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. 2020 May 1;14(4):1067–1079. doi: 10.1007/s12105-020-01166-8

Comparison of Molecular Methods and BRAF Immunohistochemistry (VE1 Clone) for the Detection of BRAF V600E Mutation in Papillary Thyroid Carcinoma: A Meta-Analysis

Kyle G Parker 1,✉, Michael G White 2, Nicole A Cipriani 1
PMCID: PMC7669962  PMID: 32358715

Abstract

The evaluation of surgically resected papillary thyroid carcinomas (PTC) by immunohistochemistry (IHC) for BRAF mutation has diagnostic, prognostic and therapeutic implications. The goal of this meta-analysis was to perform a systematic review of studies using the VE1 clone (specific for detection of the BRAF V600E mutation) on formalin-fixed paraffin embedded (FFPE) thyroid surgical resection specimens for primary papillary thyroid carcinoma. The authors’ molecular techniques, immunohistochemistry protocols, and scoring methods for VE1 immunostaining were also evaluated. This study included 4079 PTCs representing data from 23 studies. The results extracted from each study were split into two different groups, direct sequencing group or PCR group, based on the molecular “gold standard” method used to compare VE1 IHC staining. In the direct sequencing group, the IHC sensitivity was 100% (95% CI 0.97–1.00) and specificity 84% (95% 0.72–0.91). In the PCR group the sensitivity was 98% (95% CI 0.96–0.99) and specificity 89% (95% CI 0.82–0.94). Although immunohistochemical procedures varied by author, the overall performance of the VE1 clone shows that it is highly sensitive and relatively specific for detecting the BRAF V600E mutation in surgical resection specimens. However, standardization of immunohistochemical procedural method and scoring/interpretation criteria may improve the reliability and reproducibility for the use of VE1 clone for future practice.

Keywords: Papillary thyroid carcinoma, Immunohistochemistry, BRAFV600E, Meta-analysis

Introduction

The most common malignancy of the thyroid gland is papillary thyroid carcinoma (PTC). The prevalence of PTC has been reported to be increasing worldwide. Factors beyond early detection via medical surveillance and more sensitive diagnostic procedures such as environmental and unrecognized carcinogens have been implicated as potential explanations [1–3]. The most common gene mutation involving PTC (~ 60%) is a missense mutation in the BRAF oncogene, valine (V) substituting glutamic acid (E) at amino acid position 600, which ultimately drives MAPK pathway signaling [4]. Recent molecular based analytic studies have shown most PTCs are driven by two distinct signaling mechanisms of the MAPK pathway, either BRAF-like and RAS-like [5]. Further investigations correlating genotype with histologic morphology showed a strong correlation with BRAF V600E mutation to classic and tall cell PTC variants, whereas RAS mutations strongly correlated with the follicular variant of PTC [4]. A systematic review and meta-analysis by Tufano et al., showed the recurrence rate in patients with BRAF V600E mutated PTCs was twice as high as wild-type PTCs (24.9% versus 12.6%). Additionally, BRAF-mutated PTCs had increased risk for lymph node metastasis (54.1% versus 36.8%), increased risk for extrathyroidal extension (46.2% versus 23.6%), and were associated with advanced AJCC III/IV stage (35.4% versus 19.6%) [6]. The increase in prevalence of PTC along with the relative access to molecular testing has facilitated the use of therapeutic agents targeting specific mutations such as BRAF V600E. Moreover, PTCs with BRAF V600E mutation have shown to have decreased response to radioactive iodine (RAI) I-131 treatment do to loss of radioiodine avidity [7]. Currently BRAF-inhibitors (dabrafenib and vemurafenib) are being used to treat various types of cancer that harbor BRAF mutations such as colorectal carcinoma, melanoma, and various brain tumors. At the time of writing this paper, four clinical trials (three open and one closed) for patients with PTC have biomarker inclusion criteria requiring BRAF V600E mutational positivity status [8]. Thus, it is imperative that analysis of mutational status is performed efficiently and accurately to select for those patients who may in the future benefit from BRAF targeted therapy.

