Introduction
A serine/threonine kinase of the MAPK-ERK signalling pathway, B-type Raf kinase (BRAF), and other RAF family members are usually activated by GTP-bound RAS signalling downstream of the epidermal growth factor receptor (EGFR) or in response to other mitogens [
1]. Mutant BRAF, however, displays constitutive activation when affected by missense mutation [
2], most commonly V600E. Approximately 10 % of colorectal cancers (CRCs) harbour
BRAF V600E [
2‐
4], and this subset is associated with a significantly poorer survival [
5‐
7] in patients with metastatic disease.
BRAF mutation may also predict lack of benefit from anti-EGFR therapy [
8,
9] in metastatic CRC, although reports are conflicting [
10]. The current clinical value of
BRAF V600E detection is the delineation of hereditary non-polyposis colorectal cancer (HNPCC)-associated tumours (BRAF wild type) from sporadic CRCs (BRAF V600E mutant) in mismatch repair-deficient colorectal disease [
11‐
13] and rational patient enrolment to clinical trials testing BRAF inhibitors [
6].
Various methods of genotyping tumour samples for
BRAF status are currently used in diagnostic and research laboratories, ranging from traditional Sanger sequencing [
14] to quantitative pyrosequencing [
15], mutation-specific real-time polymerase chain reaction (RT-PCR) assays [
16] and mass spectrometry-based methods [
17]. Common to all these methods, however, is the requirement for DNA extraction from tissue and the need for rigorous protocols to minimise the impact of contamination of non-tumour cells on the overall tumour to non-tumour cell ratio.
Importantly, at this point in time, the expertise and infrastructure required for DNA-based genotyping methods are frequently available only at academic centres and reference laboratories. Testing for BRAF V600E therefore requires multiple steps and coordination between the primary site and reference laboratory, resulting in sample transit costs and diagnostic delays. Until genomic-based testing becomes available in routine community-based pathology laboratories, the complexity involved in such testing will continue to serve as an impediment to providing a patient’s BRAF status to their treating clinician.
A monoclonal antibody specific to the BRAF V600E kinase, VE1, has recently been described [
18] and offers the advantages of immunohistochemical determination of tumour
BRAF mutation status, no requirement for DNA purification, low cost and the ability to perform testing on formalin-fixed paraffin-embedded (FFPE) tissue in routine histopathology laboratories. To date, immunohistochemistry with VE1 has been applied to the detection of BRAF V600E in brain metastases of varied primary sites [
19], papillary thyroid carcinoma [
20,
21], Langerhans cell histiocytosis [
22,
23], ovarian carcinomas [
24,
25], melanoma [
26‐
28], lung adenocarcinoma [
29] and hairy cell leukemia [
30]. A recently published study by Sinicrope et al. explored the VE1 antibody in a carefully preselected group of 75 patients with stage III colorectal cancer, for whom
BRAF mutation status had already been determined [
31]. In another recent study examining the utility of BRAF immunohistochemistry (IHC) in microsatellite unstable CRC, Toon et al. [
32] compared BRAF IHC with conventional PCR-based molecular methods for
BRAF V600E detection in 216 patients with CRC. In a further cohort, they also performed IHC to mismatch repair (MMR) proteins and BRAF V600E in a larger cohort of 1,403 patients with CRC but failed to also validate this using conventional sequencing-based molecular techniques.
In this study, we aimed to determine the sensitivity, specificity and predictive values of VE1 immunohistochemistry for BRAF V600E in a large community-based and unselected cohort (n = 505) of patients with CRC, with the intent of determining how this could inform the use of this IHC-based antibody in routine practice. FFPE tumour samples were annotated for clinical outcomes and had been previously assessed for BRAF status by direct (Sanger) sequencing.
Discussion
Mutation of
BRAF V600E is a well-validated poor prognostic marker in metastatic CRC [
5‐
7], as well as a discriminator between HNPCC-related disease and sporadic MSI CRCs [
11‐
13]. Perhaps the greatest promise of
BRAF V600E detection, however, is in the application of targeted therapies in the emerging era of personalised medicine. While
BRAF-mutant colorectal cancer has not demonstrated the same responsiveness to single-agent BRAF inhibitors seen in metastatic melanoma [
41,
42], recent research findings show encouraging headway in the characterisation of CRC resistance mechanisms [
42‐
44]. Clinical studies combining BRAF inhibitors with EGFR inhibitors, PI3K inhibitors or MEK inhibitors are now underway based on these recent data and will require accurate, efficient identification of patients with
BRAF-mutant CRC.
