Introduction

The World Health Organization (WHO) classification of central nervous tumors, revised in 2016, classified glioblastoma (GBM) into two groups according to the genetic status of isocitrate dehydrogenase (IDH); that is, GBM-IDH mutant, and GBM-IDH wild-type (WT) [1]. In the 2021 WHO classification, IDH-mutant cases were classified as “astrocytoma, IDH-mutant grade 4,” and IDH-wild cases as “GBM, IDH-WT” [2]. Due to the poor prognosis of GBM despite standardized treatment with temozolomide (TMZ) and radiotherapy (RT), further molecular therapies targeting genetic alterations in GBMs have been explored [3].

A missense mutation at the amino acid position 600 of the Braf proto-oncogene (BRAF V600E) is frequently detected in pediatric low-grade gliomas, including 18–38.7% of gangliogliomas [4,5,6], 9–15.6% of pilocytic astrocytomas [4,5,6], and 50–66.7% of pleomorphic xanthoastrocytomas (PXAs) [4,5,6,7]. The BRAF mutation has been detected in a subset of GBMs [6, 8,9,10,11] and the majority of epithelioid GBMs (E-GBMs), which is an aggressive subtype of GBM [12, 13]. E-GBM has been described as a variant of GBM in the WHO classification in 2016 [1], and a histological pattern of “glioblastoma, IDH-wildtype” in the WHO classification in 2021 [14]. Regarding the molecular features of GBM with the BRAF mutation (BRAF-GBM), recent studies have revealed that a missense mutation in the promoter of telomerase reverse transcriptase (TERT) and homozygous deletion of cyclin-dependent kinase inhibitor 2A (CDKN2A-HD) were frequent and similar to IDH-wild GBMs [13, 15]. However, the clinical features of BRAF-GBM have not been elucidated due to the low number of cases.

The efficacy of targeting therapy for BRAF V600E using dabrafenib and trametinib have been reported in clinical experiences [16,17,18,19,20,21,22,23,24,25]. To screen the potential targeting therapy candidates for BRAF V600E, clinical sequencing of cancer-associated genes would be the most appropriate approach in clinical practice [26]. However, there are no standardized principles to recommend clinical sequencing in GBM patients, and the clinical characteristics of BRAF-GBM would have significant value for recommendation of BRAF screening. The aim of this study was to reveal the clinical and radiological features of BRAF-GBM.

Methods

Patient population

This study included patients with GBM who underwent treatment in Hokkaido University Hospital from 2000 to 2021 and whose frozen tumor tissues were available. Adult and young adult patients older than 15 years of age were included. Pathological diagnoses of GBM were provided by institutional pathologists according to the WHO classification of Central Nervous System revised 4th edition in 2016 [27]. Clinical, pathological, and radiological data of patients were retrospectively analyzed by referring to their medical record. All manipulations were performed under approval from our institutional review board. As this study was retrospective, the requirement for informed consent was waived.

Genetic analysis

DNA/RNA was extracted from frozen tumor tissues using an AllPrep DNA/RNA Mini Kit (Qiagen, Tokyo, Japan) in accordance with the manufacturer’s recommendations. Two hotspots within the TERT promoter (C228T, C250T), along with mutation hotspots at codon 132 of IDH1, codon 172 of IDH2, and codon 600 of BRAF were screened using Sanger sequencing, as previously described [28]. CDKN2A-HD was analyzed by multiplex ligation-dependent probe amplification [8], or we referred to the result of the clinical sequencing panel using FoundationOne CDx (Foundation Medicine, Cambridge, USA) or OncoGuide™ (Sysmex, Kobe, Japan).

Statistical analysis

Statistical analysis was performed using Graphpad Prism 8 (GraphPad Software, San Diego, CA). A p value less than 0.05 was considered statistically significant for a log-rank test and analysis of variance (ANOVA) with a Tukey–Kramer test as a post hoc analysis.

