Diagnostic Value of Immunohistochemistry for the Detection of the BRAF V600E Mutation in Papillary Thyroid Carcinoma: Comparative Analysis with Three DNA-Based Assays

Abstract

Background:

The aim of this study was to compare the detection of BRAF^V600E by immunohistochemistry (IHC) using a mutation-specific antibody with molecular biology methods for evaluation of papillary thyroid carcinoma (PTC) patients.

Patients and methods:

This study concerned 198 consecutive conventional PTC patients, of which the majority were women (133/198; 67%), with a mean age of 56 years (range 19–79 years). BRAF mutation analysis was performed using DNA-based (direct sequencing, pyrosequencing, and SNaPshot) and IHC (VE1 antibody) methods. The sensitivity and specificity of IHC for BRAF^V600E was compared with the molecular biology data.

Results:

A BRAF mutational result was obtained in 194 cases. A BRAF^V600E mutation was detected in 153/194 (79%) cases of PTC when using at least one molecular method, and in 151/194 (78%) cases with IHC. No false positive results were noted using IHC to detect the BRAF^V600E mutation. Besides this mutation, other rare BRAF mutations (BRAF^V600K and BRAF^K601E ), used as negative controls, were consistently negative with IHC. The sensitivity and specificity of IHC for the detection of this mutation were 98.7% and 100% respectively. The IHC test demonstrated excellent performance at a level equivalent to two DNA-based counterparts (pyrosequencing and SNaPshot). Failure to achieve a result was more frequent with the direct sequencing method than with the three other methods.

Conclusion:

IHC using the VE1 antibody is a specific and sensitive method for the detection of the BRAF^V600E mutation in PTC. IHC may be an alternative to molecular biology approaches for the routine detection of this mutation in PTC patients.

Introduction

Among the genomic alterations that occur in papillary thyroid carcinoma (PTC), the BRAF^V600E mutation is detected in 45–70% patients (1 –5). Currently, the detection of this mutation is of strong interest in daily clinical practice and is well established for different reasons. First, the presence of this mutation can be helpful to confirm specifically or make a diagnosis of PTC using fine-needle aspiration (FNA) biopsies (2,6,7). Many studies have shown an association of the BRAF^V600E mutation with aggressive clinicopathologic characteristics of PTC, including extrathyroidal extension, lymph node metastasis, advanced stage (III/IV), loss of radioiodine avidity, and, hence, resistance to radioactive iodine therapy and higher risk of tumor recurrence (2,8,9). Even if most of the PTC cases have an indolent clinical course and a very good prognosis, distant metastases can occur in some patients in association with a worse prognosis (10,11). Finally, the presence of the BRAF^V600E mutation is significantly associated with increased cancer-related mortality among patients with PTC, as recently reported (12). In this context, it is noteworthy that a BRAF^V600E mutation can be druggable using BRAF inhibitor molecules in some selected patients with an aggressive PTC or an anaplastic thyroid carcinoma (13 –15).

DNA-based assays are the standard methods for the detection of BRAF mutations in thyroid carcinomas (13). In routine clinical settings, these assays mostly include direct sequencing, pyrosequencing, SNaPshot, or Taqman technologies, although other more sensitive approaches are under investigation (13). More recently, immunohistochemistry (IHC) using a BRAF^V600E specific antibody has been used for mutation detection in several types of neoplasias, including thyroid carcinomas (16 –23). However, assessment of the specificity and the sensitivity of this antibody in comparison to well-established molecular biology methods in a large cohort of PTC patients has not been performed to date.

The purpose of this study was to evaluate the value of an IHC approach for the detection of the BRAF^V600E mutation in a consecutive series of 198 conventional PTC patients, and to determine the sensitivity and the specificity of the results in comparison to those obtained with three molecular biology methods (direct sequencing, pyrosequencing, and SNaPshot).

