areas. However, the area of SSA without cytological dysplasia still expressed MLH1 mismatch repair protein, while the cancer cells displayed loss of MLH1 protein expression. These findings support the theory of Whitehall and Leggett who hypothesized that BRAF mutation is the very first event before CIMP and MLH1 promoter hypermethylation [26]. It is also of interest to note that one of the BRAF mutated MSS colorectal carcinomas (case 8 in Table 3) arose from a TSA. This suggests that BRAF mutation is closely related to serrated morphology rather than the MSI status. In summary, our study indicates that mutation-specific immunohistochemistry for BRAF V600E mutation is a useful alternative for PCR-based assays but requires rigorously optimized staining conditions to increase the intensity and to reduce some pitfalls of nonspecific background. Our results emphasize the importance of a scoring system to identify possible borderline cases that require additional confirmation by PCR-based analysis. The use of mutation specific antibodies in the research setting is still in its early stages, but our results suggest that it may become a powerful tool to localize mutant proteins in cancer cells and allow for the correlation of tumor morphology with molecular events of carcinogenesis.
References [1] Vaughn CP, Zobell SD, Furtado LV, Baker CL, Samowitz WS. Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer. Genes Chromosomes Cancer 2011;50:307-12. [2] Patil DT, Shadrach BL, Rybicki LA, Leach BH, Pai RK. Proximal colon cancers and the serrated pathway: a systematic analysis of precursor histology and BRAF mutation status. Mod Pathol 2012;25: 1423-31. [3] Domingo E, Laiho P, Ollikainen M, et al. BRAF screening as a lowcost effective strategy for simplifying HNPCC genetic testing. J Med Genet 2004;41:664-8. [4] Pai RK, Jayachandran P, Koong AC, et al. BRAF-mutated, microsatellite-stable adenocarcinoma of the proximal colon: an
472
[5]
[6] [7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
S. -F. Kuan et al. aggressive adenocarcinoma with poor survival, mucinous differentiation, and adverse morphologic features. Am J Surg Pathol 2012;36: 744-52. Di Nicolantonio F, Martini M, Molinari F, et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol 2008;26:5705-12. Bardelli A, Siena S. Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer. J Clin Oncol 2010;28:1254-61. Capper D, Preusser M, Habel A, et al. Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody. Acta Neuropathol 2011;122:11-9. Capper D, Berghoff AS, Magerle M, et al. Immunohistochemical testing of BRAF V600E status in 1,120 tumor tissue samples of patients with brain metastases. Acta Neuropathol 2012;123:223-33. Andrulis M, Penzel R, Weichert W, von Deimling A, Capper D. Application of a BRAF V600E mutation-specific antibody for the diagnosis of hairy cell leukemia. Am J Surg Pathol 2012;36:1796-800. Adackapara CA, Sholl LM, Barletta JA, Hornick JL. Immunohistochemistry using the BRAF V600E mutation-specific monoclonal antibody VE1 is not a useful surrogate for genotyping in colorectal adenocarcinoma. Histopathology 2013;63:187-93. Capper D, Voigt A, Bozukova G, et al. BRAF V600E-specific immunohistochemistry for the exclusion of Lynch syndrome in MSI-H colorectal cancer. Int J Cancer 2013;133:1624-30. Sinicrope FA, Smyrk TC, Tougeron D, et al. Mutation-specific antibody detects mutant BRAF protein expression in human colon carcinomas. Cancer 2013;119:2765-70. Affolter K, Samowitz W, Tripp S, Bronner MP. BRAF V600E mutation detection by immunohistochemistry in colorectal carcinoma. Genes Chromosomes Cancer 2013;52:748-52. Toon CW, Walsh MD, Chou A, et al. BRAFV600E Immunohistochemistry Facilitates Universal Screening of Colorectal Cancers for Lynch Syndrome. Am J Surg Pathol 2013;37:1592-602. Bosmuller H, Fischer A, Pham DL, et al. Detection of the BRAF V600E mutation in serous ovarian tumors: a comparative analysis of immunohistochemistry with a mutation-specific monoclonal antibody and allele-specific PCR. HUM PATHOL 2013;44:329-35. Yousem SA, Nikiforova M, Nikiforov Y. The histopathology of BRAF-V600E-mutated lung adenocarcinoma. Am J Surg Pathol 2008;32:1317-21.