We report a large series of community-based colorectal cancers screened for
BRAF V600E mutation using the novel, mutation-specific monoclonal antibody VE1. In this study, blinded interpretation of VE1 IHC on CRC cores embedded in TMAs showed 98.2 % sensitivity, 98.1 % specificity, 87.3 % PPV and 99.8 % NPV against
BRAF genotype determined by direct sequencing. Similar sensitivities and specificities for VE1 have been reported in papillary thyroid cancer, lung cancer and melanoma [
20,
27‐
29]. Given that positive IHC results are likely to be verified by gene-based mutation detection methods and accurate identification of
BRAF mutation is expected to facilitate access to novel targeted therapies, the NPV of VE1 IHC is of the highest importance. Only one sample in this study was falsely negative on VE1 TMA IHC and subsequently shown to be clearly VE1 positive on whole-section staining. The resolution of discordant findings for this case, and the further six available TMA IHC false-positive cases, by additional methods is reasonable in the context of this particular study. It is important to note that whole-section IHC would be conducted in routine clinical practice, rather than via the creation of TMAs. In addition, numerous genotyping methods have been recently demonstrated to be more sensitive than Sanger sequencing [
15‐
17,
27]. The sensitivity, specificity, PPV and NPV were each recalculated as 100 % in the fully evaluable patient population. As expected, clinical correlations with BRAF V600E positivity confirmed the same patient and tumour associations in this patient cohort seen with
BRAF genotype as determined by DNA-based mutation detection methods [
38‐
40], further supporting the validity of this diagnostic approach in community-based patient cohorts. Although we restricted survival data to the subgroup of patients with metastatic disease (either de novo stage IV or recurrent stage IV disease,
n = 188), given this is the group of patients for which the presence of a BRAF V600E mutation is a clear prognostic factor, our survival data was again consistent with that seen in other series.
In studies forerunning to those using anti-BRAF antibodies, the ability to apply immunohistochemistry for the detection of mutated gene products has been recognised in the management of gliomas. A monoclonal antibody specific for the mutant protein
IDH1 R132H, important diagnostically and prognostically [
45], has been developed and applied to clinical samples [
46,
47]. Similarly, determination of
BRAF mutation status by IHC is anticipated to confer significant benefits in the clinical setting. Unlike genotyping methods, IHC is routinely performed in all hospital histopathology departments. The cost of reagents, equipment and labour is projected to be lower than that of currently available sequencing methods. Additionally, the time to results may be shorter due to the on-site sample processing and absence of requirement for DNA extraction. Routine profiling of all newly diagnosed CRCs for
BRAF V600E mutation may therefore now be feasible with an inexpensive and widely available methodology. This takes on added significance when considering
BRAF V600E’s relatively low population prevalence. Detection of
BRAF V600E mutation at the time of primary CRC resection and histopathological analysis has the advantages of excluding underlying HNPCC in MSI CRCs [
11‐
13], with resultant screening implications [
48], and prevention of lag time awaiting
BRAF mutation results in patients later diagnosed with metastatic disease. Given that patients with metastatic
BRAF-mutant CRC have a significantly poorer prognosis, early recognition of their
BRAF status may alter clinical decisions regarding disease observation and, in the future, administration of molecularly directed therapies.
As per the original description of VE1 IHC on clinical samples [
18], this study applied both VE1 and pBR1 primary antibodies to all tumours. While VE1 had been selected from 2,234 hybridoma clones as the only immunoglobulin specific for
BRAF V600E on IHC, pBR1 had been chosen from pan-BRAF clones for its demonstrated ability to bind both BRAF wild type and BRAF V600E on IHC and Western blots [
18]. The utility of positive pBR1 staining is in its confirmation of BRAF protein expression, reducing the likelihood of false-negative VE1 IHC findings, i.e. the presence of
BRAF V600E genotype but negative VE1 staining due to the absence of BRAF protein expression in the tumour. In this cohort, only 14 of 491 primary CRCs (2.9 %) were negative on pBR1 IHC, suggesting that failure to express BRAF is rare in CRC and the routine use of pBR1 concurrently with VE1 may be unnecessary for this tumour type. More important than concurrent pBR1 staining may be preservation of tissue quality, as tissue areas subject to diathermy, showing necrosis, or frozen and then formalin-fixed have all been reported to show lower antigenicity for VE1 [
18]. The use of freshly cut tissue sections is also recommended [
19]. We noted strong staining of normal colonic surface epithelial cell nuclei for VE1, which could be used as a positive internal control for VE1 IHC protocols. The reason for nuclear staining with the VE1 antibody is unknown.
