Assessment of BRAF V600E Status in Colorectal Carcinoma: Tissue-Specific Discordances between Immunohistochemistry and Sequencing.

Although sequencing provides the gold standard for identifying colorectal carcinoma with BRAF V600E mutation, immunohistochemistry (IHC) with the recently developed mouse monoclonal antibody VE1 for BRAF V600E protein has shown promise as a more widely available and rapid method. However, we identified anecdotal discordance between VE1 IHC and sequencing results and therefore analyzed VE1 staining by two different IHC methods (Leica Bond and Ventana BenchMark) in whole tissue sections from 480 colorectal carcinomas (323 BRAF wild-type, 142 BRAF V600E mutation, and 15 BRAF non-V600E mutation). We also compared the results with melanomas and papillary thyroid carcinomas (PTC). With the Bond method, among 142 BRAF V600E-mutated colorectal carcinomas, 77 (54%) had diffuse VE1 staining and 48 (33%) had heterogeneous staining, but 17 (12%) were negative. Among 323 BRAF wild-type colorectal carcinomas, 196 (61%) were negative, but 127 (39%) had staining, including 7 with diffuse staining. When positivity was defined as staining in ≥ 20% of tumor cells, VE1 IHC had sensitivity of 75% and specificity of 93% for BRAF V600E mutation. With the Ventana method, among 57 BRAF V600E-mutated colorectal carcinomas, 36 (63%) had diffuse VE1 staining, whereas 6 (11%) had no or weak (<20% of tumor cells) staining. Among 33 BRAF wild-type colorectal carcinomas, 16 (48%) had no or weak staining, whereas 15 (45%) had heterogeneous staining. In contrast with colorectal carcinoma, Bond and Ventana VE1 IHC in melanoma and PTC were highly concordant with sequencing results. We conclude that VE1 IHC produces suboptimal results in colorectal carcinoma and should not be used to guide patient management.

Estrella JS ，Tetzlaff MT ，Bassett RL Jr ，Patel KP ，Williams MD ，Curry JL ，Rashid A ，Hamilton SR ，Broaddus RR ... -  《-》

Immunohistochemistry is a feasible method to screen BRAF V600E mutation in colorectal and papillary thyroid carcinoma.

To investigate whether immunohistochemistry (IHC) could be used to screen BRAF mutation status in formalin-fixed paraffin-embedded (FFPE) colorectal carcinoma (CRC) and papillary thyroid carcinoma (PTC) samples. Eight surgical resected samples, including 2 CRCs with mutated BRAF V600E, 2 PTCs with mutated BRAF V600E, 2 CRCs with wild-type BRAF and 2 PTCs with wild-type BRAF, were selected to explore the optimized IHC conditions for BRAF V600E (VE1) immunostaining using BenchmarkXT automated immunostainer VENTANA Medical System. BRAF V600E status was tested by optimized IHC and ARMS-PCR methods in 255 samples (123 CRCs and 132 PTCs). Sanger sequencing was performed to validate the BRAF V600E status if discordant results were found between optimized IHC and ARMS-PCR methods. Antigen retrieval time in 32 min and 64 min showed satisfactory intensity and homogeneity of BRAF V600E staining in CRC and PTC samples, respectively. The concordance between IHC and ARMS/Sanger sequencing was 99.2% (122/123) in CRCs and 96.2% (127/132) in PTCs. In CRCs, the sensitivity of IHC staining for BRAF V600E was 100% (3/3) and the specificity was 99.1% (119/120). In PTCs, the sensitivity was 100% (106/106) and specificity was 80.8% (21/26). The overall concordance across all cases was 97.6% (249/255). The appropriate antigen retrieval protocol is critical for accurate analysis of BRAF V600E status by Benchmark XT automated immunostainer. IHC is a suitable method to screen BRAF V600E mutation in FFPE samples of CRCs and PTCs.