Patients and Methods

Patients and tissue samples

A total of 198 patients with PTC were enrolled in the study. Patients were surgically treated in the Department of Otorhinolaryngology (Pasteur Hospital, Nice, France) between 2004 and 2012. Written informed consent was obtained from all patients. Specimens of primary thyroid tumors were fixed (time of fixation, range 6–24 hours) in 10% formaldehyde for histological analysis and IHC. Adjacent tumor specimens were flash-frozen in liquid nitrogen and then stored at −80°C in the Human Tissue Biobank (Pasteur Hospital). A tissue section of the frozen specimen was systematically made and stained with hematoxylin and eosin (H&E) for histological control and for assessment of the percentage of tumor cells before DNA extraction. This study exclusively concerned the conventional PTC histological subtype including 160 macrocarcinomas and 38 microcarcinomas. A tumor was classified as conventional PTC if it showed >1% papillary formations and was composed of cells having the characteristic nuclear features of papillary carcinoma (24). All cases <10 mm in diameter were regarded as microcarcinoma. The epidemiological, clinical, and pathological features of this population are summarized in Table 1.

Table 1.

Clinicopathologic Features of the 198 Patients with Papillary Thyroid Carcinoma

Variables	n (%)
Age (mean)	19–82 (47)
<45 years	81 (41)
≥45 years	117 (59)
Sex
Male	49 (25)
Female	149 (75)
pT stage
T1	73 (37)
T2	77 (39)
T3	37 (19)
T4	11 (5)
pN stage
N0	118 (60)
N1a	62 (31)
N1b	18 (9)
Extrathyroid extension
Present	47 (24%)
Absent	151 (76%)
Encapsulation
Absent	135 (68)
Present	63 (32)
Thyroiditis
Absent	118 (60)
Present	80 (40)

BRAF mutation analyses

Genomic DNA from frozen tissue samples was extracted using the MagNA Pure Compact Nucleic Acid Isolation Kit Large Volume (Roche Group, Inc., Tucson, AR) according to the manufacturer's instructions. To make sure that the extractions were successful and contained DNA, polymerase chain reaction (PCR) control reactions were performed using primers designed to amplify the human GAPDH gene.

We analyzed mutations in exon 15 of the BRAF gene by direct sequencing, pyrosequencing, and SNaPshot, as previously described (Supplementary Data, available online at www.liebert.com/thy) (25 –27). Genomic DNA extracted from A375 cells (expressing BRAF^V600E mutation) was used as a positive control.

Preparation of tissue microarrays

H&E-stained sections of primary tumors were reviewed by five pathologists (M.I.I., S.L., E.L.-M., V.H., and P.H.), and tumor areas were marked on the slides. Representative carcinoma areas were selected for building tissue microarrays (TMAs), and arrays were designed as previously described (28). Three cores were selected for macrocarcinomas and two cores for microcarcinomas. In addition, five primary BRAF^V600E -mutated melanomas (six tissue cores from biopsies performed on these patients) served as positive controls and were used for regulating mark spacing between core centers; cores were spaced at intervals of 1 mm in the x- and y-axes. Tissue cores of five primary BRAF^V600K -mutated melanomas, five primary non-BRAF-mutated melanomas, and one BRAF^K601E -mutated follicular variant of PTC served as negative controls. A 4 μm H&E-stained section was reviewed to confirm the presence of each tumor sample. A histospot was considered unsuitable for analysis if it was completely absent, contained no tumor tissue (sampling error), or contained too few tumor cells (<10%) for analysis (uninformative). For each patient, the mean score of a minimum two core biopsies was calculated.

Assessment of BRAF^V600E mutation by IHC

The generation and validation of the BRAF^V600E mutation-specific antibody (clone VE1) have been reported previously (20). IHC with the VE1 antibody was performed as described earlier (22,29). Briefly, 4 μm TMA sections were freshly cut and dried at 80°C for 15 minutes. VE1 IHC (1:10 diluted VE1 hybridoma supernatant) was performed on an automated immunostainer (Ventana BenchMark XT, Ventana Medical Systems, Tucson, AR) using standard reagents provided by Ventana and the OptiView DAB IHC Detection Kit (Ventana).