Results

Patient demographics

Among the 179 cases analyzed in this study, mutations in BRAF, IDH1/2, and TERT promoter mutations were detected in 9 cases (5.0%), 9 cases (5.0%) and 96 cases (54.9%), respectively (Fig. 1a). None of the BRAF-mutant cases presented co-occurrence with the IDH mutation and one case with the BRAF mutation presented co-occurrence with the TERT promoter mutation. Pathological diagnoses in BRAF-mutant cases were classical GBM in eight cases and epithelioid GBM in one case. Eight of nine cases had a BRAF V600E mutation and one case had an in-frameshift deletion with BRAF c.1799_1801del that resulted in p.V600E and p.K601del. CDKN2A-HD was detected in seven cases (Table 1). In this study, we classified the cases into three groups according to the status of BRAF and IDH1/2: BRAF-mutant (BRAF group, 9 cases), IDH1/2-mutant (IDH group, 9 cases), and wild-type BRAF and IDH1/2 (WT group, 161 cases).

Fig. 1
figure 1

Patient demographics and clinical features of a GBM with a BRAF mutation in this study. a Genetic landscape presenting mutations of BRAF, IDH1/2, and TERT promoters in nine cases (5.0%), nine cases (5.0%), and 96 cases (54.9%), respectively. b Age at onset [median (range)] in the BRAF group, IDH group, and WT group was 37 (27–66) years, 43 (22–67) and 63 (15–89) years, respectively. Age at onset in the BRAF group and IDH group was significantly younger than in the WT group (p = 0.0005 and p = 0.0151, respectively). ANOVA: *; p < 0.05, ***; p < 0.001. c Among 46 cases with negative IDH1-R132H staining and age < 55 years, a BRAF mutation was detected in seven cases (15.2%) and no cases harbored an IDH1/2 mutation. d Kaplan–Meier survival curve in this study presenting significantly longer OS for the BRAF group and IDH group compared with the WT group (p = 0.0364 and p = 0.0162, respectively). CI = confidence interval; HR = hazard ratio

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Table 1 Summary of BRAF-GBM in this study
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Clinical characteristics of GBM with the BRAF mutation

Clinical features of nine cases with the BRAF mutation, including eight cases with classical GBM and one case with E-GBM, are summarized in Table 1. The median age at onset of the BRAF group was 37 years (range 27–66), which was significantly younger than the WT group but was similar to the IDH group (Fig. 1b). Among 46 cases with negative IDH1-R132H staining and age < 55 years, seven cases with a BRAF-mutant accounted for 15.2%, while no cases presented with the IDH1/2 mutation (Fig. 1c). Major symptoms at onset were seizure in three patients and stroke-like symptoms due to intratumoral hemorrhage in three patients. All patients except for one case had a single lesion that arose in a cerebral hemisphere.

Treatment outcome of GBM with a BRAF mutation

Five out of nine cases with BRAF-GBM showed recurrence including dissemination in four cases during observation period (Table 1). The survival curve of the BRAF group within 24 months was similar to the WT group due to early death; however, the presence of patients with long-term survival resulted in significantly longer overall survival (OS) compared with the WT group (Fig. 1d). There was no significant difference between the BRAF group and IDH group, which also presented a favorable OS compared with the WT group (Fig. 1d).

Radiological findings of GBM with BRAF mutation

Among 6 cases without hemorrhagic onset, all cases showed enhanced lesions on gadolinium-enhanced (Gd-) T1 weighted imaging (WI), while 3 cases (cases 1, 4, and 6) had mild perifocal edema on T2WI or fluid attenuated inversion recovery (FLAIR) on MRI (Supplementary Figure). A well-circumscribed border on Gd-T1WI was observed in five cases (cases 1–4 and 6, cortical involvement was observed in three cases (cases 1, 2, and 6), and a large cystic component was observed in one case (case 2). Among three cases with hemorrhagic onset, two cases had a prior lesion on FLAIR that was diagnosed at 3 and 8 years before, respectively (Table 1).

