Effective treatment of a BRAF V600E-mutant epithelioid glioblastoma patient by vemurafenib: a case report

Epithelioid glioblastoma (E-GBM) is a recently described variant of glioblastoma (GBM) which is associated with short survival and now added as a provisional entity to WHO 2016 classification of central nervous system tumors. About half of these tumors show the BRAF mutant. Therefore, this is a target of special interest for this group of patients. Meanwhile, unlike conventional glioblastoma, E-GBM lacks specific prognostic markers. We described a case of a long-term surviving 37-years-old men patient diagnosed with a BRAF V600E and TERT mutated E-GBM with wild-type in the isocitrate dehydrogenase gene (IDH wild-type). The tumor displayed atypical exophytic growth, an obvious proliferation of vascular endothelial cells, especially tumor tissue can be seen under subarachnoid space. Notably, tumor tissue was found under subarachnoid space. After postoperative conventional treatment options were exhausted, vemurafenib treatment was initiated. The patient remained clinically stable, and follow-up magnetic resonance images were consistent with stable disease for the following fifteen months up to now. Whole-exome sequencing analysis and RNA-seq results of formalin-fixed and paraffin-embedded tissue revealed nine mutant genes (AHNAK2, BFSP1, BRAF, CNTNAP3, DNHD1, MTOR, NFATC3, NOM1). For E-GBM patients, the use of BRAF inhibitors combined with inhibitors of these seven genes may be a useful remedial treatment option.


Background
Epithelioid glioblastoma (E-GBM) is a rare glioblastoma (GBM) variant newly added to IDH wild-type GBM in 2016 WHO classification [1]. The tumor is a highly invasive tumor with typical histological features, including closely arranged tumor cells with smooth and round cell boundaries, abundant eosinophilic cytoplasm and the absence of interspersed neuropil [2]. Compared with conventional GBM, E-GBM tends to occur in younger adults and exhibits more aggressive behavior, with a median survival of 5.6 months in children and 6.3 months in adults [3]. The diagnosis of E-GBM is often challenging, as there is still no specific immunohistochemical and molecular markers [4,5]. Interestingly, BRAF V600E mutation characteristically occurred in approximately 50% of all E-GBMs compared with a low frequency in conventional GBM [6].
The BRAF inhibitors targeting the V600E mutation (e.g. vemurafenib and dabrafenib) represented a breakthrough in the treatment of malignant melanoma. At present, BRAF inhibition is the treatment of choice if the V600E mutation is existent [7,8]. However, data on clinical use of BRAF inhibitors in patients with glioma are scarce [9][10][11][12][13].
Advances in high-throughput next-generation sequencing technology enable oncologists to decipher the genetic and epigenetic landscapes of tumors [14][15][16][17][18]. Aside from mutations, tumor driver genes may also be altered by transcriptional, methylation, or copy number variation (CNV), which makes the affected genes more difficult to be pinpointed as drivers. A further complicating issue is that mutations in individual gliomas can affect different genes in various combinations. This can alter prognosis and response to therapy [19,20] and poses a challenge to confidently identify genes that are truly collaborating with one another. Therefore, it is important to understand the functional genomic landscapes of glioma.
system [3]. Here, we present a rare case of an IDH wildtype E-GBM patient with BRAF V600E mutation, in whom clinical and follow-up magnetic resonance images (MRI) stability could be achieved for fifteen months by the treatment of BRAF inhibitor vemurafenib. Furthermore, we provide new insights on the combination of BRAF inhibitors with these mutant genes inhibitors (CNTNAP3, mTOR, NFATC3 and NOM1), which seems to be a valuable treatment option for E-GBM.

