Immunohistochemistry with Anti-BRAF V600E (VE1) Mouse Monoclonal Antibody is a Sensitive Method for Detection of the BRAF V600E Mutation in Colon Cancer: Evaluation of 120 Cases with and without KRAS Mutation and Literature Review

Colorectal cancer is the third most common cancer and the fourth most prevalent cause of death in the world [46]. Approximately 35–45% of patients with colorectal tumors have mutation in KRAS gene, while BRAF V600E mutation is found in about 5–15% of colorectal adenocarcinomas [8, 9, 26, 31]. Both these mutations are considered to be oncogenic driver mutations, since they are both responsible for the initiation and maintenance of the tumor [10]. Importantly, many studies have indicated that BRAF V600E mutation occurs only in tumors that do not carry mutations in KRAS gene and it is widely accepted that these two mutations are mutually exclusive [10, 23, 25, 30].

The BRAF gene encodes a cytoplasmic serine-threonine kinase that is frequently mutated in various cancers, including melanoma, papillary thyroid carcinoma, and colorectal carcinoma, among others. The oncogenic mutations in BRAF gene result in constitutive activation of the MAPK signaling pathway, leading to increased cell proliferation, resistance to apoptosis and tumor progression. The most common of these mutations, the V600E mutation, occurs in exon 15 and results in a substitution from valine to glutamic acid at position 600 within the BRAF kinase domain.

BRAF V600E mutation occurs in about 5% of microsatellite stable (MSS) CRC tumors. These tumors are associated with a distinct molecular and clinical phenotype with a poor prognosis [40]. The presence of BRAF V600E mutation in CRC is associated with poor survival [44]. BRAF V600E mutation is also detected in sporadic CRC tumors with microsatellite instability (MSI) [27]. Particularly, BRAF V600E mutation is observed in about two thirds of MSI tumors with the loss of MLH1 expression due to MLH1 promoter methylation [18]. In contrast, BRAF V600E mutation is very rare in CRC patients with Lynch syndrome [27]. In clinical practice it is much easier to detect BRAF V600E mutation than methylation status of MLH1 promoter [26]. Therefore, it was suggested that assessment of BRAF V600E mutation can be used to triage patients for mismatch repair (MMR) genetic testing to differentiate MLH1-deficient sporadic CRC from Lynch syndrome caused by germ-line MLH1 mutations [7, 14, 17, 26, 41, 43]. Currently, the American National Comprehensive Cancer Network (NCCN) guidelines recommend that BRAF V600E mutational status should be evaluated in all colorectal carcinomas to identify 1) the patients with Lynch syndrome in MMR deficient group and 2) to identify the MMR proficient/BRAF V600E group with poor prognosis [15, 38, 43].

The most common approach for the detection of BRAF mutation is sequencing of tumor DNA, for example Sanger sequencing, pyrosequencing and high resolution melting. All of these methods are able to detect a mutant allele in a background of 5–20 fold excess of wild-type alleles. In contrast, immunohistochemistry allows direct visualization of the mutant protein in the tumor cells in tissue context. The anti-BRAF V600E (VE1) antibody is currently used to evaluate the BRAF V600E mutation status in various cancers including CRC [32]. This antibody is a mutation-specific mouse monoclonal antibody that was raised against a synthetic peptide representing the BRAF V600E mutated amino acid sequence from amino acids 596 to 606 (GLATEKSRWSG) [5, 6].

The primary goal of this study was to compare the performance of the anti-BRAF V600E (VE1) antibody to detect BRAF V600E mutation by IHC in colon cancer cases with/without KRAS mutation. Since concomitant KRAS and BRAF tumor mutations are considered mutually exclusive we wanted to confirm that the CRC cases carrying KRAS mutation show negative BRAF V600E staining by IHC with anti-BRAF V600E (VE1) antibody. In addition, we performed a review of 25 studies that compared IHC using anti-BRAF V600E (VE1) antibody with molecular testing for BRAF V600E mutation.

This study evaluated 120 CRC cases with and without KRAS mutation to access the performance of IHC using anti-BRAF V600E (VE1) antibody for detection of BRAF V600E mutation. Overall, the results of these experiments demonstrates that IHC using the anti-BRAF V600E (VE1) antibody with the VENTANA OptiView DAB detection system and BenchMark ULTRA platform is a highly specific and sensitive method for the detection of BRAF V600E in colon cancer.

There is strong evidence from multiple studies that the IHC using anti-BRAF V600E (VE1) antibody is highly concordant with molecular tests for the BRAF V600E mutation. Table 2 shows a summary of 25 studies that evaluated sensitivity and specificity of IHC with anti-BRAF V600E (VE1) antibody in comparison with molecular tests using different methods (Sanger, pyroseqencing, SNapShot PCR, NGS, etc.). Altogether, 4041 patient samples were evaluated in these studies, the overall sensitivity and specificity of IHC assay using anti-BRAF V600E (VE1) antibody compared to molecular tests was 93% (934/1008) and 96% (2922/3033), respectively.

