BRAFnon-V600E more frequently co-occurs with IDH1/2 mutations in adult patients with gliomas than in patients harboring BRAFV600E but without a survival advantage

Background The effects of BRAFnon-V600E and BRAFV600E on the outcomes and the molecular characteristics of adult glioma patients are unknown and need to be explored, although BRAFV600E has been extensively studied in pediatric glioma. Methods Co-occurring mutations and copy number alterations of associated genes in the MAPK and p53 pathways were investigated using data from The Cancer Genome Atlas (TCGA) public database retrieved by cBioPortal. The prognosis of available adult glioma cohorts with BRAFV600E and BRAFnon-V600E mutations were also investigated. Results Ninety patients with BRAFV600E or BRAFnon-V600E were enrolled in this study, and data from 52 nonredundant patients were investigated. Glioblastoma multiform was the most common cancer type, with BRAF non-V600E and BRAFV600E. TP53 (56.00% vs. 7.41%), IDH1/2 (36.00% vs. 3.70%), and ATRX (32.00% vs. 7.41%) exhibited more mutations in BRAFnon-V600E than in BRAFV600E, and TP53 was an independent risk factor (56.00% vs. 7.41%). Both BRAFnon-V600E and BRAFV600E frequently overlapped with CDKN2A/2B homozygous deletions (HDs), but there was no significant difference. Survival analysis showed no difference between the BRAF non-V600E and BRAFV600E cohorts, even after excluding the survival benefit of IDH1/2 mutations and considering the BRAFnon-V600E mutations in the glycine-rich loop (G-loop) and in the activation segment. The estimated mean survival of patients with BRAFnon-V600E & IDH1/2WT with mutations in the G-loop groups was the shortest. Conclusions BRAFnon-V600E exhibited a stronger association with IDH1/2 mutations than BRAFV600E, but no survival advantage was found. Supplementary Information The online version contains supplementary material available at 10.1186/s12883-021-02224-6.

Background BRAF (v-raf murine sarcoma viral oncogene homolog B1) is a serine-threonine kinase in the Ras/Raf/mitogenactivated protein kinase (MAPK) pathway [1,2] that transduces mitogenic stimuli after the activation by growth factor receptors that are involved in cell survival, proliferation, and differentiation [3]. MAPK pathway activation is common in various neoplasms. Active RAS mutations have been detected in approximately 15% of malignant human tumors.
Compared with ARAF and RAF1, BRAF plays a critical role in kinase activity [4]. A previous study showed that RAF1 is activated by BRAF through direct interactions between proteins and phosphorylation [5]. BRAF participates in the pathological mechanism of 7% of human neoplasms, especially in patients with melanoma and colorectal, thyroid, and lung cancer [6,7]. The expression of BRAF is highly restrained [1,8]. The high expression of BRAF in neural cells indicates that it is a vital MEK kinase in neuronal tissues [9,10]. BRAF mutations are found in some central nervous system neoplasms. In pediatric low-grade gliomas (LGGs), these alterations correlate with oncogenic senescence, which may contribute to an improved prognosis [11]. The BRAF V600E mutation is rare in adult LGGs and glioblastomas and can only be found in 1 to 5% of samples [12,13]. While BRAF activation contributes to tumor development and progression in the neural stem cells and progenitor cells of Homo sapiens, BRAF mutations are detected in adult diffuse gliomas and are associated with poor outcomes [14].
Most studies have focused on the BRAF V600E mutation, although more than 70 BRAF mutations have been reported to date. Mutations in BRAF at V600 can activate ERK, which plays a critical role in the G1/S transition by adjusting the expression of cyclin D, cyclin E, and p21Cip1 [15]. The BRAF V600E mutation is the most potent MAPK pathway activator, whereas BRAF non-V600E mutations are low-activity kinases that slightly stimulate the MAPK pathway [16]. However, these low-activity BRAF mutants could activate MAPK signaling in COS-1 cells to a high level by activating RAF1 [16].
Isocitrate dehydrogenase (IDH) is a frequent mutation associated with a survival benefit in glioma patients and it has been defined as a molecular parameter to define the categories of brain tumors in the updated 2016 edition of the World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS) [17]. IDH1 and BRAF V600E mutations are associated with infiltrative gliomas or circumscribed gliomas and glioneuronal tumors, respectively [18,19], and they are exclusive in most cases [20]. The exact effect of BRAF non-V600E and BRAF V600E on the prognosis of glioma patients and whether there are unique molecular characteristics in their MAPK and p53 pathways remain largely unknown.
In this study, co-occurring mutations and copy number alterations of 35 associated genes in the MAPK and p53 pathways were retrieved and investigated, and the prognosis of the available adult glioma cohorts with BRAF V600E and BRAF non-V600E were evaluated by using The Cancer Genome Atlas (TCGA) public database. We determined that BRAF non-V600E exhibited a stronger association with the IDH1/2 mutation than BRAF V600E , but no survival advantage was found.