The intensity and cellular localization of the VE1 staining were independently analyzed by five pathologists (M.I., S.L., E.L., V.H., and P.H.) who were blinded to the molecular BRAF mutation data. Discrepancies were resolved using a multihead microscope. VE1 staining was scored based on signal intensity (0–3) and proportion (0–100%). Positively stained tumors were then stratified into three intensity categories: 1+, 2+, and 3+ (Supplementary Fig. S1). For statistics, histospots containing <10% of tumor cells were excluded from additional analyses. In parallel, in order to evaluate the topography and the intensity of the immunostaining of the positive-stained tumors observed on the TMAs, we selected 35 positive VE1 PTCs and performed IHC on whole paraffin sections using the same IHC protocol.

Statistical analysis

All cases were analyzed with the four different methods. To determine the specificity and sensitivity of each method, we compared the results of each method for each specimen, and when there was discordance, we used the consensus mutation to calculate each parameter as a reference test, as described (30). A consensus mutation was defined as that detected by at least one molecular method. The receiver operating characteristic (ROC) curve was used to estimate the cutoff value for VE1 immunostaining. A Kappa (κ) analysis was performed to evaluate the agreement between pyrosequencing, SNaPshot, and direct sequencing, and the consensus mutation, and to compare IHC data from whole-tissue sections and TMA histospots. Kaplan–Meier survival analysis and the log-rank test were used to evaluate the effect of BRAF^V600E mutation on PTC recurrence. Recurrence was defined as the reappearance of disease after complete ablation of thyroid remnants as confirmed by cytological and/or histopathological examination or by the presence of definite ¹³¹I uptake outside of the thyroid bed on post-treatment whole body scan after administration of 5.55 GBq ¹³¹I (31).

Results

The DNA extracted from the 198 frozen samples was screened for a BRAF^V600E mutation using pyrosequencing, SNaPshot, and direct sequencing methods. The diagnostic accuracy of each test in terms of sensitivity and specificity is presented in Table 2. One hundred ninety-four samples had a mutational result with at least one DNA-based analysis. A BRAF^V600E mutation was found in 153/194 cases (79%) with pyrosequencing, in 147/194 cases (76%) with SNaPshot, and in 142/194 cases (73%) with direct sequencing (Fig. 1). No BRAF mutation other than V600E was found in PTC cases. By using these three methods, the genotype results were concordant in 183/194 cases (94%). Finally, a BRAF^V600E mutation was detected in 79% (153/194) of PTC cases when using at least one DNA-based method.

FIG. 1.

Representative BRAF^V600E analyses of a mutated papillary thyroid carcinoma (PTC) by direct sequencing (A), pyrosequencing (B), and SNaPshot (C). (A) Chromatogram of exon 15 of the BRAF gene sequenced with a forward primer (top) and a reverse primer (bottom). Arrows: the transversion from T to A detected in the sense and A to T in the antisense direction. (B) Pyrogram with the differential peak heights of T and A (blue rectangle) that show the percentage of each nucleotide at position 1799. (C) SNaPshot for the BRAF genotyping assay. (i) Sense pattern for BRAF c.1799T> A mutated DNA. Two peaks were detected at the BRAF c.1799 position: one corresponds to the T wild-type nucleotide (red peak) and the other to the mutated A nucleotide (green peak). (ii) Antisense pattern. The red peak corresponds to the T mutated antisense nucleotide and the green peak to the A antisense wild-type nucleotide. Color images available online at www.liebertpub.com/thy

Table 2.