Immunohistochemistry may be an optimal method for determining the
BRAF V600E mutation status of tissue samples with low cellularity due to its single-cell-level resolution. In contrast, for DNA-based detection methods, even microdissected tissue samples contain significant proportions of stromal cell-derived DNA, posing challenges in the detection of diluted tumour cell-derived mutant alleles. BRAF V600E-specific IHC has some limitations, however. Samples collected by fine-needle aspiration (FNA) may pose a challenge because of the fixatives used. Caution is urged until the validity of this approach in FNA samples has been adequately investigated. Due to the heterogeneity of VE1 staining seen in a proportion of CRCs, 1.7 % of the evaluable population in this study, small biopsy (e.g. core biopsies) and tissue specimens may be erroneously interpreted due to sampling error. This phenomenon was documented in our patient cohort with the initial use of TMAs for VE1 IHC; for one sample, IHC findings were changed from positive to negative, and for three samples, the reverse occurred when TMA IHC findings were compared to staining on whole mount sections. Although in our patient cohort <3 % of CRCs were negative on pan-BRAF IHC, other malignancies with low BRAF protein expression may not be amenable to IHC testing. VE1 is highly specific for
BRAF V600E and does not detect the protein products of other
BRAF aa.600 mutations [
19,
27,
28]; hence for diseases with a higher burden of
BRAF non-V600E mutations, DNA-based genotyping or alternative monoclonal antibodies may be indicated, particularly should these mutations demonstrate drug sensitivity. In CRC, <4 % of
BRAF mutations are non-V600E [
4,
9], and no other aa.600 mutations were detected by sequencing in the 505 patients of this cohort. However, we cannot exclude the possibility that a small number of patients may have carried other non-aa.600
BRAF mutations, as DNA sequencing was limited to the region of aa.600.
Two recently published studies exploring the VE1 antibody in colorectal cancer have also shown a high degree of specificity and sensitivity, but included either small series [
32] and/or a highly preselected patient population [
31]. However, the consistent findings in both of these studies add considerable weight to our findings in supporting the incorporation of the antibody as the initial method of detection of
BRAF V600E status in patients with colorectal cancer. As opposed to the findings of strong concordance between the VE1 IHC antibody and sequencing-based techniques in these two studies and ours, another study examining VE1 IHC in colorectal cancer was also published recently and reports low sensitivity (71 %) and specificity (74 %) of VE1 tested on 52 FFPE CRC samples [
49]. Methodological differences in the antigen retrieval, staining and signal amplification protocols resulting in poor signal intensity and background staining likely account for this discrepancy [
49]. We found that the use of OptiView DAB IHC Detection Kit resulted in clear discrimination between background tissue and
BRAF V600E-positive tumour cell cytoplasmic staining using the VE1 antibody. In our experience, patchy focal staining, rather than the level of staining intensity, can lead to difficulties with interpretation, particularly in tissue core samples. Patchy focal staining was occasionally seen (1.7 %) in otherwise clearly negative tumours and could lead to false-positive results (Fig.
3). Patchy focal non-staining was occasionally seen in otherwise clearly positive tumours and could lead to false-negative results. These staining anomalies were often present in the same areas in restained sections and did not correspond to any anatomical features such as tumour nodules. Interpretive difficulties were often resolved by examining additional tumour areas by staining whole mount sections. In such cases, confirmation of
BRAF status should be verified by IHC on a different specimen from that resection or by a sensitive molecular method.
The observation that the vast majority of CRCs stained in this study showed either homogeneously negative or positive BRAF V600E expression argues against the clonal acquisition of
BRAF activating mutations in primary CRCs. Interestingly, lack of clonality for BRAF V600E expression has also been noted in other malignancies including metastatic lesions [
19‐
21,
28], despite suggestions of polyclonality for
BRAF genotype in melanoma and thyroid cancer using alternative experimental methods [
50‐
52]. The use of mutation-specific antibodies to address this controversial cancer biology question is but one of their potential research applications. Busam et al. [
53] and Sahm et al. [
23] utilised VE1 IHC in co-staining experiments to further characterise
BRAF mutation-positive melanocytic lesions and Langerhans cell histiocytosis respectively. Exploration of staining intensity has also been suggested as a potentially fruitful translational research application [
54].
In conclusion, the BRAF V600E VE1 antibody is an accurate immunohistochemical diagnostic assay in patients with CRC and should serve as a simple method for the detection of the BRAF V600E mutation in routine practice.