Adult classical glioblastoma with a BRAF V600E mutation

The B-Raf proto-oncogene serine/threonine kinase (B-Raf) is a member of the Raf kinase family. The BRAF V600E mutation occurs frequently in certain brain tumors such as pleomorphic xanthoastrocytoma, ganglioglioma, and pilocytic astrocytoma, and less frequently in epithelioid and giant cell glioblastoma. BRAF V600E mutation in these cases has been canonically detected using Sanger sequencing or immunohistochemistry but not with next-generation sequencing (NGS). Moreover, to our knowledge, there is no detailed report of the BRAF V600E mutation in an adult glioblastoma with classical histologic features (c-GBM). Therefore, we performed NGS analysis to determine the mutational status of BRAF of 13 glioblastomas (GBMs) (11 primary and 2 secondary cases) and detected one tumor harboring the BRAF V600E mutation. We report here the detection of the BRAF V600E mutation in a patient with c-GBM and describe the patient’s clinical course as well as the results of histopathological analysis.


Case presentation
A 49-year-old man was admitted to the hospital complaining of headache, vomiting, and mild left hemiparesis. Magnetic resonance imaging (MRI) showed a huge multicystic mass in the right occipitoparietal area with marked surrounding edema and a shift of the midline structures to the left side ( Figure 1A). The cyst wall and adjacent cortical mass were enhanced with contrast medium ( Figure 1B). 18F-Fluorodeoxyglucose (FDG) and methionine (MET) positron emission tomography (PET) revealed high accumulation in the right occipitoparietal area ( Figure 1C, D).
Near-total resection of the tumor was performed. After glioblastoma (GBM) was pathologically diagnosed, the patient had local radiation using tomotherapy (60 Gy/30 fractions), with concomitant chemotherapy consisting of temozolomide (75 mg/m 2 /day). After a 4-week break, the patient received 19 cycles of adjuvant temozolomide (150 mg/m 2 /day) for 5 days every 28 days. A small contrast-enhancing lesion was seen on MRI close to an extraction cavity 22 months after the first operation. Because MET-PET showed a high accumulation in the mass, although none was detected using FDG-PET ( Figure 1F, G), a second operation was performed, and the recurrence of GBM was diagnosed. Furthermore, the patient continues to receive 31 cycles of adjuvant temozolomide (200 mg/m 2 /day) for 5 days every 28 days and is living without recurrence 4 years after the first operation ( Figure 1H).

Pathological findings
Numerous atypical spindle cells were interspersed with gemistocytes ( Figure 2A, D), and microvascular proliferation and pseudopalisading were present ( Figure 2B, C). Tumor cells were highly positive for glial fibrillary acidic protein (GFAP; Figure 2E), and the Ki67 index was approximately 10% ( Figure 2F). Expression of cytokeratins was undetectable in EMA + tumor cells ( Figure 2G, H). Findings of tumor cells negative for epidermal growth factor receptor (EGFR) but positive for P53 are typical of secondary GBM ( Figure 2I, J). Expression of the IDH1 R132H mutant or the IDH1 R132H mutation was not detected using immunohistochemistry or NGS analysis, respectively ( Figure 2K). In contrast, expression of the BRAF V600E mutant was detected using immunohistochemistry, and the BRAF V600E mutation was detected using NGS ( Figure 2L).  instructions. The target enrichment library pool was sequenced using a MiSeq (Illumina, San Diego, CA, USA). The sequence data were aligned, analyzed, and visualized using SureCall 2.0 software (Agilent Technologies).

Conclusions
The BRAF V600E mutation may occur at low frequency in adult c-GBM. This mutation was detected by the direct sequence method of Sanger method or immunohistochemistry. NGS techniques are in wide use because mutations are detected with greater sensitivity compared with Sanger sequencing [7,8]. Therefore, we have performed NGS analysis to detect BRAF mutations in tissues of 13 patients with c-GBM (11 primary and 2 secondary cases) who were treated at Hokuto Hospital. However, the BRAF V600E mutation was detected in only one case (7.7%; Table 1). The validity of the data is supported by the detection of BRAF V600E in the tumor tissue of the same patient after the tumor recurred.
When we reviewed the pathology of this case, the tumor was not an epithelioid or a giant cell GBM because foci with glandular and ribbon-like epithelial structures and multinucleated giant cells were not present. Classical GBM was confirmed by two expert neuropathologists (H.N. and S.T).
Although there is no detailed report of a BRAF V600E-positive adult c-GBM, to our knowledge, there is a study of two such cases [9]. The tumors of both patients were located within the right parietal lobe. Interestingly, the tumor of our present patient was located in the right occipitoparietal lobe. Moreover, our patient with BRAF V600E-positive adult c-GBM was alive when this manuscript was submitted, 4 years after the first operation. In contrast, the patients studied by Chi et al. [9] survived for 19 and 36 months. The IDH1 mutation serves as a good prognostic factor for patients with GBM but was not detectable using NGS or immunohistochemical analyses. These data are consistent with the results of a study of two patients with BRAF V600Epositive c-GBM [9]. Although the number of patients was small, these three patients with BRAF V600E-positive GBM survived relatively longer compared with patients without this mutation. Therefore, tumors with the BRAF V600E mutation may represent a more favorable subtype of GBM. More patients with GBM must be analyzed to conclude that the BRAF V600E mutation is a potential prognostic marker for GBM. Moreover, inhibitors of B-Raf protein kinase activity may serve as efficacious drugs for treating patients with BRAF V600E-positive GBM [10,11]. Therefore, we suggest that we should perform routine genetic testing of BRAF V600E mutation, which might provide effective alternatives to treat patients with GBM.