Representative cases

Case #1 (non-hemorrhagic onset, long-term survival without recurrence)

A 27-year-old female presented with convulsive and repeated dysosmia, which was considered as partial seizures. MRI showed a mass lesion in the mesial temporal lobe, in which Gd-T1WI showed ring-like enhancement with a well-circumscribed border and cortical involvement and FLAIR showed mild perifocal edema (Fig. 2a, Supplementary Figure). Gross total removal (GTR) was achieved at tumor resection. A pathological examination indicated a necrotic area (Fig. 2b) and dense tumor cells with nuclear atypia and mitosis (Fig. 2c). The Ki-67 labeling index was 50% and the pathological diagnosis was GBM. The patient underwent oral administration of TMZ (75 mg/m2), 60 Gy (Gy) of local RT, and a subsequent maintained administration of TMZ (150 mg/m2/4 weeks). She had no neurological deficit and no recurrence for 69 months.

Fig. 2
figure 2

Radiological and pathological findings of representative cases with non-hemorrhagic onset (a–c: case 1, d–f: case 4). a Preoperative Gd-T1WI of MRI of case 1 who had a tumor with ring-like enhancement in the left mesial temporal lobe. b HE staining of case 1 (original magnification 4 × , scale bar = 200 μm) showing global necrosis. c HE staining of case 1 (original magnification 20 × , scale bar = 50 μm) showing dense tumor cells with nuclear atypia. d Preoperative Gd-T1WI of MRI of case 4 who had a tumor with ring-like enhancement in the right frontal lobe. e HE staining of case 4 (original magnification 4 × , scale bar = 200 μm) showing global necrosis and microvascular proliferation. f HE staining of case 4 (original magnification 40 × , scale bar = 20 μm) showing dense tumor cells with nuclear atypia with gemistocytic tumor cells supporting the diagnosis of E-GBM

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Case #4 (non-hemorrhagic onset, early recurrence)

A 42-year-old male presented with a grand mal seizure. MRI indicated a tumor in the right frontal lobe with ring-like enhancement and a well-circumscribed border on Gd-T1WI and mild perifocal edema on FLAIR (Fig. 2d, Supplementary Figure). GTR was achieved at tumor resection. A pathological examination identified dense tumor cells with nuclear atypia, microvascular proliferation, and necrosis (Fig. 2e). Gemistocytic tumor cells were also observed (Fig. 2f) and the pathological diagnosis was E-GBM. He underwent oral administration of TMZ (75 mg/m2), 60 Gy of RT, and a subsequent maintained administration of TMZ. However, local recurrence and spinal dissemination were detected at 3 months after surgery and he died five months after surgery.

Case #7 (hemorrhagic onset)

A 44-year-old female presented with sudden onset of headache and aphasia. Head CT showed a high-density lesion on the left insula and FLAIR on MRI showed a high-intensity lesion surrounding the hematoma, which suggested hemorrhagic onset of an intra-axial tumor (Fig. 3a). Emergent surgery was performed, and the pathological specimens exhibited hematoma with a small amount of diffuse astrocytoma showing the Ki-67 labeling index of 5% (Fig. 3b). Based on the postoperative MRI presenting no apparent residual lesion (Fig. 3c) and low-grade histological malignancy, the patient was carefully observed without additional treatment; however, MRI showed recurrence at nine months after surgery (Fig. 3d). She underwent additional surgery and subtotal removal was achieved. Pathological examinations presented increased tumor cell density and nuclear atypia. The Ki-67 labeling index was 25% and the diagnosis was GBM (Fig. 3e). She underwent oral administration of TMZ (75 mg/kg) and 60 Gy of irradiation followed by 24 courses of maintained administration of TMZ. She had no apparent recurrence for 39 months after treatment.