Various molecular methods have been used for the detection of BRAF V600E mutation including DNA sequencing methods (Sanger, pyrosequencing, mass spectroscopy and direct) and PCR based methods [real-time PCR (rt-PCR), allele-specific locked nucleic acid PCR (ASLNA-qPCR), peptide nucleic acid clamping polymerase chain reaction, and SNaPshot]. However, these methods are labor intensive, time-consuming, generally more expensive than immunohistochemistry and often subject to the quality of the DNA within formalin-fixed paraffin embedded tissue (FFPE). In 2011 Capper et al., developed the first BRAF V600E specific antibody, the VE1 clone, which has allowed the potential role of immunohistochemistry (IHC) to act as a surrogate marker for the detection of BRAF V600E, thus, ideally making an immunohistochemical method more attractive for general pathology practice [9]. Since the development of this antibody, numerous studies have correlated the performance of the VE1 antibody on various tissue types, including papillary thyroid carcinoma, to current molecular gold standard techniques for the detection of the BRAF V600E mutation.

In 2015 Pyo et al., published the first systematic review of the diagnostic test accuracy using clone VE1 in 1141 PTC cases from 11 different studies [10]. This meta-analysis included tissue from surgical resection specimens and core needle biopsies as well as cytology specimens from fine needle aspirates. Review of the various studies included in the meta-analysis showed a wide variation in immunohistochemical protocols (i.e., incubation times and antibody dilution), antibody (commercially available vs laboratory developed), and grading methods used to assess scoring of antibody positivity. In light of these data, the current study aimed to evaluate the sensitivity and specificity of VE1 IHC in detecting BRAF V600E mutations in surgically resected PTC compared to molecular methods (gold standard). In addition, immunohistochemical method protocols and scoring systems used in the various studies were reviewed, in an attempt to further standardize testing and interpretation in the future.

Material and Methods

Selection and Search Criteria

A literature search was performed using MEDLINE databases (PubMed search interface) up to October 31, 2019. Keywords used in the search included the following: “papillary thyroid carcinoma,” “immunohistochemistry,” and “BRAF.” Inclusion criteria for our study included: papillary thyroid carcinoma and variants of PTC, formalin-fixed paraffin embedded surgically resected thyroid specimens, immunohistochemistry performed using a commercially available VE1 clone, comparison testing performed using a molecular method (gold standard) with correlation data, and written in the English language. Exclusion criteria included: non-FFPE tissue (i.e., cytology specimens and fine needle aspirates), non-commercially available VE1 clones (i.e. individual laboratory developed antibody), and non-PTC carcinomas. Malignant thyroid neoplasms other than PTC; such as anaplastic thyroid carcinoma, follicular thyroid carcinoma, medullary carcinoma, etc., were not included within the data set (see Fig. 1 for details).

Fig. 1.

Fig. 1

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Flow chart—final study selection

Evaluation of Articles and Data Extraction

The title and abstract of each article were screened for exclusion. If initial inclusion criteria were meet, the materials and methods section of the article were screened for further inclusion criteria (FFPE thyroid tissue, surgical resection specimens, and commercially available VE1 clone). Data from all eligible studies were then extracted [9, 11–37]. Extracted data included the following; first author’s name, title of article, year of publication, number of PTC and PTC variant specimens, type of molecular method for the detection of BRAF V600E, immunohistochemistry protocol (including dilution and incubation time), VE1 clone manufacturer, and method used for interpretation of antibody staining. For the meta-analysis the following data was extracted: all molecular (wild type and mutation) and immunohistochemistry (positive and negative) results.

Studies from the selected articles were then divided (Tables 1 and 2) based on the gold standard comparison testing used in each study to assess for BRAF V600E mutation. Those using direct sequencing methods (Sanger, Pyrosequencing) were placed in the “Direct Sequencing group” and those using PCR-based methodologies (rt-PCR, PNA-Clamp, SNaPshot, etc.) were placed in the “PCR group.”

Table 1.