[17] Chiosea S, Shuai Y, Cieply K, Nikiforova MN, Dacic S. EGFR fluorescence in situ hybridization-positive lung adenocarcinoma: incidence of coexisting KRAS and BRAF mutations. HUM PATHOL 2010;41:1053-60. [18] Radu OM, Nikiforova MN, Farkas LM, Krasinskas AM. Challenging cases encountered in colorectal cancer screening for Lynch syndrome reveal novel findings: nucleolar MSH6 staining and impact of prior chemoradiation therapy. HUM PATHOL 2011;42: 1247-58. [19] Hartman DJ, Brand RE, Hu H, et al. Lynch syndrome-associated colorectal carcinoma: frequent involvement of the left colon and rectum and late-onset presentation supports a universal screening approach. HUM PATHOL 2013;44:2518-28. [20] Pileri SA, Roncador G, Ceccarelli C, et al. Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 1997;183:116-23. [21] Emoto K, Yamashita S, Okada Y. Mechanisms of heat-induced antigen retrieval: does pH or ionic strength of the solution play a role for refolding antigens? J Histochem Cytochem 2005;53: 1311-21. [22] Kawahara A, Taira T, Azuma K, et al. A diagnostic algorithm using EGFR mutation-specific antibodies for rapid response EGFR-TKI treatment in patients with non-small cell lung cancer. Lung Cancer 2012;78:39-44. [23] Arends MJ. Pathways of colorectal carcinogenesis. Appl Immunohistochem Mol Morphol 2013;21:97-102. [24] Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996;87:159-70. [25] Hawkins NJ, Bariol C, Ward RL. The serrated neoplasia pathway. Pathology 2002;34:548-55. [26] Leggett B, Whitehall V. Role of the serrated pathway in colorectal cancer pathogenesis. Gastroenterology 2010;138:2088-100. [27] Ogino S, Cantor M, Kawasaki T, et al. CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies. Gut 2006;55:1000-6. [28] Weisenberger DJ, Siegmund KD, Campan M, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet 2006;38:787-93.
www.elsevier.com/locate/humpath
Original contribution
Immunohistochemical detection of BRAF V600E mutant protein using the VE1 antibody in colorectal carcinoma is highly concordant with molecular testing but requires rigorous antibody optimization☆ Shih-Fan Kuan, Sarah Navina, Kristi L. Cressman, Reetesh K. Pai ⁎ Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA Received 16 July 2013; revised 7 October 2013; accepted 24 October 2013
Keywords: Colorectal carcinoma; BRAF; V600E; VE1; Lynch syndrome; Immunohistochemistry
Summary The BRAF V600E mutation occurs in 15% of colorectal carcinomas (CRCs) and has important genetic, prognostic, and therapeutic implications. A monoclonal antibody (VE1) targeting the BRAF V600E mutant protein has become available with variable efficacy in literature reports. We investigated the utility of the VE1 antibody in detecting BRAF V600E mutant protein in two cohorts: (1) a retrospectively accrued series of 103 resected CRCs with (N = 57) and without (N = 46) known BRAF V600E mutation status by PCR and (2) a prospective series of 25 CRCs requiring BRAF analysis during routine screening for Lynch syndrome. All 74 cases with positive BRAF V600E mutation demonstrated cytoplasmic positivity with the VE1 antibody with most tumors (70/74, 95%) demonstrating moderate to strong staining. Of the 54 BRAF V600E–negative cases, 51/54 CRCs (94%) were negative with the VE1 antibody while 3 CRCs (6%) demonstrated weak cytoplasmic staining. The sensitivity and specificity of VE1 was 100% and 94%, respectively. Ten BRAF V600E–mutated CRCs had adjacent precursor lesions including 7 sessile serrated adenomas associated with CRCs with high-level microsatellite instability (MSI-H). All 10 precursor adenomas were positive for VE1 staining with the 7 sessile serrated adenomas maintaining preserved MLH1 expression. Our results indicate that VE1 immunohistochemistry is a useful surrogate for the detection of the BRAF V600E mutation in CRC, although weak staining must be evaluated by BRAF PCR analysis to exclude a false positive result. In addition, the BRAF V600E mutation appears to be an early event before the divergent development into MSS and MSI-H pathways. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Activating mutations of BRAF are found in 15% to 17% of colorectal carcinomas (CRCs) with most occurring in a ☆
The authors have no conflict of interest and have received no funding for this manuscript. ⁎ Corresponding author. Department of Pathology, University of Pittsburgh, Presbyterian Hospital, Pittsburgh, PA, USA. E-mail address: [email protected] (R. K. Pai). 0046-8177/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.10.026
hotspot of amino acid position 600 by a missense substitution of valine by glutamic acid, known as the BRAF V600E mutation [1]. Detection of the BRAF V600E mutation has important genetic, prognostic, and therapeutic implications for patients with CRC. First, most BRAF V600E–mutated CRCs exhibit sporadic high levels of microsatellite instability (MSI-H) and arise from a sessile serrated adenoma (SSA) [2]. Thus, BRAF mutation status helps to further stratify MSI-H CRCs into Lynch syndrome– associated CRCs which are negative for the BRAF V600E