Fig. 3
figure 3

Radiological and pathological findings of a representative case with hemorrhagic onset (Case 7). a Radiological findings at presentation (left: CT, right: FLAIR) indicating a high-density lesion in the left insular cortex and high intensity lesion surrounding the hemorrhage. b HE staining (original magnification 20 × , scale bar = 50 μm) showing diffusely proliferating astrocytic tumor cells with a prominent eosinophilic cytoplasm and multinucleated giant cells and occasional microcalcification. c Postoperative FLAIR of MRI indicating removal of the right putaminal hemorrhage. d Gd-T1WI of MRI taken at nine months after the initial surgery indicating the recurrence of a ring-like enhanced lesion. e HE staining (original magnification 20 × , scale bar = 50 μm) showing dense pleomorphic tumor cells with nuclear atypia

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Case #8 (hemorrhagic onset with a prior lesion)

This male patient was shown to have an intra-axial lesion on the right occipital lobe due to headache at the age of 28 years (Fig. 4a), and he was observed at another institution. At the age of 31 years, he presented with a sudden onset headache. Head CT showed a high-density lesion in the right occipital lobe, and MRI showed mild perifocal edema and heterogenous enhancement in the lesion (Fig. 4b). Emergent surgery was performed and GTR was achieved. The pathological diagnosis was GBM (Fig. 4c) and he underwent oral administration of TMZ and 60 Gy of RT followed by maintenance TMZ. However, he underwent additional resection surgery for local recurrence at 10 months after the initial surgery. Although he was treated with TMZ and bevacizumab, distant recurrence was observed 7 months later. Clinical sequencing revealed BRAF V600E and he started combined treatment with dabrafenib and trametinib, but he died due to progression of the disease at 29 months after initial treatment.

Fig. 4
figure 4

Radiological and pathological findings of representative cases with a prior lesion and hemorrhagic onset (a–c: case 8, d–f: case 9). a FLAIR of case 8 taken at the age of 28 years showing a high intensity lesion in the right occipital lobe. b Radiological findings of case 8 at 31 years of age (left: CT, middle FLAIR, right: Gd-T1WI) showing a high-density lesion with hemorrhage surrounding a high intensity lesion and heterogeneous enhancement in the wall of the lesion in the right occipital lobe. c HE staining of case 8 (original magnification 10 × , scale bar = 100 μm) showing highly cellular tumor cells with palisading necrosis. d FLAIR of case 9 taken at the age of 30 years showing a high intensity lesion in the right temporal lobe. e Radiological findings of case 9 at 31 years of age (left: CT, right: FLAIR) showing a high-density lesion with hemorrhage and a surrounding high intensity lesion in the right temporal lobe. f HE staining (original magnification 40 × , scale bar = 20 μm) indicating astrocytic tumor cells with a monomorphic population

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Case #9 (hemorrhagic onset with a prior lesion)

This male patient was diagnosed in other institution with a right temporal lobe abnormality on MRI at the age of 30 years due to epilepsy (Fig. 4d); however, surgical confirmation was not performed, because the patient did not consent. At the age of 38 years, he presented with sudden onset of headache. Head CT showed a high-density lesion in the right temporal lobe, FLAIR on MRI showed a high-intensity lesion around the hemorrhage, and Gd-T1WI showed ring-like enhancement (Fig. 4e). Emergent surgery was performed and the pathological diagnosis was GBM (Fig. 4f). He underwent standardized treatment with TMZ and 60 Gy of irradiation, and salvage surgery for the remaining lesion was performed after irradiation. He continued TMZ maintenance without evidence of recurrence for 30 months after the initial surgery.