Direct sequencing group—methods

Study (year) Method Number of specimens Clone VE1 vendor Dilution/incubation Interpretation Note
Bullock (2012) Sanger 96 Spring Bioscience NA/30 min No intensity score. Positive if staining > 20% of tumor cells
Dvorak (2014) Sanger 73 Ventana NA/16 min Intensity 0–3+; strong (3+), medium (2+), weak (1+) and no staining (0). Positive if unequivocal staining in > 85% tumor cells
Fisher (2013) Pyrosequencing 25 Spring Bioscience 1:40/30 min Intensity 0–3+; Positive if > 10% of tumor cells and 2+ or 3+. Anything less considered negative Study also used pan-BRAF antibody. These were excluded from study
Ghossein (2013) Mass spectrometry 31 Spring Bioscience 1:50/NA Intensity 0–3+; 0 (no staining), 1+ (faint), 2+ (moderate), 3+ (strong) Considered positive if 2+
Kim (2014) Sanger 91 Spring Bioscience 1:300/15 min Allred
Kim (2014) Pyrosequencing 91 Spring Bioscience 1:300/15 min Allred
Kim (2018) Sanger 697 Spring Bioscience NA/32 min No staining negative; weak/faint to strong/diffuse considered positive
Loo (2018) Sanger 12 Ventana NA/32 min NA
Martinuzzi (2016) Sanger 85 Spring Bioscience 1:50/NA Capper et al. method
Oh (2018) Sanger 23 Ventana 1:4/16 min Diffuse homogenous staining in all tumor cells considered positive. Non-spec staining of colloid and equivocal weak or focal as negative
Qui (2015) Sanger 127 Ventana NA/16 min NA
Zagzag (2013) Sanger 37 Spring Bioscience NA/NA Intensity 0–3+ of 0–100% of tumor cells; < 1+ and < 20% was considered negative. > 80%, stratified into 1–3+ as positive Some molecular results used to determine true positivity in weakly staining samples
Zhao (2019) Sanger 185 Ventana NA/16 min Intensity 0–3; 1–3+ staining considered positive. No or only "slight" staining as negative
Zhu (2016) Sanger 118 Spring Bioscience NA/40 min Intensity 0–3+; 0 if no or faint staining in 10% or fewer cells, 1+ faint > 10% cells, 2+ mod > 10% cells, 3+ strong > 10%. Positive when score 1+
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Table 2.

PCR group—methods

Study (year) Method Number of specimens Clone VE1 vendor Dilution/incubation Interpretation Note
Abd Elmageed (2017) rt-PCR 130 Spring Bioscience 1:100/60 min Capper et al. method
Chen (2018) Quantitative rt-PCR 40 GBI Biotechnology NA/NA Capper et al. method
de Biase (2014) ASLNA-qPCR 20 Spring Bioscience NA/NA NA
Jung (2015) PCR 467 Spring Bioscience 1:50/NA No or weak staining considered negative. Moderate or strong staining considered positive
Kim (2014) PNA-clamp 91 Spring Bioscience 1:300/15 min Allred
Kim (2014) rt-PCR 91 Spring Bioscience 1:300/15 min Allred
Martinuzzi (2016) PNA-clamp 85 Spring Bioscience 1:50/NA Capper et al. method
McKelvie (2013) C-PCR 71 Spring Bioscience NA/12 min Unequivocal diffuse cytoplasmic staining in > 85% of tumor cells considered positive SNaPshot used in 9 discordant cases. 8/9 VE1+/C-PCR- were confirmed positive by SNaPshot, the last case VE1+, C-PCR- and SNaPshot-
Na (2015) rt-PCR 104 Spring Bioscience 1:100/NA Intensity 0–3+; strong (3+) when staining of tumor cells was stronger than or equivalent to surrounding follicular colloid
Qiu (2015) rt-PCR 127 Ventana NA/16 min NA
Routhier (2013) SNaPshot 15 Spring Bioscience 1:100/NA Capper et al. method Study included 4 PTC mets to LNs and 4 Follicular carcinomas. These were removed from the data set
Sun (2015) rt-PCR 556 Ventana NA/NA Capper et al. method
Szymonek (2017) rt-PCR #1 137 Ventana 1:100/16 min Intensity 0–3+; Positive if staining was ≥ 25% of tumor cells
rt-PCR #2 137 Ventana 1:100/32 min Same as above
Zhang (2018) ARMS-PCR 132 Ventana NA/32 min Intensity 0–3+; weak (1+), requiring 10× or greater objective to observe staining, 2+ moderate, easy to recognize "yellow" staining w/10×. 3+ strong, “brown” with 4× Sanger sequencing used in discrepant cases
Zhao (2019) PCR 185 Ventana NA/16 min Intensity 0–3+; positive 1–3+. No or only slight staining, negative
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Statistical Analysis

The meta-analysis was performed using all extracted data and analyzed using STATA Version 13 (College Station, TX) software [38]. Rates of BRAF V600E mutation according to the immunohistochemical results were included in the meta-analysis. Forrest plots and summary receiver operating characteristic (SROC) curves were obtained. Both the direct sequencing and PCR groups were assessed for publication bias using Begg’s funnel plots (data not shown).