Performance for the Detection of the BRAF Mutations with Immunohistochemistry and Molecular Biology Methods

		V600		V600E
		(Among 153 cases for whom the 4 methods were conclusive)
Method	Number (%) of test failures (out of 198 cases)	Sensitivity, %	Specificity, %	Sensitivity, %	Specificity, %
Sanger sequencing	19 (9.6)	93	100	91.5	100
Pyrosequencing	4 (2)	100	100	100	100
SNaPshot	4 (2)	96	100	97.5	91.2
IHC	5 (2.5)	83	100	98.7	100

The detection of other rare BRAF mutations with the DNA-based techniques determines lower sensitivity for IHC among the 198 analyzed patients.

IHC, immunohistochemistry.

According to the ROC curve (AUC=0.993; Supplementary Fig. S2), a tumor was considered positive for the BRAF^V600E mutation as determined by the VE1 immunostaining when a distinct, strong, and homogenous signal (score 3+) was observed in the cytoplasm of all carcinoma cells (Fig. 2). Positive immunolabeling for the BRAF^V600E mutation detected by IHC on TMAs was noted in 78% (151/194) of all cases with molecular analysis (Fig. 2). The BRAF^V600E expression levels observed on the TMA spots faithfully reflected the staining intensity of the protein on whole-tissue sections from corresponding tumor blocks (κ=0.92), with no variation between TMA cores (Supplementary Fig. S1). All five observers who interpreted the IHC both on TMA spots and whole-tissue sections showed perfect agreement (κ=0.99). No variation in VE1 immunostaining was observed between the leading/invasive edge versus the center of the tumor or with the allele peak height on DNA-based assays. No statistical difference in the frequency of V600E BRAF protein expression was seen between macrocarcinomas and microcarcinomas with positive molecular analysis (124/158 vs. 27/36 respectively; p=0.889, Fisher's exact test). We also observed six wild-type tumor samples that stained weakly or moderately (scores 1+ or 2+) on IHC (Supplementary Fig. S1), whereas none of the wild-type cases detected by a molecular method showed positive immunostaining (Fig. 2). None of the control tissue samples (including melanoma and follicular variant of PTC) having a BRAF^V600K or a BRAF^K601E mutation were positively immunostained (not shown), whereas melanoma samples harboring a BRAF^V600E mutation, used as positive controls, were strongly immunostained (Fig. 2). The comparison of the four methods showed that failure to achieve a result was more frequent with direct sequencing than with the three other methods, including IHC.

FIG. 2.

(A–C) Conventional PTC with BRAF^V600E mutation. (A) 3+ diffuse homogenous immunostaining with the VE1 antibody (immunoperoxidase, original magnification 200×); (B) strong cytoplasmic immunostaining with VE1 (immunoperoxidase, original magnification 1000×); (C) corresponding hematoxylin and eosin (H&E; original magnification 100×). (D, E) Conventional PTC negative for the BRAF^V600E mutation. (D) Absent immunostaining with the VE1 antibody (immunoperoxidase, original magnification 400×); (E) corresponding H&E (original magnification 100×). (F) Immunohistochemistry of a melanoma harboring the BRAF^V600E mutation (immunoperoxidase, original magnification 200×).

Furthermore, we explored the relationship between the BRAF mutational status and the outcome of PTC patients. Patients were followed up for a median of 4.5 years (range 0.8–8.6 years) after thyroidectomy. Follow-up data were available for 178 patients, and all had a positive BRAF genotype. During this time, 12 (7%) patients showed locoregional recurrence. Among the 12 specimens (primary T2 stage, n=3; T3 stage, n=5; and T4 stage, n=4) with locoregional recurrence, 8 (67%) harbored the BRAF^V600E mutation. However, the presence of the BRAF^V600E mutation was not associated with tumor recurrence, as demonstrated by the univariate Kaplan–Meier curve analysis (Supplementary Fig. 3, p=0.165).