Discussion

Previous reports indicated that the BRAF mutation accounted for 1.6–6.3% of adult GBM cases [6, 8,9,10] and 15 of 633 (2.4%) adult high-grade gliomas including WHO grade 3 cases [8]. The BRAF mutation has been observed more frequently with 16 of 107 (15.0%) cases in the study of young adult GBM cases [11]. Our results also indicated younger ages at onset in BRAF-mutated cases as well as IDH-mutant cases, which was consistent with previous reports [29, 30]. Because of the low frequency of IDH mutations in the elderly, the current WHO classification recommends sequencing analysis of IDH1/2 in cases with age < 55 years if immunostaining for IDH1-R132H is negative [1]. Although cases with co-occurrence of BRAF and IDH1/2 mutations have been reported [5, 12, 29], most of the BRAF-mutated cases did not harbor the IDH1/2 mutation [8, 10, 11]. Thus, we consider that analysis of the BRAF mutation would be valuable, especially in patients who are less than 55 years old and are negative for IDH1-R132H as presented in our study.

Dissemination, which is frequent in E-GBM cases [13, 15], was observed in four out of five recurrent cases including one E-GBM case in this study. Classical GBM with BRAF mutation, as well as E-GBM cases, would tend to present dissemination at recurrence. Although BRAF-GBM tended to present favorable treatment outcomes compared to other IDH-wild GBMs in this study, the BRAF mutation has also been detected in E-GBMs that are associated with aggressive clinical behaviors [13, 15]. Indeed, early death after initial treatment was observed in a subset of cases in this study (Case 4), and another cohort including a higher percentage of E-GBMs indicated no survival benefit with a BRAF mutation compared with BRAF-WT cases [31]. Thus, further molecular biomarkers associated with prognoses should be clarified in BRAF-GBMs. Previous reports have revealed frequent combinations of TERT promoter mutation both in epithelioid GBMs and classical GBMs with a BRAF mutation [13, 15, 16, 29]. In our series, a combination of BRAF mutation and TERT promoter mutation was detected only in one patient with a classical GBM. Although the TERT promoter mutation has been reported as a poor prognosis marker of IDH-wild GBMs [32], it is still unclear whether the TERT promoter mutation is a prognostic marker in BRAF-mutant GBMs. CDKN2A-HD has also been detected in a large fraction of BRAF-mutant GBMs, which suggests the important role of tumorigenesis in BRAF-GBM. However, the diagnostic value of CDKN2A-HD in BRAF-GBMs is still unclear.

A well-circumscribed border and mild perifocal edema on MRI were frequent in non-hemorrhagic cases in this study and similar findings have been reported in previous studies [9, 29, 33]. However, a recent study suggested that a combination of well-circumscribed borders, large cysts, and cortical involvement was more definitive in BRAF-mutant cases rather than the extent of perifocal edemas [31]. Although one patient in our study had multifocal lesions and cases with diagnosis of gliomatosis cerebri harboring a BRAF mutation have been reported [6, 29, 34], the majority of BRAF-GBMs show a single lesion on MRI [9, 20, 22, 25, 29, 31].

In our series, a prior lesion and hemorrhagic onset were observed in two and three cases, respectively, both of which have not been reported as characteristics of BRAF-GBM previously. Prior lesions would suggest the presence of low-grade pathology, which supports the hypothesis that a subset of BRAF-GBMs arise from low-grade BRAF-associated tumors, such as PXA [35, 36]. Hemorrhagic onset is a rare clinical presentation in GBMs and several underlying mechanisms such as perivascular necrosis with subsequent loss of vessel support, thin walled or poorly formed vessels, endothelial proliferation with subsequent obliteration of the lumen, and the presence of intratumoral arteriovenous fistulae have been reported [37]. Among previous cases with BRAF-GBM, one patient with hemorrhagic onset has been reported [38]. Although the specific mechanism of hemorrhage associated with the BRAF mutation is unclear in pathological and radiological examinations in our cases, considering the frequency in this study, hemorrhagic onset may be frequent in BRAF-GBM cases.

Conclusions

Analysis of BRAF in GBM cases is recommended, especially in patients younger than 55 years of age and IDH1-R132 negative cases. Considering the clinical course, a prior lesion and hemorrhagic onset suggest the possible presence of a BRAF mutation.