Out of these 25 studies, 4 publications reported lower sensitivity and/or specificity of anti-BRAF V600E antibody compared to sequencing [1, 12, 20, 21]. However, there were several problems with these studies. First, the study by Adackapara et al. analyzed 52 colorectal carcinomas with known BRAF mutation status determined by pyrosequencing and found that IHC had a low sensitivity (71%) and specificity (74%) for detecting BRAF V600E mutation compared to pyrosequencing (Table 2). They concluded that IHC using anti-BRAF V600E (VE1) antibody is not a useful surrogate for detecting BRAF mutation in colorectal carcinoma. However, in their experiment, manual staining with citrate buffer as antigen retrieval was employed. In our experience and the experience of others the use of acid for antigen retrieval step results in suboptimal staining that is difficult to interpret [19]. TRIS or EDTA buffers at pH = 8 proved to be retrieval agents that produced the most robust and homogenous cytoplasmic staining with anti-BRAF V600E (VE1) antibody. Similarly, Lasota et al. used in their studies Bond Epitope Retrieval Solution 1 (pH = 6) which is not an optimal solution for antigen retrieval for this assay [20].

Another important factor that may contribute to the different outcome of the studies is the interpretation of the IHC results. As multiple studies have highlighted, a proper scoring system is necessary to reduce false-positive and false-negative cases. Since BRAF V600E mutation is a driver mutation, a majority of tumor cells should express this mutated protein. The scoring criteria shown in Table 1 were used in the individual studies presented in Table 2 that compare IHC using anti-BRAF V600E (VE1) antibody with molecular testing for BRAF V600 E mutation. Overall, 14 out of 25 studies scored cases positive for BRAF V600E mutation when uniform or nearly uniform, diffuse staining was present in tumor cells or when the majority (≥ 75%) of tumor cells exhibited unequivocal cytoplasmic staining (Table 2). All studies that used these interpretation criteria (and appropriate protocol using antigen retrieval at alkaline pH) reached close to 100% sensitivity and specificity compared to sequencing. Additional 7 studies did not include scoring criteria in the method section, however they reported that homogeneous, diffuse staining pattern was observed in cases with confirmed BRAF V600E mutation. Three studies provided no details. In one study the cases were scored as positive for BRAF V600E staining when only ≥20% tumor cells showed positive signal in one study [12]. The sensitivity and specificity reported in this study was only 89% and 57% (when IHC assay on BenchMark ULTRA platform was used) and 75% and 93% (when IHC assay on Leica Bond was used). Several studies suggested that additional analysis is required for minority of equivocal cases with ambiguous, focal, heterogeneous staining (Table 2). False positive staining was noted in signet ring tumor cells [33]. Importantly, the nuclear staining was described as the most common artifact (Table 2) [22]. For example, Bledsoe et al. noted that BRAF-mutant cases showed homogeneous, finely granular, cytoplasmic staining with varying intensities, however, non-specific nuclear and heterogeneous non-diffuse cytoplasmic staining of variable intensity was observed in a minority of non-BRAF mutant cases [3]. Therefore, it was recommended by Marin et al. that “the interpretation should be made with caution in the presence of nuclear staining” [22]. Our study also suggests that the cases showing the presence of heterogeneous cytoplasmic staining with or without nuclear staining should be carefully interpreted.

Overall, the evidence from the publications presented in Table 1 and from the current study suggests that the cases should be scored as positive for BRAF V600E mutation if they display unequivocal, diffuse, uniform, granular, cytoplasmic staining in the majority of tumor cells at stain intensity ≥1. They should be scored as negative for BRAF V600E mutation if they exhibit no staining or weak, cytoplasmic, non-granular, non-uniform staining (stain intensity <1). The cases with staining of isolated tumor cells in a tumor that otherwise showed no staining should be considered negative. The cases should be considered as equivocal if they display ambiguous, heterogeneous, cytoplasmic staining with or without nuclear staining in tumor cells. If these interpretation criteria are followed the IHC with anti-BRAF V600E (VE1) antibody using recommended protocol with OptiView detection is optimal for detection of BRAF V600E mutation in CRC. In our study all 30 cases with BRAF V600E mutation showed unequivocal positive cytoplasmic staining in 85–100% tumor cells; all 30 cases with wild-type KRAS and BRAF were negative; 6.7% (4/60) cases with KRAS mutation showed heterogeneous, cytoplasmic/nuclear staining at stain intensity 1. However, the staining was heterogeneous and the presence of distinct nuclear staining was noted in these four cases along with cytoplasmic staining. Therefore, these cases were assigned as equivocal for BRAF V600E mutation. These cases were sequenced and confirmed to be negative for BRAF V600E mutation.

In summary, this study indicates that IHC with the anti-BRAF V600E (VE1) antibody performed on the Benchmark ULTRA automated stainer is a highly sensitive and specific detection method for determination of BRAF V600E mutation status in CRC. The results presented in this study are consistent with previous reports indicating that KRAS and BRAF V600E mutation are mutually exclusive. Based on our findings and consistent with other literature reports, the majority of BRAF V600E positive cases demonstrate a uniform or nearly uniform, diffuse staining pattern present in the majority of tumor cells. We propose that in the minority of cases with an equivocal staining pattern, additional molecular testing should be done to assess BRAF mutational status.