Discussion

This study, performed on a large series of 198 conventional PTC patients, shows that IHC using the VE1 clone was a specific and sensitive method to detect the BRAF^V600E mutation. Several studies have previously evaluated the performance of the VE1 antibody in PTC cases (Table 3) (19,20,32 –35). However, an important limitation in the majority of these studies was the use of direct sequencing as the standard reference for BRAF genotyping. Such settings were bound to yield a relatively high number of false positive results with VE1 when compared to direct sequencing. Thus, in our study, we avoided a low quality gold standard method (i.e., direct sequencing) to assess the performance of the VE1 antibody by taking advantage of the comparison with highly sensitive DNA-based methods. The comparative analysis of the IHC approach with molecular biology methods for BRAF^V600E detection demonstrated that IHC performed very well and at a level equivalent to the pyrosequencing and SNaPshot methods, but was more sensitive than the direct sequencing method. Moreover, the use of an IHC approach on TMA allowed us to perform an analysis of a large number of PTC cases. Although one recently published study employed the same approach using TMA, the immunostaining was first validated on whole sections with high agreement for immunostaining between TMA cores and the corresponding whole sections. One limitation related to the design of our study is the lack of PTC variants other than conventional type that have a lower prevalence of BRAF mutations such as follicular variant (3,33,35). This is likely to inflate estimates of the VE1 antibody sensitivity and specificity in low-prevalence subtypes. However, several studies have previously reported a strong relationship between VE1 immunostaining and the subtypes of PTC or other types of thyroid carcinoma (33,35).

Table 3.

Previously Published Work on the Study of VE1 Immunohistochemistry in Papillary Thyroid Carcinoma

Study (ref.)	Number of cases	Histology	Antibody clone	Scoring scheme	IHC cutoff	DNA-based techniques	Type of samples
Bullock et al. 2012 (19)	101	PTC NOS	VE1 hybridoma supernatant	Positive or negative	Not defined	Direct sequencing	FFPE
Capper et al. 2012 (20)	6	PTC NOS	VE1 hybridoma supernatant	Nonambiguous cytoplasmic staining on viable tumor cells	Not defined	Direct sequencing	FFPE
Capper et al. 2011 (31)	21	PTC NOS	VE1 hybridoma supernatant	Weak, moderate, strong intensity	Not defined	Direct sequencing	FFPE
Koperek et al. 2012 (32)	144	PTC (conventional, follicular, diffuse sclerosing, solid, oncocytic and tall cell type variants)	VE1 hybridoma supernatant	Distinct, homogenous immunoreaction in the cytoplasm of all cancer cells	No quantitative cutoff	Direct sequencing	FFPE
Ghossein et al. 2013 (34)	91	PTC (conventional, tall cell, oncocytic cell variants) ; poorly differentiated thyroid carcinoma; anaplastic thyroid carcinoma	Commercialized VE1 (Springer-Bio)	Staining intensity score, 0 (no staining), 1+ (faint), 2+ (moderate), 3+ (strong)	Staining intensity of 2+ or 3+ irrespective of the number of tumor cells stained	Mass spectrometry Sequenom-based genotyping assay	FFPE
Zagzag et al. 2013 (33)	34	Conventional PTC	Commercialized VE1 (Springer-Bio)	Staining intensity score (0−3), and proportion (0−100%) scale	Score 3+ in ≥80% of tumor cells	Direct sequencing	FFPE
Present study	198	Conventional PTC (micro/macrocarcinoma)	VE1 hybridoma supernatant	Staining intensity score, 0 (no staining), 1+ (weak), 2+ (moderate), 3+ (strong)	Score 3+ in all tumor cells (determined by the ROC curve)	Direct sequencing, pyrosequencing, SNaPshot	FFPE

FFPE, formalin-fixed paraffin embedded; NOS, not otherwise specified; PTC, papillary thyroid carcinoma.

