BRAF, a serine/threonine protein kinase belonging to the RAF family, is a key intermediary in the RAS-RAF-MEK-ERK-MAP kinase signaling pathway. It is also a key regulator of cellular functions, including cell proliferation, cell-cycle arrest, terminal differentiation, and apoptosis. BRAF V600E mutation have been detected in several types of cancer, including a variety of tumors in central nervous system. The detection of BRAF V600E is essential in the diagnosis of brain tumors for the following reasons: 1) Some circumscribed gliomas (GGs and PXAs) are difficult to be differentiated from diffuse gliomas via histopathology alone, especially on biopsy specimens as they have different molecular characteristics. Therefore, BRAF-V600E detection has important differential diagnostic value; 2) Detection of BRAF-V600E has a certain stratification effect on the prognosis of adult invasive glioma; 3) With the clinical development of BRAF-targeted therapeutics, the detection of BRAF-V600E has important diagnostic and prognostic value. However, the current molecular biology techniques are expensive and not yet widely available. If the BRAF-V600E expression status can be clarified through immunohistochemistry, it will be more efficient in the diagnosis and treatment evaluation for brain tumors. In melanoma, papillary cholangiocarcinoma, thyroid cancer, and colorectal cancer, VE1 detection has the specificity and sensitivity of more than 95%[2; 8; 9], but few study has evaluated the sensitivity and specificity of BRAF VE1 immunochemical staining in central nervous system tumors. This study aimed to elucidate the current bias in the histopathological interpretation of BRAF VE1 staining, and provide the clinical insight into the potential future use of BRAF VE1 immunochemical staining in the diagnosis of brain tumor.
The gold standard for BRAF mutation analysis is direct sequencing of tumor DNA, but real-time qPCR tests were considered more effective and widely accepted in recent year. In this study, the reliability of BRAF VE1 immunohistochemical staining was verified by real-time qPCR on the routinely processed formalin-fixed, and paraffin-embedded (FFPE) tumor tissue.
Our results suggested that all of the immunoreactivity patterns of diffuse positive cases for real-time qPCR detection were further confirmed to have BRAF V600E mutation (55/55,100%). None of the VE1-negative cases carried a BRAF V600E mutation (35/35,100%). It is important to note that reliable positivity within the plasma in single tumor cells generally suggested BRAF V600E mutations. What is more interesting is that the pattern of reliable positive of single tumor cell this single cell positive pattern predominantly occurred in neuronal tumor cells of GG. It is similar as previously reported. Circumspection was required for interpreting the immunohistochemical staining of BRAF VE1, especially for GC/GG. Our study indicated the pattern of completely negative IHC appeared to preclude the need mutational testing for BRAFV600E. And diffuse strongly positive cases in our experience equate with the presence of BRAF V600E mutation, reliable positive of single tumor cell in GG preferentially harbored BRAFV600E mutations, which might be exempt from molecular test for BRAF V600E in our practice.
In addition, uniform "coating" positivity with a heavy background occurred in some cases despite every attempt at optimization of the protocol. Our experience paralleled those reported by others[16; 17]. And uniform "coating" positivity was different from reliable diffuse positivity under a microscope, which was weak-moderate intensity positive in cytoplasmic and varying degrees of nuclear expression cell with a heavy background. In our study, the Image-Pro Plus 6.0 was used to measure the area, IA of staining cells in every sample. And the results showed that there were Significant differences in IA/area between DP groups and u-CP groups(P = 0.000002), also between DP and negative groups(P < 0.000001). Although there was a difference between u-CP group and negative group(P = 0.029), we prefer to interpret them as equivocal positivity. Similar to previous reports[16; 17], the u-CP group is more likely to be interpreted without BRAF V600E mutation in our study (18/20,90%). Given the importance of accurate documentation of the BRAF mutational status prior to potential targeted therapies[18; 19], we propose to continue molecular testing to further determine the BRAF gene status in these ambiguous cases.
What’s the reason that the u-CP immunoreactivity pattern of BRAF VE1 fail to interpret as gene mutation was unknow. Some argued that this is mainly due to the limitations of commercial antibodies, while others speculated that the epitope of BRAF VE1 antibody shared structural homolog to the epitope of completely unrelated molecules in some protein-rich cells, such as ganglionic-like or ganglion cells and pituitary adenoma cells. Therefore, while staining of BRAF VE1 protein may appear as a feasible method for evaluating BRAF mutation status, it is important to confirm the presence of BRAFV600E mutation by molecular test when the Immunoreactivity Patterns is u-CP, especially in some types of tumors not known to, or rarely harbor such mutations.
It was very interesting that p-CPGs typically showed weak to moderate staining for BRAF VE1, while always harbored BRAF V600E mutations. On the contrary, adamantinomatous craniopharyngioma was always negative for VE1, while aberrant nuclear accumulation of beta-catenin could be detected by immunohistochemistry. Therefore, we can distinguish p-CPG from adamantinomatous craniopharyngioma by BRAF VE1 staining. When the craniopharyngioma is associated with cystic degeneration due to the cyst wall epithelium atrophies caused by the pressure of the cyst, it is difficult to differentiate from Rathke cleft cysts in sellar region with epithelial squamous metaplasia, which can be resolved by conducting immunohistochemistry. Unlike craniopharyngioma, Rathke cleft cysts are known for the lack of BRAF V600E mutations and cell membrane accumulation of beta-catenin localizes. In addition, BRAF-V600E status can provide an important evidence for the targeted therapies of recurrent and refractory craniopharyngioma.
In summary, we found that BRAF VE1 staining could replace molecular detection to some extent, on the premise of mastering the key points in the interpretation of BRAF VE1 immunostaining. Key points of interpretation including: 1) as long as the positive signal was accurately located in the cytoplasm of tumor cells, the sample was considered to have BRAF V600E mutation, disregarding the number of staining cells, especially for GG; 2) for craniopharyngioma, even a weak positive for BRAF VE1 staining, a diagnosis of papillary craniopharyngioma harbored BRAF V600E mutation should be considered. 3) Tissue samples that had no signal of BRAF VE1 expression with clear background could be confirmed without BRAF-V600E mutation.4) Some equivocal positive with uniform “coating” positive cases, which were often considered as false-positive and usually required further molecular detection. Otherwise, the models for predicting BRAF mutations of BRAF VE1 by tumor type, positive area and average IA of stained area may provide a possible scheme for artificial intelligence interpreting of immunohistochemical staining.