Zhang X ，Wang L ，Wang J ，Zhao H ，Wu J ，Liu S ，Zhang L ，Li Y ，Xing X ... -  《-》

Value of immunohistochemistry in the detection of BRAF(V600E) mutations in fine-needle aspiration biopsies of papillary thyroid carcinoma.

Fine-needle aspiration biopsy (FNAB) is important in the diagnostic establishment of suspicious thyroid nodules. In thyroid neoplasms, mutation of the BRAF gene occurs rather exclusively in papillary thyroid carcinoma (PTC) and results in>98% of the cases in V600E amino acid substitution. In the current study, the authors investigated the diagnostic value of a recently described monoclonal antibody that detects this specific mutation on FNAB specimens from patients with PTC. BRAF(V600E) status of FNAB cell blocks from 55 patients with PTC was analyzed by immunohistochemistry (IHC) with the new BRAF(V600E) antibody (clone VE1) and by Sanger sequencing (SaS). In discrepant cases, ultra-deep sequencing was also performed. Available corresponding histological specimens were investigated by IHC and, in selected cases, with SaS as well. All cases yielded evaluable IHC staining results of the cell block sections with good interobserver agreement (kappa value, 0.650). Ten tumors (18.2%) demonstrated no staining, 10 tumors (18.2%) demonstrated equivocal staining, 25 tumors (45.4%) demonstrated moderate staining, and 10 tumors (18.2%) demonstrated strong staining. SaS was able to be performed in 48 cases. Nineteen cases demonstrated wild-type BRAF and 29 cases were found to have the BRAF(V600E) mutation. After performing ultra-deep sequencing 1 false-positive and 2 false-negative VE1 IHC cases remained, resulting in a sensitivity of 93.8% and a specificity of 93.8%. BRAF(V600E) mutations in FNAB specimens from patients with PTC can be reliably detected in most cases by IHC with a new mutation-specific antibody. Interpretation of VE1 IHC staining results on cell block slides of PTC can be difficult in some cases.

Zimmermann AK ，Camenisch U ，Rechsteiner MP ，Bode-Lesniewska B ，Rössle M ... -  《-》

Comparison of 2 monoclonal antibodies for immunohistochemical detection of BRAF V600E mutation in malignant melanoma, pulmonary carcinoma, gastrointestinal carcinoma, thyroid carcinoma, and gliomas.

BRAF mutation is seen in a variety of human neoplasms including cutaneous malignant melanoma, papillary thyroid carcinoma, colorectal carcinoma, non-small cell lung carcinoma, pleomorphic xanthoastrocytoma, and others. Currently, there are 2 commercially available monoclonal antibodies for the detection of BRAF V600E mutation; however, a full and practical comparison of their performance in various tumor types on an automated staining platform has not been done. We investigated their sensitivity and specificity in detecting the BRAF V600E mutation in a series of 152 tumors including 31 malignant melanomas, 25 lung carcinomas, 32 gastrointestinal carcinomas, 23 thyroid carcinomas, 35 gliomas, and 6 other malignancies. In this series, the concordance rate between immunohistochemistry (IHC) and mutational analyses was 97% (148/152) for VE1 and 88% (131/149) for anti-B-Raf. The sensitivity and specificity were 98% (60/61) and 97% (88/91) for monoclonal VE1 and 95% (58/61) and 83% (73/88) for anti-B-Raf, respectively. There were 4 cases with discordant IHC and mutational results for monoclonal VE1 in contrast to 18 cases for anti-B-Raf. Our studies showed that IHC with monoclonal VE1 has a better performance compared with anti-B-Raf in an automated staining platform and confirmed that clone VE1 provides excellent sensitivity and specificity for detecting the BRAF V600E mutation in a variety of tumor types in a clinical setting.

Routhier CA ，Mochel MC ，Lynch K ，Dias-Santagata D ，Louis DN ，Hoang MP ... -  《-》

Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF V600E mutations in colorectal carcinoma.

Rössle M ，Sigg M ，Rüschoff JH ，Wild PJ ，Moch H ，Weber A ，Rechsteiner MP ... -  《-》