BRAF VE1 immunohistochemistry in CRC mutation from sporadic MSI-H CRCs which are often positive for the BRAF V600E mutation. BRAF mutation analysis in MSI-H tumors has become a cost-effective assay in selecting patients who may benefit from further germline testing to evaluate for Lynch syndrome [3]. In addition, knowledge of BRAF mutation status has prognostic value in CRC. The presence of a BRAF V600E mutation is an unfavorable prognostic factor in microsatellite stable (MSS) CRCs as patients with BRAF–mutated MSS tumors typically follow an aggressive clinical course and often present with widespread metastatic disease [4]. Finally, the presence of a BRAF V600E mutation in CRC predicts resistance to anti-EGFR therapy in patients with a wild-type KRAS genotype [5]. BRAF and KRAS are downstream molecules of the Ras-Raf-MAPK signaling pathway lying downstream of EGFR. Current therapies for patients with CRC include anti-EGFR antibodies such as cetuximab and panitumumab as well as conventional chemoradiation. Several studies have shown that KRAS or BRAF mutations lead to constitutive activation of the MAPK pathway and result in the failure of anti-EGFR therapy [6]. Due to the high cost of anti-EGFR treatment, testing for the presence of KRAS and BRAF mutations before instituting therapy is recommended to predict therapeutic efficacy. The conventional polymerase chain reaction (PCR)– based methods for BRAF mutation detection are relatively time-consuming and expensive. Recently, an antibody (VE1) targeting the BRAF V600E mutant protein has become commercially available to detect its presence in formalinfixed, paraffin-embedded tissue [7–15]. This monoclonal antibody was raised against a synthetic peptide containing BRAF-mutated amino acid sequence from amino acid 596 to 606 (GLATEKSRWSG). A few studies using this antibody in various types of malignant tumors have demonstrated promising results [7–9]. However, the findings in CRC have been conflicting [10–14], likely due to the small number of cases included in the analyses and the lack of a standardized antibody optimization protocol. Herein, we investigated the immunohistochemical expression of BRAF V600E protein using the VE1 antibody in a series of resected CRCs with known BRAF V600E mutation status order to (1) determine the optimum assay conditions for BRAF V600E immunohistochemistry in formalin-fixed paraffin-embedded tissue from the resection specimens of CRCs, (2) assess the sensitivity and specificity of VE1 immunohistochemistry, and (3) analyze the presence of BRAF V600E in precursor lesions of CRC.
2. Materials and methods 2.1. Study group and molecular analysis We collected 2 cohorts of CRCs: (1) a retrospective series of 103 resected CRCs for the years 2009 through 2012 and (2) a prospectively accrued series of 25 resected CRCs analyzed for BRAF V600E during routine screening for
465 Lynch syndrome between June 2013 through September 2013. All tumors were analyzed for the presence of KRAS codon 12 and 13 mutations, BRAF V600E mutation, and MSI by either PCR or DNA mismatch repair (MMR) protein immunohistochemistry. This study was conducted under the guidelines of the University of Pittsburgh Institutional Review Board (IRB# PR012020335). For surgical resection specimens, manual microdissection was performed. Only specimens with a minimum of 50% tumor cellularity in a microdissection target were accepted for analysis. DNA was extracted from paraffin sections, using the DNeasy tissue kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. Detection of BRAF mutations was performed using real-time PCR and post-PCR fluorescence melting curve analysis on LightCycler (Roche Applied Science, Indianapolis, IN), as previously described [16]. Briefly, a pair of oligonucleotide primers flanking the mutation site was designed, together with 2 fluorescent probes, with the sensor probe spanning the nucleotide position 1799. Post-amplification FMCA was performed by gradual heating of samples at a rate of 0.2°C from 45°C to 95°C. All PCR products that deviated from the wild-type melting peak (placenta DNA) were sequenced to verify the presence of mutation. KRAS codon 12 and 13 mutations were assessed by direct sequencing in both directions (forward and reverse) as previously described [17]. Detection of MSI was performed using a National Cancer Institute (NCI)–recommended panel of microsatellite markers (BAT25, BAT26, D2S123, D5S346, and D17S250) as well as one novel quasi-monomorphic mononucleotide marker CAT25, as previously described [18].
2.2. Immunohistochemistry Immunohistochemical staining using VE1 mouse monoclonal antibody (Spring Biosciences, CA) was performed on 4-micron formalin-fixed paraffin-embedded tissue sections either by manual method or Ventana BenchMark Ultra automated stainer (Tucson, AZ). The manual staining method was used to evaluate the factors affecting the staining results including the pH of heat-induced antigen retrieval solutions and antibody concentrations. Briefly, 4-μm paraffin sections on negatively charged glass slides were deparaffinized in xylene solutions and rehydrated in graded alcohol solutions followed by washes in distilled water. Antigen retrieval was performed in a pressure cooker for 15 minutes in the following buffers: 10 mmol/L citric buffer (pH 6.0), 1 mmol/L EDTA (pH 8.0) or 20 mmol/L Tris-EDTA buffer (pH 9.0). The sections were allowed to cool to room temperature and then washed with PBS, 0.05% Tween 20, pH 7.0. The sections were incubated overnight in a humidified chamber at room temperature with various dilutions of VE1 antibody (1:50, 1:100, and 1:200). After washing with PBS, the sections were incubated with HRP-labeled polymer anti-mouse secondary antibody (DAKO Envision+ system, Carpinteria, CA) for 1
466
S. -F. Kuan et al.
Fig. 1 Effects of pH and antibody concentrations on the staining of BRAF V600E VE1 antibody in colorectal carcinoma. A-C, Heat-induced antigen retrieval was performed in a pressure cooker for 15 minutes in the following buffers: 10 mmol/L citric buffer pH 6.0 (A), 10 mmol/L EDTA buffer, pH 8.0 (B) and 20 mmol/L Tris-EDTA buffer, pH 9.0 (C). The antibody dilution was 1:200 in A, B, and C. D-F, Antigen retrieval was performed using 10 mmol/L EDTA buffer, pH 8.0, for 15 minutes, then incubated with VE1 antibody in following dilutions: 1:200 (D), 1:100 (E), and 1:50 (F). All images are at 100× magnification.