Data collection and enrollment
All data were collected and generated from the TCGA public database using the TCGA data mining tool cBio-Portal (https://www.cbioportal.org/) [21,22]. We strictly followed the TCGA publication guidelines (https://www. cancer.gov/about-nci/organization/ccg/research/ structural-genomics/tcga/using-tcga/citing-tcga). In multiple patient cohorts of all twenty available CNS/brain studies (6164 samples), the available data were queried, including the gene mutations, copy number alterations, mRNA expression, and protein expression data of patients with BRAF gene mutations. In each study, the mutations were selected for genomic profiles. Samples with mutation data were selected for the patient/case set and entered into three groups: (1) General: Ras-Raf-MEK-ErK/JNK signaling (26 genes), including KRAS, HRAS,  BRAF, RAF1, MAP 3 K1, MAP 3 K2, MAP 3 K3, MAP 3  K4, MAP 3 K5, MAP 2 K1, MAP 2 K2, MAP 2 K3, MAP 2  K4, MAP 2 K5, MAPK1, MAPK3, MAPK4, MAPK6,  MAPK7, MAPK8, MAPK9, MAPK12, MAPK14, DAB2,  RASSF1, and RAB25; (2) General: p53 signaling (6 genes), including TP53, MDM2, MDM4, CDKN2A, CDKN2B, and TP53BP1; (3) Other frequently mutated genes, including IDH1, IDH2, and ATRX, were then submitted for query. Among the downloadable data files, the available data regarding the mutations, copy number alterations, mRNA expression, and protein expression were downloaded. In the type of genetic alterations across all samples, samples harboring the BRAF mutation were chosen. Data regarding mutations and copy number alterations on the summary page and the patient and sample data on the clinical data page were downloaded. All of the data were recorded in a chart for further analysis (Supplementary Dataset S1).
Major characteristics of the BRAF V600E and BRAF non-V600E cohorts using univariate logistic regression analysis The enrolled populations were divided into BRAF V600E and BRAF non-V600E groups. The numbers and percentages of categorical variables were calculated. Their demographic characteristics, including sex, diagnosis age, cancer type, and overall survival status, were analyzed using univariate logistic regression analysis. The odds ratios (ORs) and 95% confidence intervals (CIs) were estimated.
Co-occurring mutations of the BRAF V600E and BRAF non-V600E cohorts using univariate and multivariate logistic regression analysis The numbers and percentages of categorical variables were calculated in the BRAF V600E and BRAF non-V600E groups. The available data for co-occurring mutated genes in these two groups were analyzed using univariate logistic regression analysis. Thereafter, significant variables (P < 0.10) were analyzed using multivariate logistic regression analysis. The ORs and 95% CIs were estimated.
Co-occurring copy number alterations in the BRAF V600E and BRAF non-V600E cohorts using heatmap and univariate logistic regression analysis The available copy number alterations of the BRAF V600E and BRAF non-V600E cohorts were retrieved and displayed using a heatmap by Morpheus (https://software. broadinstitute.org/morpheus). The putative copynumber alterations are as follows: − 2 = homozygous deletion; − 1 = hemizygous deletion; 0 = neutral/no change; 1 = gain; 2 = high-level amplification. Univariate logistic regression analysis was used to calculate the numbers and percentages of CDKN2A homozygous deletion (HD) and CDKN2B HD. The ORs and 95% CIs were estimated.
Crossover analysis with Kaplan-Meier survival curves and the log rank (mantel-Cox) test The overall survival rates of the BRAF V600E and BRAFnon-V600E cohorts were compared using Kaplan-Meier curves and the log rank (Mantel-Cox) test [23]. To exclude the benefit of IDH1/2 on survival, we referred to the BRAF V600E & IDH1/2 WT group as the BRAF V600E group minus those with IDH1/2 mutations, as well the BRAF non-V600E & IDH1/2 WT group as the BRAF non-V600E group minus those with IDH1/2 mutations. The survival of the BRAF V600E & IDH1/2 WT group was compared with that of the BRAF non-V600E & IDH1/2 WT groups. There were two clusters of mutations, one in the glycine-rich loop (referred to as the G-loop) and the other in the activation segment. To evaluate the effect of the mutation site on survival, we defined two subgroups in the BRAF non-V600E & IDH1/2 WT group. One subgroup was the BRAF non-V600E & IDH1/2 WT group with the mutation site in the G-loop, and the other subgroup was the BRAF non-V600E & IDH1/ 2 WT group with the mutation site in the activation segment. The BRAF V600E & IDH1/2 WT group was compared with those two subgroups. Furthermore, the G-loop BRAF non-V600E & IDH1/2 WT subgroup was compared with the remaining patients in the BRAF non-V600E & IDH1/2 WT group.