Some solid tumors harboring other rarer BRAF mutations (i.e., the BRAF^K601E and the BRAF^V600K mutations) were consistently negative using the VE1 clone, as were the tumors without the BRAF^V600E mutation (36 –38). With regard to the different clinical significance of these latter mutations, it is important to note that the VE1 antibody will not detect BRAF mutations other than V600E (39).

Interestingly, the positive (score 3+) immunostained tumors showed a diffuse and strong staining occurring throughout the tumor area. Moreover, we found no correlation between the expression of the BRAF^V600E -mutated protein and the allele peak height on DNA-based assays, suggesting an “on-off” method of detection not suitable for quantitative purposes. Moreover, we also observed six cases that stained weakly or moderately (scores 1+ or 2+) on IHC, but these tumors had a wild-type BRAF genotype. As previously suggested, ambiguous, focal, or moderate immunostaining may cause uncertainty in the diagnostic setting (33,35). Therefore, the staining score should be validated in house. In such cases, additional genetic analysis may be required to clarify the BRAF status (24,35). Finally, no variation in staining intensity was observed between the leading/invasive edge versus the center of the tumor, as previously reported in PTC harboring a BRAF^V600E mutation (32).

Although our results concerning the correlation between the BRAF status and patient outcomes were not consistent given the limited follow-up time, BRAF mutations were present in a higher proportion of recurrent tumors. Although several studies have reported that the presence of a BRAF mutation in PTC may be associated with poor clinicopathologic features, radioiodine resistance, and a higher risk for disease recurrence and shorter survival, the real impact of the BRAF mutation on the outcome of PTC patients still remains a controversial issue (9,12,40,41).

The advent of molecular therapeutics targeting some genomic alterations in solid tumors requires detection of gene mutations and rearrangements in tissues and cells. In this regard, the management of biological resources by the surgical pathology laboratories must be optimal both for the pre-analytical and analytical phases. IHC using the anti-BRAF^V600E antibody can be potentially associated with false negative results in the case of a weak control of the pre-analytical steps with hypo- or hyperfixation of the biological specimen in formaldehyde.

In a few patients with thyroid cancer, the detection of the BRAF^V600E mutation leads to the administration of specific inhibitor molecules targeting this mutation. In clinical daily practice, the detection of this mutation is performed by molecular biology technologies from extracted DNA obtained from thyroid FNA or from formalin-fixed paraffin-embedded tissue sections and more rarely from frozen specimens. In this context, the detection of a BRAF mutation is mostly performed in a molecular biology laboratory and not in the surgical pathology laboratory. However, surgical pathologists can take advantage of a growing list of available mutation-specific antibodies, including the VE1 BRAF^V600E -specific antibody, to use IHC as an efficient screening tool for the detection of druggable genomic alterations, and thus to enhance the competitiveness relative to the molecular biology approaches (42).

The strengths and the advantages of detection of the BRAF^V600E mutation using IHC can be commented on briefly. One of the main interests is to obtain simultaneously the BRAF^V600E status and the morphological features corresponding to the mutated cells and to ensure that the presence or the absence of this mutation is strictly linked to the carcinoma cells. The fact that detection of the mutation can be done by the observation of a tissue section also reduces the possibility of sampling errors and thus increases traceability. False negative results can be obtained according to the sensitivity of the molecular biology method (such as with the direct sequencing method) and according to the percentage of mutated tumoral cells in the specimen submitted to DNA extraction. A false positive result obtained with the molecular biology methods is mainly due to contamination between different samples. Using IHC, this false positive result should be considerably limited and eliminated by using a negative immunostaining control simultaneously. Moreover, only an intense and diffuse cytoplasmic immunostaining in carcinoma cells (score=3), without nuclear staining should be considered to give a positive result. IHC gives a quicker result than other molecular biology technologies and thus reduces the turnaround time between the physician's request and the result. This would be of particular interest in urgent cases that require targeted therapy against an aggressive thyroid carcinoma harboring the BRAF^V600E mutation (14). Moreover, one potential advantage of VE1 IHC over DNA-based assays is the possibility of testing decalcified specimens.