hour at room temperature. Color visualization was performed with liquid DAB chromogen in imidazole-HCl buffer (pH 7.5) containing hydrogen peroxide until the brown color fully developed. The sections were counterstained with hematoxylin and coverslipped with permanent mounting media. After optimization of staining conditions by the manual method, all cases were immunostained in a Ventana BenchMark Ultra stainer to avoid variation by manual processing. The automated program for immunohistochemistry included deparaffinization at 72°C, pre-primary oxidase inhibition for 4 minutes, cell condition (CC1, pH 8.0) for 56 minutes at 100°C; incubation with 1:200 diluted VE1 antibody at 37°C for 20 minutes, incubation with OptiView HRP Linker for 8 minutes, incubation with OptiView HRP multimer for 8 minutes, and incubation with hematoxylin and bluing reagent for 4 minutes each.
For all cases, VE1 immunohistochemistry was independently read as positive and negative blinded to the BRAF mutation status by PCR. For the retrospective series, each case was reviewed by 3 gastrointestinal pathologists (S.F.K., S.N., and R.K.P.). For both the retrospective and prospectively accrued cases, a semiquantitative assessment of intensity was given by one pathologist (S.F.K.) using the following scoring criteria: 0, negative with no cytoplasmic staining visualized at any magnification; 1+, weak, difficult to recognize brown color on the slide requiring a 10× or greater objective for confirmation; 2+, moderate, easy to recognize staining seen with a 2× or 4× objective; and 3+, strong color on glass slides by naked eyes with confirmation of strong staining using 2× microscopic objective. MMR protein immunohistochemistry for MLH1, PMS2, MSH2, and MSH6 was performed as previously described [19]. Normal expression was defined as nuclear staining within
BRAF VE1 immunohistochemistry in CRC tumor cells, using infiltrating lymphocytes as positive internal control. Negative protein expression was defined as complete absence of nuclear staining within tumor cells with concurrent positive labeling in internal non-neoplastic tissues.
3. Results 3.1. Optimization of staining conditions by manual method We investigated the optimal staining conditions including pH of retrieval solutions and antibody concentrations for the VE1 antibody using 10 resection cases of CRC that had been confirmed to be BRAF V600E–mutated by PCR. When heatinduced antigen retrieval was performed in citric acid buffer, (pH 6.0), BRAF-mutated CRC showed heterogeneous and weak cytoplasmic staining in some cases (Fig. 1A) and complete absence of staining in some cases. EDTA buffer (pH 8.0) pretreatment provided satisfactory intensity and homogeneity of VE1 staining in all cases (Fig. 1B). TrisEDTA buffer (pH 9.0) further increased the intensity but also created nonspecific staining of nuclei (Fig. 1C). We then tested the effects of various dilutions of VE1 antibody after antigen retrieval in EDTA (pH 8.0) solution. Although all 3 dilutions (1:50, 1:100, and 1:200) of the antibody generated recognizable brown color in tumor cytoplasm (Fig. 1D-F), we identified a nonspecific nuclear staining in some areas of normal colonic mucosa at 1:50 or 1:100 dilutions. A 1:200 dilution of antibody reduced the chance of nonspecific nuclear staining. During the experimentation, we also noticed that the automated stainer produced more consistent and satisfactory staining quality than the manual method. We therefore decided to conduct our study on the Ventana automated stainer because (1) the automated stainer has been adapted as the sole instrumentation by many clinical laboratories including our institution and (2) it was the only staining method recommended by the vendor.
3.2. Automated BRAF VE1 immunohistochemistry in colorectal carcinoma study groups The retrospective study group included 103 cases of surgically resected CRCs with known BRAF and KRAS Table 1
467 mutation by PCR (Table 1). Among these cases, 57 were BRAF V600E–mutated, 21 displayed KRAS codon 12 or 13 mutations, and the remaining 25 cases were BRAF/KRAS wild type. All 57 cases with a known BRAF V600E mutation were positive by VE1 antibody. The intensity was scored strong (3+) in 35 cases, moderate (2+) in 19 cases, and weak (1+) in 3 cases (Fig. 2A-C). All 46 cases of BRAF wild-type CRC, including 21 KRAS mutants, were negative for VE1 staining (Fig. 2D). The independent evaluation (positive vs negative) by 3 GI pathologists showed 100% concordance of the reading and scoring. It is noteworthy that a diffuse and homogeneous cytoplasmic staining pattern was observed in all ranges of tumor differentiation and histological subtypes such as mucinous (Fig. 2E) and large cell neuroendocrine carcinomas (Fig. 2F). Nine cases of CRCs with known history of Lynch syndrome were stained negative. The 25 CRCs included in the prospective study group of CRCs were identified during routine screening of CRC for DNA MMR protein expression at our institution over a 3month period. All 25 CRCs demonstrated loss of immunohistochemical expression for MLH1 and PMS2 requiring evaluation for BRAF V600E in an attempt to distinguish sporadic MSI-H CRC from possible Lynch syndrome– associated CRC. BRAF VE1 immunohistochemistry was independently evaluated using the 0-3+ scoring scheme and without knowledge of the BRAF PCR results. All 16 CRCs with 2+ or 3+ VE1 staining were positive for BRAF V600E by PCR (Table 2). All 5 CRCs with negative (0) staining were negative for BRAF V600E by PCR. Of the four cases of CRCs demonstrating weak (1+) staining, three cases were negative for BRAF V600E by PCR while one case was positive for BRAF V600E by PCR.