Results

Study Selection and Characteristic Review

Using the search criteria as described above, a total of 265 articles were identified in the database search. Initial evaluation of studies eliminated 236 articles. Twenty-nine remaining articles were then evaluated and six additional studies were excluded. Twenty-three studies were ultimately included in the meta-analysis [11–15, 17–26, 28–30, 32–35]. The characteristics of the selected 23 studies are included in Tables 1 and 2. The reason for exclusion of the six studies included the use of a non-commercially available BRAF V600E antibody clone in 4 studies [9, 16, 27, 31], one study using a non-VE1 clone [37], and one study using only core needle biopsy specimens [36]. The majority of the studies used the anti-human BRAF V600E (clone VE1) monoclonal antibody by Spring Bioscience (Spring Bioscience Pleasanton, CA, USA) [11, 13–15, 18, 21, 25, 26, 28–30, 32, 39, 40] or Ventana medical systems (Ventana Medical Systems, Roche Diagnostics Indianapolis, IN, USA) [12, 17, 19, 20, 22, 33–35]. Only one study used a BRAF V600E antibody from GBI Biotechnology (Beijing Zhongshan-GBI Biotechnology Co., Beijing, China) [24]. Of the twenty-thee included studies, a total of 3669 surgically resected FFPE PTC specimens were included in the meta-analysis. Some studies used the same specimen tested on different molecular platforms, bringing the total number of molecular tests performed to 4079 [14, 34]. The number of tissue samples tested in the various studies ranged from 12 [17] to 697 [15] samples.

Immunohistochemistry Method and Scoring

As stated above, the majority of the studies used the VE1 clone from either Spring Bioscience or Ventana, only one study used a VE1 antibody from GBI Biotechnology. However, processing of the slides varied in terms of dilution of antibody, incubation time, and temperature. Although some studies did not include a dilution factor in their method description, studies that did include a dilution factor varied from 1:4 to 1:300. Likewise, incubation times, if included in the method description, ranged from 12 to 60 min. A variety of immunohistochemical automatic staining platforms were used that included; Ventana Benchmark (Ventana Medical Systems Tucson, AZ), DAKO (DAKO, Carpinteria, CA), and Leica Bond (Leica Microsystems, Bannockburn, Il) systems for the staining of tissue sections. Method comparison of the various studies is included in Tables 1 and 2.

Immunohistochemical (IHC) staining interpretation criteria varied widely amongst the included studies, see Tables 1 and 2. Some studies used a previously described interpretation criteria developed by Capper et al [18, 24, 26, 32, 33]. This method used the following criteria: staining must be unambiguous, cytoplasmic and observed in a substantial fraction of viable tumor cells. Secondly, positivity was graded as weak, moderate, or strong based on illustrations provided in the paper (from 0–3+, respectively). Lastly, faint diffuse staining, any isolated nuclear staining, or weak staining of single cells was scored as negative [9]. However, some authors set more specific quantitative (percentage) criteria for the number of positively staining tumor cells ranging from ≥ 20% (11) to ≥ 85% (12, 29) to determine positivity. Yet other authors used a cutoff of ≥ 10% positivity of tumor cells staining, but only considered staining intensity of 2+ or greater as truly positive [13]. On the other hand, negative staining was considered by some authors to be weak or focal [13, 19, 22, 28]. Others considering weak (1+) staining as positive, but did not have a cutoff value for the number of positively staining tumor cells [15, 35]. Only one group, two separate published studies, used the Allred scoring system which combines the percentage of cells staining positive and overall intensity [14, 15]. Interpretation of IHC staining was performed by at least two pathologists for most studies (all pathologists were blinded to the molecular results in each study), whereas, one study used only one pathologist [21] and another had as many as five pathologists [27].