Still, the question remains about the clinical scenario in which BRAF testing by IHC would be of clinical benefit. The preoperative identification of BRAF positivity on FNA may guide initial surgical management (43). For instance, some authors have proposed BRAF testing of suspicious thyroid nodules ≥1 cm and lymph nodes, and a total thyroidectomy as the initial surgical approach for a BRAF-positive tumor (43,44). With regard to cytology analyses that do not providing a definitive diagnosis, such as those in the atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) category, this may avoid repeating FNAs and eliminate the need for two-step surgery, that is, diagnostic lobectomy followed by completion thyroidectomy (44). The role of BRAF testing in PTC that is already metastatic to the lymph nodes has not been clearly defined. Moreover, in current clinical practice, the performance of central neck dissection in patients with PTC varies. A recent study suggested that once selected, this procedure may be needed to a greater extent to reduce disease recurrence. According to the authors, this extent may be defined by the combined use of preoperative BRAF mutation testing with conventional risk factors of PTC (45).

Finally, cost should be taken into consideration, since an IHC method is more cost-effective than other molecular biology methods. For instance, in our institution, the total cost ratio between IHC and a molecular biology technique is in the order of 1:3.4. Taken together, these points highlight that the IHC approach can be a good alternative to the molecular biology approach for BRAF^V600E detection in PTC patients. However, this approach has to be done in surgical pathology laboratories with dedicated expertise and quality-control procedures established for accredited laboratories.

Interestingly, this study demonstrates that the different BRAF^V600E -mutated PTC samples were diffusively and strongly immunostained with the VE1 antibody in all the carcinoma cells. Both whole sections and TMA sections of the mutated tumors were strongly immunostained in their carcinoma cell components. Previous studies have discussed the fact that BRAF^V600E could or could not be a clonal event in PTC (46,47). Even if the data obtained in the present study need to be confirmed in a larger series, the IHC results underline that the studied PTC homogenously expressed the BRAF^V600E mutation. These findings confirm previous reports demonstrating the concordance in BRAF^V600E mutation status between different metastatic sites within the same patient using genotyping (40). Therefore, BRAF^V600E tumor homogeneity rather than heterogeneity may have important therapeutic implications (48).

The present work was performed in one laboratory and must be validated in other surgical pathology laboratories and in other large cohorts of PTC patients in order to demonstrate high reproducibility of the results. The interobserver analysis made by the pathologists in this study showed perfect concordance. This is in accordance with a recent multicenter study performed with the VE1 antibody but used on melanoma specimens, which showed a high concordance of the results of a blinded analysis by different surgical pathologists from different institutions (49).

In conclusion, we show that IHC is a valuable tool to detect the BRAF^V600E -mutated protein in PTC patients. Whilst DNA-based assays are still the standard method for detection of the BRAF^V600E mutation in thyroid carcinomas, they require specialized equipment. We believe that IHC is a promising screening modality showing excellent sensitivity and specificity for the detection of BRAF^V600E -mutated PTC. In this context, IHC should be considered as a valid alternative to the molecular biology methods for the detection of this mutation, particularly as it is able to provide a quick result in daily clinical practice.

Footnotes

Author Disclosure Statement

The authors declare no conflict of interest.

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Diagnostic Value of Immunohistochemistry for the Detection of the BRAF V600E Mutation in Papillary Thyroid Carcinoma: Comparative Analysis with Three DNA-Based Assays

Abstract

Background:

Patients and methods:

Results:

Conclusion:

Introduction

Patients and Methods

Patients and tissue samples

BRAF mutation analyses

Preparation of tissue microarrays

Assessment of BRAFV600E mutation by IHC

Statistical analysis

Results

Discussion

Footnotes

Author Disclosure Statement

References

Assessment of BRAF^V600E mutation by IHC