Case report
A 37-year-old man was admitted to our hospital with sudden dizziness and headache for 5 days (Fig. 1). The first MRI showed that there was a lump of abnormal signal shadow with a size of about 4.5 × 3.0 × 3.0 cm in the left temporal lobe. Severe edema regions around the lesion were present. To enable a histological diagnosis and treatment decision, tumor resection was performed. The operation was performed from the posterior part of the inferior temporal gyrus, and the old hematoma and grayish red tumor tissue were seen. Extended resection of the tumor was performed along the lateral side of the relative boundary, deep to the temporal horn of the lateral ventricle. After the surgery, the pathological diagnosis was E-GBM. The surgical specimen showed that cancer cells were distributed diffusely, and the cells were arranged closely, which were polygonal, round or triangular, with dense nuclei, visible local nucleoli, partial nuclear deviation and rich red cytoplasm. Besides, there were obvious cell atypia, visible mitotic cells, obvious proliferation of vascular endothelial cells, vasodilatation and congestion and flaky necrosis and hemorrhage. Notably, tumor tissue was found in the local subarachnoid space. The immunohistochemical results were listed as the following: Ki-67 (label index: 15%), GFAP (positive), MGMT (unmethylated), EMA (negative), EGFR (negative), VEGF (positive), ATRX (wild-type), Olig-2 (positive), IDH1 (wild-type), BRAF V600E (mutant), CIC (positive), FUBP1 (positive), H3K27M (negative), Desmin (negative) and CK (negative). Fluorescence in situ hybridization detection showed that chromosome (chr) 1p was deleted and 19q was intact. Eight days after the operation, the second MRI showed that the left temporal lobe tumor changed after resection, with effusion and a little hematocele in the operation area, and the ependyma of the right lateral ventricle body was slightly thickened.
On the eleventh day after the operation, temozolomide (TMZ, Merck Sharp & Dohme Ltd.) 75 mg/m 2 was used as neoadjuvant chemotherapy for 17 days. Compared with the second MRI, the third MRI (28 days after the operation) showed a new slice-like enhancement in the operation area and tumor recurrence was not ruled out. The fourth MRI of cervical, thoracic and lumbar showed GBM cerebrospinal fluid spread and metastasis, and the dura mater in the spinal canal and spinal cord surface was widely thickened and strengthened (35 days after the operation). Cerebrospinal fluid liquid-based cytology showed that individual malignant tumor cells could be seen (35 days after the operation). The third and fourth MRI showed that the patient's condition was still progressing after TMZ treatment. Therefore, the patient received 36 Gy total central radiotherapy (37 days after the operation) for eighteen days, and then locally pushed for 24 Gy for seven days. During concurrent radiotherapy Overview of the patient's course of disease and therapeutic regimens. and chemotherapy, the patient developed third-degree bone marrow suppression. Besides, there are persistent low back pain, a sharp deterioration of mental and dietary conditions and bed rest and inability to go to the fields. Therefore, the patient stopped radiotherapy and chemotherapy. Since all other conventional treatment options had been exhausted, and to find a target for experimental salvage therapy, we adopt genomic technology and whole-exome sequencing (WES) analysis to study the molecular characteristics of this case. The WES sequencing analysis results revealed a V600E mutation of the BRAF kinase. Consecutively, vemurafenib (Roche, BRAF inhibitor) therapy was initiated (960 mg twice daily).
Following vemurafenib treatment, the patient was in stable condition and moves freely. However, seven gastrointestinal reactions (malignant, vomiting, etc.) occurred two weeks after the start of dose treatment. Therefore, we changed the dose of vemurafenib to 480 mg, twice daily. So far, patients have been well-tolerated and the clinical follow-up was stable. Compared with the fourth MRI, the fifth MRI found that the extensive thickening and enhancement of the endocranium on the spinal canal and spinal cord surface were significantly reduced. WES sequencing analysis and RNA-seq results of formalin-fixed and paraffin-embedded tissue revealed nine mutant genes (AHNAK2, BFSP1, BRAF, CNTNAP3, DNHD1, MTOR, NFATC3, NOM1) in both DNA-seq data and RNA-seq data (Table 1 and Fig. 2a). The protein-protein interaction analysis (PPI) of these nine mutant genes suggested that NFATC3, mTOR and BRAF interact with each other (Fig. 2b). The result of CNV analysis revealed deletion of copy number on chromosome 1 and 6, and amplification of copy number of chromosome 19q and chromosome 21 in this case of E-GBM, as shown in Fig. 3.