Results of the Meta-Analysis

Tables 1, 2, and 3 list the included studies broken down into two groups based on the gold standard molecular method used to compare VE1 IHC staining. These were termed as the following: those studies using direct sequencing (Sanger, Pyrosequencing) were termed the “direct sequencing group” and those using PCR-based methodologies (rt-PCR, PNA-Clamp, SNaPshot, etc.) were termed the “PCR group.” One study using ASLNA-qPCR (by de Biase et al. [25]), which studied 20 PTC specimens, was excluded from the PCR group as the study’s specificity was unable to be calculated given no true negatives or false positives recorded in their results. The remaining studies included in the meta-analysis showed the pooled sensitivity for IHC in detecting a BRAF V600E mutation in the direct sequencing group was 100% (95% confidence interval [CI] 0.97–1.00) and for the PCR group was 98% (95% CI 0.96–0.99) and overall combined methods 99% (95% CI 0.98–1.00). The pooled specificity of the direct sequencing group was 84% (95% CI 0.72–0.91) and for the PCR group was 89% (95% CI 0.82–0.94), and overall combined methods was 87% (95% CI 0.81–0.91) (Figs. 2, 3, 4 and 5). The values of AUC on the SROC curves for the direct sequencing group and PCR group were 0.97 and 0.98, respectively (Figs. 6 and 7). The accuracy rates for the direct sequencing and PCR groups were 91.7% and 93.4%, respectively.

Table 3.

Direct sequencing and PCR group—combined statistics

Direct sequencing group
Study Direct method Total PTCs TP TN FP FN Sensitivity Specificity Accuracy PPV NPV
Bullock (2012) Direct (Sanger) 96 59 28 9 0 100.00% 75.68% 90.63% 86.76% 100.00%
Capper (2011) Direct (Sanger)
Dvorak (2014) Direct (Sanger) 73 52 19 2 0 100.00% 90.48% 97.26% 96.30% 100.00%
Ghossein (2013) Mass spectrometry 31 15 16 0 0 100.00% 100.00% 100.00% 100.00% 100.00%
Fisher (2013) Pyro 25 10 11 4 0 100.00% 73.33% 84.00% 71.43% 100.00%
Kim (2014) Direct (Sanger) 91 56 16 12 7 88.89% 57.14% 79.12% 82.35% 69.57%
Kim (2014) Pyro Seq 91 56 14 12 9 86.15% 53.85% 76.92% 82.35% 60.87%
Kim (2018) Direct (Sanger) 697 581 70 46 0 100.00% 60.34% 93.40% 92.66% 100.00%
Koperek (2012) Direct (Sanger)
Loo (2018) Direct (Sanger) 12 5 7 0 0 100.00% 100.00% 100.00% 100.00% 100.00%
Martinuzzi (2016) Direct (Sanger) 85 54 23 8 0 100.00% 74.19% 90.59% 87.10% 100.00%
Oh (2018) Direct (Sanger) 23 16 5 2 0 100.00% 71.43% 91.30% 88.89% 100.00%
Qiu (2015) Direct (Sanger) 127 100 25 2 0 100.00% 92.59% 98.43% 98.04% 100.00%
ZagZag (2013) Direct (Sanger) 37 25 9 0 3 89.29% 100.00% 91.89% 100.00% 75.00%
Zhao (2019) Direct (Sanger) 185 140 43 1 1 99.29% 97.73% 98.92% 99.29% 97.73%
Zhu (2016) Direct (Sanger) 118 73 37 8 0 100.00% 82.22% 93.22% 90.12% 100.00%
PCR based group
Study PCR method Total PTCs TP TN FP FN Sensitivity Specificity Accuracy PPV NPV
Chen (2018) quant-rtPCR 40 34 4 2 0 100.00% 66.67% 95.00% 94.44% 100.00%
de Biase (2014) ASLNA-qPCR 20 18 0 0 2 90.00% 90.00% 100.00% 0.00%
Elmageed (2017) rt-PCR 130 97 28 2 3 97.00% 93.33% 96.15% 97.98% 90.32%
Ilie (2014) SNaPshot
Jung (2015) PCR 467 392 65 9 1 99.75% 87.84% 97.86% 97.76% 98.48%
Kim (2014) PNA clamp 91 56 17 12 6 90.32% 58.62% 80.22% 82.35% 73.91%
Kim (2014) rt-PCR 91 58 15 10 8 87.88% 60.00% 80.22% 85.29% 65.22%
Martinuzzi (2016) PNA-Clamp 85 58 21 2 4 93.55% 91.30% 92.94% 96.67% 84.00%
McKelvie (2013) C-PCR (SNaPshot) 71 49 21 1 0 100.00% 95.45% 98.59% 98.00% 100.00%
Na (2015) rt-PCR 104 71 29 4 0 100.00% 87.88% 96.15% 94.67% 100.00%
PajaFano (2017) rt-PCR
Qiu (2015) rt-PCR 127 100 25 2 0 100.00% 92.59% 98.43% 98.04% 100.00%
Routhier (2013) SNaPshot 15 9 6 0 0 100.00% 100.00% 100.00% 100.00% 100.00%
Sun (2015) rt-PCR 556 414 137 0 5 98.81% 100.00% 99.10% 100.00% 96.48%
Szymonek (2017) rt-PCR#1 137 69 46 9 13 84.15% 83.64% 83.94% 88.46% 77.97%
rt-PCR#2 137 80 49 6 2 97.56% 89.09% 94.16% 93.02% 96.08%
Zhang (2018) ARMS-PCR 132 106 21 5 0 100.00% 80.77% 96.21% 95.50% 100.00%
Zhao (2019) PCR 185 142 35 4 4 97.26% 89.74% 95.68% 97.26% 89.74%
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Fig. 2.