3.3. BRAF V600E protein expression in precursor lesions in the study group Ten separate BRAF V600E–mutated CRC cases also contained precursor lesions adjacent to the main tumor (Table 3). Seven cases were MSI-H, and 3 were MSS. All 7 precursors adjacent to those 7 MSI-H CRC cases were classified as sessile serrated adenomas and stained moderately to strongly positive by VE1 antibody (Fig. 3A and B). Of the three precursor lesions adjacent to MSS CRCs, one was a traditional serrated adenoma (Fig. 3C) and 2 were conventional tubular adenomas. All 3 also demonstrated positive staining with VE1 antibody.
Correlation between VE1 immunophenotype and BRAF/KRAS genotype in the retrospective study group
BRAF V600E genotype
KRAS genotype
No. with positive VE1 staining
Staining intensity
Mutant
Wild type
57/57
Wild type Wild type
Mutant Wild type
0/21 0/25
3+ (N = 35); 2+ (N = 19); 1+ (N = 3) All negative All negative
468
S. -F. Kuan et al.
Fig. 2 Immunohistochemistry of BRAF V600E VE1 antibody in colorectal carcinoma. A-D, The intensity of staining was scored as weak (1+, A), moderate (2+, B), strong (3+, C) and negative (0, D). E and F, Diffuse and homogenous staining were seen in different histological subtypes such as mucinous (E) and large cell neuroendocrine (F) colorectal carcinomas. All images are at 100× magnification.
To further investigate the sequence of BRAF V600E mutation and loss of MLH1 expression, we performed immunostaining of VE1 and anti-MLH1 antibodies on the serial sections of 7 pairs of SSA-associated MSI-H tumors. In all 7 cases, the areas of SSA without cytological dysplasia expressed both MLH1 and BRAF V600E mutant protein, while the areas of invasive carcinoma expressed BRAF mutant protein but lost MLH1 expression (Fig. 3E and F). These findings support the notion that BRAF V600E
Table 2
mutation occurs before the loss of MLH1 mismatch repair protein in the SSA-MSI-H pathway for CRC.
4. Discussion Our results indicate that VE1 immunohistochemistry is highly sensitive and specific for detecting colorectal carcinomas harboring a BRAF V600E mutation. However,
Prospective comparison of BRAF VE1 immunohistochemistry with BRAF V600E PCR
BRAF VE1 immunohistochemistry
Negative Weak staining (1+) Moderate to strong staining (2+ or 3+)
No. of cases
BRAF PCR V600E positive (%)
V600E negative (%)
5 4 16
0 1 (25) 16 (100)
5 (100) 3 (75) 0
BRAF VE1 immunohistochemistry in CRC Table 3
469
Analysis of VE1 immunohistochemistry in colorectal carcinomas with adjacent precursor lesions
Case
Age
Sex
Cancer histology
Carcinoma MSI status
Precursor histology
Cancer VE1
Precursor VE1
1 2 3 4 5 6 7 8 9 10
81 85 75 83 80 62 77 66 83 67
F F F F F F F F F M
Medullary carcinoma PD adenocarcinoma MD adenocarcinoma MD adenocarcinoma MD adenocarcinoma Mucinous/SRC MD adenocarcinoma LC-NEC MD adenocarcinoma LC-NEC
MSI-H MSI-H MSI-H MSI-H MSI-H MSI-H MSI-H MSS MSS MSS
SSA SSA SSA SSA SSA SSA SSA TSA TA TA
3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+
2+ 3+ 3+ 2+ 2+ 2+ 3+ 2+ 3+ 2+
Abbreviations: MD, moderately differentiated; PD, poorly differentiated; SRC, signet-ring cell; LC-NEC, large cell neuroendocrine carcinoma; TSA, traditional serrated adenoma; TA, tubular adenoma.
our study highlights several important points concerning the implementation of BRAF V600E VE1 immunohistochemistry. First, optimization of the staining protocol is essential
before VE1 can be reliably applied in a clinical or research setting. Second, nonspecific nuclear staining is not uncommon, especially in high antibody concentrations and high pH
Fig. 3 CRC and adjacent precursors using the BRAF V600E VE1 antibody. A and B, Two colorectal carcinomas with MSI-H and adjacent SSA were strongly stained with the VE1 antibody. C, A microsatellite stable colorectal carcinoma and its adjacent traditional serrated adenoma (TSA) were positively stained by VE1. D-F, Serial sections of a MSI-H colorectal carcinoma (left lower) arising in a sessile serrated adenoma (right upper) which were stained with hematoxylin and eosin (D), BRAF V600E VE1 antibody (E) and MLH1 (F). All images are at 40× magnification.