Discussion
In the IDH wild-type, TERT mutant and BRAF V600E mutant E-GBM patient treated with vemurafenib as salvage therapy, we achieved clinical stability over 15 months, which is remarkable at that point of the clinical course after postoperative conventional treatment options were exhausted. Therefore, targeted therapy with BRAF inhibitors may constitute a valuable salvage treatment option. Furthermore, the case showed that when the postoperative conventional treatment options were exhausted in E-GBM patients, especially young patients, it may be helpful to assess whether there is a BRAF mutation. Venn diagram (a) shows the common mutant genes in both DNA-seq data and RNA-seq data. The PPI network (b) of the common mutant genes in both DNA-seq data and RNA-seq data. PPI, protein-protein interaction.
In malignant melanoma patients with a BRAF V600E mutation, targeted BRAF inhibitors had significantly improved the prognosis [7,8]. In contrast, there was little data on the curative effect of patients with brain tumors, particularly GBM. Targeted therapies such as vemurafenib or dabrafenib have been used in a limited number of brain tumor patients with predominantly pleomorphic xanthoastrocytoma, ganglioglioma and E-GBM [10][11][12][13]21]. Meletath et al. reported a case of malignant gliomas arising from ganglioglioma, which produced significant clinical and radiologic responses within 24 months through the use of dabrafenib in combination with tumor-treating fields [10]. Chamberlain [11] treated three adult BRAF V600E mutant ganglioglioma patients with dabrafenib. The median progression-free survival was seven months (range: 4-10 months). In another case series by the same author [12], similar results were observed in four patients with BRAF V600E-mutated and recurrent pleomorphic xanthoastrocytoma treated with vemurafenib. In a recently published case series, Carry [21] reported a secondary E-GBM patient who survived for 16 months after dabrafenib treatment initiation. The E-GBM patient was arising from an anaplastic astrocytoma after ten years of first-line and second-line treatment. Liangliang successfully treated a BRAF V600E-mutated extraneural metastatic anaplastic oligoastrocytoma with vemurafenib and everolimus (mTOR inhibitor) [22].
Up to now, our patients still show clinical stability over 15 months with vemurafenib. The survival rate of our patients is comparable to or even longer than that of other patients with BRAF V600E mutation [6,23,24]. A recent meta-analysis in glioma patients demonstrated an improved overall survival (hazard ratio: 0.6) if a BRAF mutation was present [25]. That meta-analysis also revealed that a BRAF V600E mutation improved the survival of children and young adults with gliomas but did The CNV analysis of the E-GBM sample. CNS, central nervous system; E-GBM, epithelioid glioblastoma.
not have prognostic value in older adults. On the other hand, children with a newly diagnosed epithelioid glioblastoma suffer from an overall poor prognosis, independent of a BRAF V600E mutation [3].
The patient is a 37-year-old young and middle-aged man with sudden headache and dizziness as the main symptom. The postoperative pathology showed E-GBM, and WES test showed IDH wild-type, TERT and BRAF mutant. The postoperative TMZ chemotherapy and radiotherapy were insensitive and severely intolerant, and the tumor cerebrospinal fluid spread. We used BRAF inhibitor for salvage treatment. Now the patient has stable clinical symptoms and no progress on MRI.

Conclusion
The cases of glioma treated with BRAF inhibitors are rare. We reported the first case of IDH wild type, E-GBM with TERT and BRAF V600E mutations, and effectively treated this patient with vemurafenib (up to now: 15 months). For E-GBM patients, the use of BRAF inhibitors may be a useful remedial treatment option.

Acknowledgements
A particular acknowledgment to the medical staff involved in the study and genomic biotechnology (Shanghai) co. ltd. for its support. Informed consent was granted from the patient to publish the present data as a case report. The Institutional Review Board of Nanfang Hospital affiliated with Southern Medical University approved the study.
Written informed consent was obtained from the patients for publication of this case report. All the authors listed have approved the enclosed manuscript.
The data analyzed in this study can be accessed by sending a request to the corresponding author.

Conflicts of interest
There are no conflicts of interest.