Statistical analysis
Major characteristics, co-occurring mutations and copy number alterations of the BRAF V600E and BRAF non-V600E cohorts were analyzed using univariate logistic regression analysis. Significant variables (P < 0.10) of co-occurring mutations of the BRAF V600E and BRAF non-V600E cohorts were analyzed using multivariate logistic regression analysis. Kaplan-Meier curves were generated for glioma patients with BRAF mutations and were compared using the log-rank (Mantel-Cox) test. A P value < 0.05 was considered statistically significant.

Data enrollment in the study
In all 20 CNS/brain studies (6164 samples), 4674 samples with mutation data were queried; 90 samples (90 patients) with BRAF mutations, including 53 samples (53 patients) with BRAF V600E and 37 samples (37 patients) with BRAF non-V600E , are shown in Table 1. The cancer types of 20 CNS/brain studies included diffuse glioma, glioblastoma, oligodendroglioma, embryonal tumor, encapsulated glioma, and miscellaneous neuroepithelial tumor. The scheme for the final enrolled and investigated data is shown in Fig. 1. Ninety patients with BRAF V600E or BRAF non-V600E were enrolled in this study, and data from 52 nonredundant patients were investigated. The integrated data of their major patient characteristics, including sex, age, diagnosis age, cancer type, data of co-occurring mutations, copy number alterations, and overall survival time and status, were collected for further analysis.
Major characteristics of the cohorts with BRAF V600E and BRAF non-V600E The study populations were divided into two groups, BRAF V600E and BRAF non-V600E . The major demographic characteristics and clinical data of the two groups are summarized in Table 2. The patients' ages ranged from 20 to 85 years and were divided into early adulthood, midlife, mature adulthood, and late adulthood (aged 20-35, 35-50, 50-80, and > 80 years, respectively). The two groups had comparable proportions of male patients, diagnosis age, cancer type, and overall survival status. Glioblastoma multiform was the most common cancer type in both cohorts (74.07% vs. 56.00%; P = 0.175; Table 2).
Co-occurring copy number alteration in the BRAF V600E and BRAF non-V600E cohorts using heatmap and univariate logistic regression analysis There were no available copy number data for five patients with BRAF V600E and five patients with BRAFnon-V600E . The copy number alterations of the available co-occurring genes included BRAF, RAF1, MAP 3 K1, MAP 2 K1, MAP 2 K2, MAP 2 K4, MAPK1, MAPK3, TP53, MDM2, MDM4, TP53BP1, IDH1, IDH2, ATRX, CDKN2A, and CDKN2B. The HD copy number was frequently retrieved for these two genes, including CDKN2A and CDKN2B (Fig. 2), and the HD of both CDKN2A (77.27.00% vs. 60.00%; P = 0.032) and CDKN2B (77.27.00% vs. 60.00%; P = 0.032) was more frequent in Table 1 The CNS/brain projects of TCGA database enrolled in the study retrieved by cBioPortal  (Fig. 3). The numbers at risk of Kaplan-Meier survival curves were shown in Supplementary Dataset S2.

Discussion
BRAF mutations critically affect cancer growth and progression and are supposed to be a founder event for mutations occurring early in the initiation process of cancer. However, BRAF mutations must cooperate with other mechanisms for a fully cancerous state, as they are insufficient to induce cancer alone [5]. BRAF V600E has  been the mutation of interest in previous studies on glioma, especially in pediatric glioma patients, for the available molecule-targeted drugs. However, various BRAF non-V600E cells exert different activation effects on the MAPK pathway. The exact impact on the clinical prognosis and possible molecular mechanism of associated co-occurring genes with mutations or copy number alterations co-occurring with BRAF mutations remains unclear in adult glioma patients. In this study, the available data of patients with BRAF non-V600E and BRAF V600E in the TCGA CNS/brain database were investigated to determine the possible mechanisms of BRAF gene mutations in adult glioma patients. Our data indicated that in adult glioma patients with BRAF mutations, including both BRAF non-V600E and BRAF V600E cohorts, glioblastoma multiform was the most common cancer type. A previous study showed that all BRAF V600E glioblastomas were primary tumors in both pediatric and adult patients [44]. Tabouret et al. [20] reported a case the co-occurrence of both IDH1 mutation and BRAF V600E although those two mutations are mutually exclusive in glial tumor. The available cooccurring mutated genes in the MAPK and p53 pathways showed that mutated genes frequently co-occurred in the BRAF non-V600E cohort, and there were more TP53, IDH1/2, and ATRX mutations in BRAF non-V600E than in BRAF V600E . Lai et al. [45] found that a TP53 point mutation at position 273 (Arg to Cys) was more common than IDH1 mutations at position 132 (Arg to His). They hypothesized that the TP53 mutation (C → T) occurred in the nontranscribed strand, while the IDH1 mutation existed in the transcribed strand, which is a strand asymmetry pattern [46]. Another study indicated that IDH1/2 mutations represent early events in brain tumor Fig. 2 The co-occurring copy number alterations of the BRAF V600E cohort and BRAF non-V600E cohort using a heatmap. The cohorts of BRAF V600E (red) or BRAF non-V600E (green) are shown, and putative copy-number alterations change from light to dark with value enhancement formation [47]. Liu et al. [48] found that ATRX alterations correlated with mutations in IDH1/2 and TP53 in glioma of all grades. It has been reported that ATRX deletions/mutations are correlated with TP53 and IDH1 mutations [49,50]. Somatic TP53, ATRX, and IDH1/2 mutations have been found in adult LGGs [51]. ATRX mutations are detected in adult diffuse gliomas and astrocytomas harboring both TP53 and IDH1/2. The cooccurrence of these three mutated genes, including TP53, IDH1/2, and ATRX, facilitates the growth of an adult diffuse astrocytoma subgroup [48]. All of the studies above indicate that ATRX mutations frequently overlap with IDH1/2 and TP53 mutations. In the present study, we also found the co-occurrence of these three mutations, which were frequently detected in the BRAFnon-V600E cohort but not in the BRAF V600E cohort. Our findings indicated that in adult glioma patients, a possible correlation between BRAF non-V600E and these three common mutations simultaneously occurred in glioma.