470
S. -F. Kuan et al.
antigen retrieval solutions. Finally, a scoring system needs to be established for identifying borderline cases which require additional testing. Heat-induced antigen retrieval (HIAR) has been widely used in many laboratories, although the mechanism is not yet fully understood [20,21]. Citric buffer, pH 6.0 is the most popular solution for HIAR. However, pre-treatment of sections in citric buffer (pH 6.0) only revealed weak, heterogeneous and inconsistent staining in the cytoplasm with the VE1 antibody. Adackapara and colleagues analyzed a series of 52 colorectal carcinomas with known BRAF mutation status by PCR and found that VE1 immunohistochemistry had a low sensitivity (71%) and specificity (74%) for detecting BRAF V600E mutation (Table 4) [10]. They argued that VE1 immunohistochemistry is not a useful surrogate for detecting BRAF mutation in colorectal carcinoma. However, in their analysis, manual staining with citrate buffer pre-treatment was employed which, in our experience, results in weak heterogeneous immunoreactivity that is difficult to interpret. EDTA buffer (pH 8.0) proved to be a retrieval agent that produced robust and homogenous cytoplasmic staining by VE1 antibody. The remaining literature reports analyzing the utility of VE1 immunohistochemistry in detecting BRAF mutation all employed EDTApretreatment and automated staining and identified a sensitivity and specificity of VE1 immunohistochemistry approaching 100% [11–14], similar to our series (Table 4). Tris-EDTA buffer (pH 9.0) further increased the positive signals, but it also significantly increased the chance of nonspecific nuclear staining. A recent study by Emoto and colleagues demonstrated that heat treatment cleaves the intraand intermolecular methylene bridge induced by formaldehyde [21]. Once the antigen molecule is denatured by heat, the polypeptide extends to expose the hydrophilic and hydrophobic portions. In the setting of solution around neutral pH (4.5-7.5), the antigenic determinants are conTable 4
cealed after the solutions cool because the peptides are ready to self-associate. In contrast, in more basic pH solutions, the extended peptides are charged negatively which prevents the refolding of peptide. This hypothesis may explain our observation that optimal staining can only be achieved by EDTA (pH 8.0) or higher pH solutions but not by near neutral solutions (pH 6.0). The presence of nonspecific nuclear staining is a troublesome but intrinsic property of the VE1 antibody. We speculate that there is a low-affinity nuclear protein that may cross-react with the 11-amino-acid peptide that was used to produce VE1 antibody. First, higher antibody concentration increased the chance of nuclear staining because more free antibody was available in the solution to bind this low-affinity protein. Second, retrieval solutions with more basic pHs also enhanced nuclear staining, suggesting that this nuclear protein required more basic retrieval solutions to better expose its antigen determinants, as discussed previously. Finally, in order to correctly interpret the results, users of VE1 antibody need to be aware of this and other nonspecific staining. These include the occasional background staining in smooth muscle and in degraded tissue, either due to tumor necrosis or drying artifacts during the staining procedure. When an immunohistochemical assay is utilized to guide clinical management, it is crucial to develop a standard scoring system to define the positivity. Various scoring criteria have been employed in early literature reports using the VE1 antibody. Two reports used 80% and 75% positive tumor cells respectively as the cut-off for positivity, although both admitted their staining results were diffuse and homogeneous [11,14]. Two other groups used a 4-tiered system (negative, weak, moderate, strong) to assess the intensity [10,12]. Our findings indicate that a scoring system relying on a cut-off percentage of positive tumor cells may not apply to BRAF mutant protein. We believe that a scoring
Comparison of literature reports of VE1 BRAF V600E immunohistochemistry in colorectal carcinoma
Authors
Adackapara et al
Affolter et al
Sinicrope et al
Capper et al
Toon et al
Kuan et al
Reference No. of cases Sensitivity (%) Specificity (%) Tissue type
[10] 52 71 74 Whole sections
[13] 31 100 100 Whole sections
[12] 75 100 100 Whole sections
[11] 91 100 98.8 Whole sections
Current Study 128 100 94 Whole sections
HIAR solution/ instrument VE1 concentration VE1 incubation/ method
Citrate pH 6.0 Pressure cooker 1:50
EDTA pH 9.0 Pressure cooker 1:600
CC1 pH 8.0 Ventana Stainer 1:45
CC1 pH 8.0 Ventana Stainer 1:5
[14] 201 98 a 100 a TMA and Whole sections Leica ER2 pH 9.0 Leica Bond III 1:80
Overnight, 4°C Manual
1 hour, 35°C Ventana Stainer
16 min, 37°C Ventana Stainer
32 min, 37°C Ventana Stainer
Strong, Mod, Weak, Neg
Not done
3+; 2+; 1+; 0
Positive (N80% cancer cells +)
Scoring a
30 min, room temperature Leica Stainer Positive (N75% cancer cells +)
Using the reported combined BRAF V600E multiplex PCR assay and real-time BRAF PCR assay results [14].