Fig. 2

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Forest plot—direct sequencing group—sensitivity

Fig. 3.

Fig. 3

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Forest plot—direct sequencing group—specificity

Fig. 4.

Fig. 4

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Forest plot—PCR group—sensitivity

Fig. 5.

Fig. 5

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Forest plot—PCR group—specificity

Fig. 6.

Fig. 6

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Receiver operator curve—direct sequencing group

Fig. 7.

Fig. 7

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Receiver operator curve—PCR group

To assess for publication bias, a set of Begg’s funnel plots were developed for each group (data not shown). The direct sequencing group showed relative symmetry (p = 0.071), signifying no-publication bias. However, the PCR group showed more asymmetry (p = 0.010), suggesting the existence of some publication bias.

Discussion

The first published meta-analysis review of the utility of BRAF IHC in PTCs by Pyo et al. was performed in 2015, and since then more studies have emerged comparing IHC to various gold standard methods for the detection of BRAF V600E [10]. All studies included in the aforementioned meta-analysis used the VE1 (BRAF V600E specific) antibody clone. Additionally, non-surgical resection specimens (core needle biopsies and cytology FNAs) were included and a critical review of the scoring systems was not performed. Recently, a second systematic review and meta-analysis has also been published on the topic by Singarayer et al. [41]. This meta-analysis included surgical resection and cytology specimens, and other histologic diagnoses beside PTCs [i.e., Follicular Thyroid carcinoma (FTC), Medullary Thyroid Carcinoma (MTC), Poorly Differentiated Thyroid Carcinoma (PDTC), Anaplastic Thyroid Carcinoma (ATC), ectopic thyroid tissue, and benign thyroid tissue]. Additionally, two studies included in this meta-analysis used non-VE1 clones. In order to standardize data, the current meta-analysis focused only on studies using PTCs from surgical resection specimens comparing VE1 IHC to molecular “gold standard” methods with additional evaluation of IHC methodology and scoring criteria.

Pooled sensitivity and specificity combining the direct sequencing and PCR groups were 99% (95% CI 0.98–1.00) and 84% (95% CI 0.72–0.91) respectively. However, there were slightly more false positives amongst the direct sequencing group (6.3%) compared to the PCR group (2.8%). This difference may partially be explained by the need for a sufficient quantity of tumor cells and high-quality DNA to perform successful direct sequencing assays. Conversely, rt-PCR based assays such as the Cobas 4800 BRAF Mutation Test (Roche Diagnostics, Indianapolis, IN), which has been approved by the United States Food and Drug Administration for the in-vitro detection in patients with papillary thyroid carcinoma, can detect BRAF V600E mutations at > 5% allele frequency with 100% accuracy [42]. However, molecular methods are often more labor intensive and costly, making immunohistochemistry a more attractive method for the detection of BRAF V600E mutation.