CC1 pH 8.0 Ventana Stainer 1:200 20 min, 37°C Ventana Stainer 3+; 2+; 1+; 0
BRAF VE1 immunohistochemistry in CRC assessment based on staining intensity such as that was established in EGFR mutation-specific antibodies is probably more appropriate for the current BRAF V600E VE1 immunoassay [22]. In our retrospective analysis, we identified three cases with weak (1+) but diffuse staining that was clearly distinguishable from the surrounding nonneoplastic tissue, and confirmed by PCR to harbor the BRAF V600E mutation. However, in our prospective analysis, we identified three cases of colorectal carcinoma with weak (1+) staining that lacked BRAF V600E by PCR. Our results indicate that weak (1+) staining with VE1 is not diagnostic of BRAF V600E protein expression and requires additional testing by PCR analysis. In our practice, we routinely perform BRAF PCR analysis on any tumor demonstrating weak (1+) staining with VE1. During our prospective evaluation, we also encountered variability in staining with the VE1 antibody, particularly between new antibody lots purchased from the vendor or when new reagents are used in the assay. This indicates that rigorous antibody optimization must be performed if any new reagents are to be used. The staining variability with different conditions and reagents is a major limitation of the VE1 antibody as it requires considerable time to fully validate the antibody. Until such variability in staining quality is reduced by the vendor, use of the VE1 antibody for clinical and diagnostic purposes may not be entirely practical. Another interesting issue is whether immunohistochemistry or PCR-based method is more sensitive in detecting BRAF V600E mutation. Currently, PCR-based assays are the gold standard to detect BRAF mutation. However, PCR-based methods require the presence of an adequate number of cancer cells to detect the mutation. Capper and colleagues recently reported a case which demonstrated strong staining for VE1, but no BRAF mutation could be detected by repeated Sanger sequencing analysis, which they attributed to the rare cancer cells within abundant stroma in that specimen [11]. Similarly, Toon and colleagues found that BRAF VE1 immunohistochemistry outperformed the PCR MassArray assay used in their analysis for detecting BRAF V600E mutation in colorectal carcinoma using real-time PCR as a gold standard [14]. In addition, 15 cases failed to amplify by BRAF PCR indicating that PCR-based methods may not always provide a result when assessing for BRAF mutation status. Since proteins could sometimes be more abundant or better preserved than DNA molecules in certain conditions of tissue processing, it is not surprising that immunohistochemistry may be more sensitive than PCR-based assay in detecting BRAF mutation in small specimens such as needle aspiration. However, the application of VE1 antibody in small biopsies or aspiration samples should be more fully validated. The 2 major pathways for colorectal carcinogenesis are chromosomal instability and microsatellite instability (MSI) [23]. In the chromosomal instability pathway, CRCs often follow the classic adenoma-carcinoma sequence of tumorigenesis. According to Kinzler and Vogelstein, mutation of the APC tumor suppressor gene is frequently an early event
471 while the activating mutation of the KRAS oncogene occurs at the intermediate stage of adenoma progression [24]. In contrast, CRCs within the MSI pathway commonly arise in serrated adenomas (so-called “serrated neoplastic pathway”) [25,26]. MSI-H CRCs often involve BRAF mutation and hypermethylation of CpG islands (CIMP) for several genes, such as MLH1, CDKN2A, and CACNA1G [27]. The coexpression of BRAF mutation and CIMP phenotypes has an odds ratio of 203 [28]. However, the time sequence or cause-effect relationship between BRAF mutation and CIMP is not well defined. In the past, the sequence of various gene mutations in the carcinogenesis pathway could only be analyzed by microdissection and PCR-based technology. With the development of mutation-specific immunohistochemistry, it becomes possible to dissect the sequence of mutation events on a single slide for a particular tumor. Indeed, in our series, we identified 7 colorectal carcinomas arising in SSAs. All 7 cases demonstrated BRAF V600E–mutated protein in the precursor SSA as well as cancerous areas. However, the area of SSA without cytological dysplasia still expressed MLH1 mismatch repair protein, while the cancer cells displayed loss of MLH1 protein expression. These findings support the theory of Whitehall and Leggett who hypothesized that BRAF mutation is the very first event before CIMP and MLH1 promoter hypermethylation [26]. It is also of interest to note that one of the BRAF mutated MSS colorectal carcinomas (case 8 in Table 3) arose from a TSA. This suggests that BRAF mutation is closely related to serrated morphology rather than the MSI status. In summary, our study indicates that mutation-specific immunohistochemistry for BRAF V600E mutation is a useful alternative for PCR-based assays but requires rigorously optimized staining conditions to increase the intensity and to reduce some pitfalls of nonspecific background. Our results emphasize the importance of a scoring system to identify possible borderline cases that require additional confirmation by PCR-based analysis. The use of mutation specific antibodies in the research setting is still in its early stages, but our results suggest that it may become a powerful tool to localize mutant proteins in cancer cells and allow for the correlation of tumor morphology with molecular events of carcinogenesis.