Immunohistochemistry scoring criteria varied amongst the studies. The most consistently used criteria described by Capper et al., details described above, was specifically used in four separate studies [18, 24, 26, 32, 33]. A scoring system similar to the one describe by Capper et al., was used in the majority of studies: a numerical 4-tier intensity score, ranging from 0 (no staining) to 3+ (strong), details included in Tables 1 and 2. However, various other studies used a quantified number (percentage) of positively staining tumor cells as a cut-off value to determine positivity. For example, Bullock et al. required > 20% of positively staining tumor cells, Zagzag et al. required > 80%, and McKelvie et al. used a cut off of > 85% of tumor cell staining to determine positivity. When assessed for poor performance characteristics, assuming staining methods as equal across all studies, one study performed lower than the overall cohort in both specificity and accuracy in both the direct sequencing and PCR groups. The study performed by Kim et al. 2014, compared VE1 IHC to four different molecular methods (Sanger, Pyrosequencing, PNA clamp and rt-PCR) [14]. Assessment of the supplemental material showed that all four methods had relatively low specificity (Direct 57.1%, Pyro 54.0%, PNA-clamp 59.0%, and rt-PCR 60.0%). Calculated kappa values between the molecular methods were quite poor ranging from 0.53 to 0.79 (all P values < 0.001). When all molecular tests were negative for a single specimen (15 of 91 specimens), only two specimens had positive VE1 IHC staining. Interestingly, this study used the Allred scoring system, using a cutoff of < 4 as negative and ≥ 6 as positive. This Allred scoring system, first developed for the interpretation of estrogen receptor (ER) immunohistochemical stain on breast tissue, combines a 5-tier percentage cutoff for the amount positively staining tumor cells with a 1–3+ intensity score [no staining = 0, < 1% = 1, 1–10% = 2, 10–33% = 3, 33–66% = 4, > 66% = 5; no staining = 0, weak = 1, moderate = 2, strong = 3] [14, 43]. In this study, Allred score of 5 [weak (1+) expression in 33–66% of cells (score 4)] was not predictive of BRAF V600E mutation, as 7 cases were mutated by at least one molecular method and 4 were negative for all molecular methods. Whether the low accuracy in this study is specific to the Allred scoring system or other factors related to molecular methods is uncertain. However, the Allred method was not used in other studies, all of which had higher specificities. Therefore, this method may not be ideal for interpretation of VE1. In the authors’ unpublished experience, positive VE1 staining is almost always diffuse with equal intensity expression in all tumor cells. Comparatively, when assessing the Martinuzzi et al. study, which used the Capper et al. interpretation criteria, only two false positive IHC results were obtained by PCR method showing 2+ and 3+ positivity (2 of 85 specimen, wild-type for both molecular methods) [18]. It is worth noting that both Kim et al. [14] and Martinuzzi et al. [18] studies provided accessible supplementary data that included the results of all molecular studies with immunohistochemical interpretation and score for each individual tested sample. Further studies assessing scoring methods may be helpful, and individual results of the molecular tests in comparison to IHC scores should be provided in the supplementary material to allow for future meta-analysis assessment.

Additional factors such as type of tissue, length and type of fixation are also important to consider when validating immunohistochemical antibodies. The study by Dvorak et al. 2014, included comparative data assessing the effects of time to fixation, length of fixation and type of fixative used on xenograft (A2058 and LS411N xenografts) tissue. They concluded that a cold ischemic time of > 6 h (ideal conditions would be < 2 h) followed by fixation in 10% neutral buffered formalin (NBF) produced membranous and patchy staining in the xenograft tissue (A2058 and LS411N, respectively). Fixation for less than 12 h in 10% NBF produced weaker VE1 staining compared to xenograft tissue that was fixed for 12–72 h. Lastly, fixatives other than 10% NBF, such as 95% ethanol, Prefer, AFA, and Z-5 were recommended not to be used due to poor quality in staining regardless of fixation time [12]. Additional guidance provided by this paper suggested validating each mutation-specific immunohistochemical marker to a specific tumor type. Similar insight was provided by Szymonek et al. and Zhang et al. [34, 35]. Both studies evaluated the performance characteristics when simply doubling the immunohistochemical incubation time and performing the study in duplicate. Szymonek et al. showed improved sensitivity and slightly better specificity by doubling the incubation time from 16 to 32 min (sensitivity 84% to 98%, specificity 84% to 89%) [34]. Zhang et al. 2018 evaluated select PTC specimens positive for BRAF V600E by molecular analysis and concluded that VE1 staining intensity for PTC specimens was improved by doubling the incubation time from 32 to 64 min, thus reducing the number of falsely negative staining PTCs. This study also looked at VE1 staining in colorectal carcinomas (CRC) and concluded that optimal incubation times for VE1 antibody in PTC and CRC differed, CRC best at 32 min and PTC best at 64 min [44]. Similar to Dvorak et al., these findings suggest VE1 antibody validation may be tumor specific and the selection of control tissue used to compare patient specimens may not be as simple as comparing “apples to apples.” Thus, additional consideration for future studies or internal laboratory validation would include the effect of incubation times on sensitivity and specificity.

Although no specific methodology for scoring has been established for the interpretation of VE1 IHC, it is worth mentioning that reproducibility in the form of standardization in IHC protocol may be warranted. Clinical trials are underway using BRAF-inhibitors, vemurafenib and dabrafenib, for patients with BRAF V600E mutated PTCs. It is likely that future clinical management of such patients will depend on their tumors BRAF V600E mutational status. Thus, if BRAF V600E (VE1 clone) IHC is used for mutational screening of PTC, it must be reproducible (good interobserver variability), reliable, and accurate for routine practice. As an ideal screening test, high sensitivity would be desired. The results of this review show the VE1 antibody clone to be highly sensitive and specific for the detection of BRAF V600E mutation in PTC. The findings of this meta-analysis are consistent with previous reports by Pyo et al. [10] and Singarayer et al. [41]. In our opinion as well as stated by Dvorak et al., staining should be considered positive when cytoplasmic, uniform (of any intensity), and diffuse throughout the tumor (Fig. 8).

Fig. 8.

Fig. 8

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Papillary thyroid carcinoma, classic type (a) showing diffuse cytoplasmic VE1 expression with moderate intensity (b); Papillary thyroid carcinoma, invasive follicular variant (c) showing negative VE1 immunostain (d). Evaluation of VE1 immunostain in papillary thyroid carcinoma at low power (e) may be deceptive. Higher power examination confirms diffuse but weak VE1 positivity (f) in a case with confirmed BRAF V600E mutation by next generation sequencing (unpublished data). These examples utilized prediluted Roche Ventana VE1 monoclonal antibody (Indianapolis, IN, USA) on the Leica Bond Refine System (Buffalo Grove, IL, USA) at 60 min incubation time

In addition to the BRAF V600E mutation in papillary thyroid carcinoma another actionable genetic target, NTRK, is currently being investigated in thyroid carcinomas. Constitutive activation or overexpression of the NTRK1, NTRK2, and NTRK3 transmembrane tyrosine kinases has been implicated in oncogenesis [45]. Constitutive activation or overexpression of these Trk receptors have been implicated in oncogenesis [45]. NTRK fusions are more commonly seen in pediatric PTC (up to 26%) compared to adult [46]. Larotrectinib, a selective TRK inhibitor, was approved by the FDA in November 2018 for the treatment of TRK fusion-positive advanced solid tumors [47]. Recently a pan-Trk monoclonal antibody (Abcam, Cambridge, MA) has been developed. In two studies, pan-Trk was positive 2/4 and 9/11 NTRK-rearranged thyroid carcinomas (sensitivities 50% and 81.8%, respectively), with reported pan-tumor-type specificities ranging from 81.1 to 100% [45, 48, 49]. Additional larger studies to assess the reliability of pan-Trk IHC for the detection of NTRK fusion in thyroid carcinoma are warranted.

Although mutation-specific IHC may not be performed in routine pathology practices as of yet, detection of actionable genetic alterations such as BRAF V600E mutation will likely become incorporated into treatment algorithms for recurrent/metastatic PTC. Recommendations based on this meta-analysis and personal observation include: Fixation should only be performed using 10% neutral buffered formalin, with minimal cold ischemic time (preferably less than 2 h), and at least 12 h of total fixation time (no longer than 72 h). Incubation time for the VE1 antibody should be at least 32 min (up to 64 min) for optimal results. Staining should be considered positive when cytoplasmic, uniform (of any intensity), and diffuse throughout the tumor with ≥ 90% of tumor cells staining (Fig. 8). Isolated nuclear staining or single cell staining should be interpreted as negative. Patchy or inconsistent staining should be repeated and may represent fixation artifact. In summary, VE1 IHC is a rapid, cost effective, sensitive, and specific method for the detection of BRAF V600E mutation and can be used as a reliable screening test. However, individual validation studies should be performed with institutional molecular methods and tissue-specific controls for the tumor of interest, for use in routine clinical practice.

Acknowledgements

The authors would like to give special thanks to Dr. Girish Venkataraman (University of Chicago, Director of the Immunohistochemistry Lab in the Department of Pathology, Section of Hematopathology) for his guidance regarding BRAF immunohistochemical techniques.

Funding

No funding was received for this study.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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