References [1] Vaughn CP, Zobell SD, Furtado LV, Baker CL, Samowitz WS. Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer. Genes Chromosomes Cancer 2011;50:307-12. [2] Patil DT, Shadrach BL, Rybicki LA, Leach BH, Pai RK. Proximal colon cancers and the serrated pathway: a systematic analysis of precursor histology and BRAF mutation status. Mod Pathol 2012;25: 1423-31. [3] Domingo E, Laiho P, Ollikainen M, et al. BRAF screening as a lowcost effective strategy for simplifying HNPCC genetic testing. J Med Genet 2004;41:664-8. [4] Pai RK, Jayachandran P, Koong AC, et al. BRAF-mutated, microsatellite-stable adenocarcinoma of the proximal colon: an
472
[5]
[6] [7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
S. -F. Kuan et al. aggressive adenocarcinoma with poor survival, mucinous differentiation, and adverse morphologic features. Am J Surg Pathol 2012;36: 744-52. Di Nicolantonio F, Martini M, Molinari F, et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol 2008;26:5705-12. Bardelli A, Siena S. Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer. J Clin Oncol 2010;28:1254-61. Capper D, Preusser M, Habel A, et al. Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody. Acta Neuropathol 2011;122:11-9. Capper D, Berghoff AS, Magerle M, et al. Immunohistochemical testing of BRAF V600E status in 1,120 tumor tissue samples of patients with brain metastases. Acta Neuropathol 2012;123:223-33. Andrulis M, Penzel R, Weichert W, von Deimling A, Capper D. Application of a BRAF V600E mutation-specific antibody for the diagnosis of hairy cell leukemia. Am J Surg Pathol 2012;36:1796-800. Adackapara CA, Sholl LM, Barletta JA, Hornick JL. Immunohistochemistry using the BRAF V600E mutation-specific monoclonal antibody VE1 is not a useful surrogate for genotyping in colorectal adenocarcinoma. Histopathology 2013;63:187-93. Capper D, Voigt A, Bozukova G, et al. BRAF V600E-specific immunohistochemistry for the exclusion of Lynch syndrome in MSI-H colorectal cancer. Int J Cancer 2013;133:1624-30. Sinicrope FA, Smyrk TC, Tougeron D, et al. Mutation-specific antibody detects mutant BRAF protein expression in human colon carcinomas. Cancer 2013;119:2765-70. Affolter K, Samowitz W, Tripp S, Bronner MP. BRAF V600E mutation detection by immunohistochemistry in colorectal carcinoma. Genes Chromosomes Cancer 2013;52:748-52. Toon CW, Walsh MD, Chou A, et al. BRAFV600E Immunohistochemistry Facilitates Universal Screening of Colorectal Cancers for Lynch Syndrome. Am J Surg Pathol 2013;37:1592-602. Bosmuller H, Fischer A, Pham DL, et al. Detection of the BRAF V600E mutation in serous ovarian tumors: a comparative analysis of immunohistochemistry with a mutation-specific monoclonal antibody and allele-specific PCR. HUM PATHOL 2013;44:329-35. Yousem SA, Nikiforova M, Nikiforov Y. The histopathology of BRAF-V600E-mutated lung adenocarcinoma. Am J Surg Pathol 2008;32:1317-21.
[17] Chiosea S, Shuai Y, Cieply K, Nikiforova MN, Dacic S. EGFR fluorescence in situ hybridization-positive lung adenocarcinoma: incidence of coexisting KRAS and BRAF mutations. HUM PATHOL 2010;41:1053-60. [18] Radu OM, Nikiforova MN, Farkas LM, Krasinskas AM. Challenging cases encountered in colorectal cancer screening for Lynch syndrome reveal novel findings: nucleolar MSH6 staining and impact of prior chemoradiation therapy. HUM PATHOL 2011;42: 1247-58. [19] Hartman DJ, Brand RE, Hu H, et al. Lynch syndrome-associated colorectal carcinoma: frequent involvement of the left colon and rectum and late-onset presentation supports a universal screening approach. HUM PATHOL 2013;44:2518-28. [20] Pileri SA, Roncador G, Ceccarelli C, et al. Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 1997;183:116-23. [21] Emoto K, Yamashita S, Okada Y. Mechanisms of heat-induced antigen retrieval: does pH or ionic strength of the solution play a role for refolding antigens? J Histochem Cytochem 2005;53: 1311-21. [22] Kawahara A, Taira T, Azuma K, et al. A diagnostic algorithm using EGFR mutation-specific antibodies for rapid response EGFR-TKI treatment in patients with non-small cell lung cancer. Lung Cancer 2012;78:39-44. [23] Arends MJ. Pathways of colorectal carcinogenesis. Appl Immunohistochem Mol Morphol 2013;21:97-102. [24] Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996;87:159-70. [25] Hawkins NJ, Bariol C, Ward RL. The serrated neoplasia pathway. Pathology 2002;34:548-55. [26] Leggett B, Whitehall V. Role of the serrated pathway in colorectal cancer pathogenesis. Gastroenterology 2010;138:2088-100. [27] Ogino S, Cantor M, Kawasaki T, et al. CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies. Gut 2006;55:1000-6. [28] Weisenberger DJ, Siegmund KD, Campan M, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet 2006;38:787-93.