Multivariate logistic regression revealed that TP53 was an independent risk factor in the BRAF non-V600E cohort vs. the BRAF V600E group. Our data demonstrated a correlation between BRAF non-V600E and TP53 mutations in adult glioma patients. Previous findings have shown that active Ras can induce heterodimerization of BRAF and RAF1 [52] and that this event may be critical for RAF1 activation [53]. RAF1 directly regulates cell apoptosis, which does not depend on MAPK signaling [54,55], but occurs through direct interaction with Bcl-2 [54]. TP53 can regulate Bcl-2 by suppressing Bcl-2 transcription [56]. We proposed that the BRAF non-V600E mutation might activate the BRAF-RAF1 heterodimer, which shows antiapoptotic properties via the activation of Bcl-2 through RAF1 phosphorylation. Mutant TP53, which is frequently accompanied by IDH1/2 mutation by a strand asymmetry mechanism, fails to regulate Bcl-2. Therefore, with both activated RAF1 and mutated TP53, an enhanced antiapoptotic effect, which promotes cancer growth, might be predicted.
Compared to BRAF fusions, BRAF V600E tends to be more aggressive, more likely to be associated with CDKN2A/B deletions, and can transform cancers into higher-grade tumors [57,58]. Our data showed that CDKN2A and CDKN2B HDs were more frequent in the BRAF V600E cohort than in the BRAF non-V600E cohort. Concomitant CDKN2A and CDKN2B HDs could be detected in patients with glioblastoma multiform cancer, astrocytoma, and gliosarcoma. A previous report indicated that five of seven pediatric grade II-IV astrocytomas with BRAF V600E had concomitant CDKN2A HD [59] and CDKN2A deletions combined with BRAF V600E alterations, constituting a subgroup of secondary high-grade gliomas [60]. We found that in adult glioma patients, BRAF V600E and BRAF non-V600E frequently co-occurred with CDKN2A HDs combined with CDKN2B HDs, especially in patients with BRAF V600E . Except for astrocytoma, glioblastoma multiform cancer was the most common cancer type with these combined alterations. Robinson et al. [61] indicated that activated Akt or Ink4a/ARF deletions are necessary for high-grade brain neoplasms with BRAF mutations in a Cre/lox animal model. Our results showed the possible synergy of CDKN2A and CDKN2B HDs with BRAF mutations, especially in adult glioma patients with BRAF V600E and BRAF non-V600E .
BRAF V600E reportedly enhances BRAF kinase activity 500-fold [62]. According to its kinase viability, BRAFnon-V600E mutations can be classified into three groups: high activity (130-700 times), intermediate activity (1.3-64 times), and impaired activity (30-80%) [16]. Theoretically, the higher the BRAF kinase activity, the worse the prognosis. To clarify whether there is a difference between BRAF V600E and BRAF non-V600E , we compared the overall survival of these two cohorts, and no statistical significance was found.
In addition, the status of IDH mutations in glioblastomas definitely influences the prognosis of patients with glioblastomas; therefore, IDH-wildtype glioblastomas are defined as primary tumors, while IDH-mutant glioblastomas are classified as secondary tumors [63]. To exclude the benefit of IDH mutations on survival, we compared the BRAF V600E & IDH1/2 WT and BRAF non-V600E & IDH1/2 WT cohorts, and no difference was detected. The positions of the G-loop and the activation segment are 458-470 aa and 577-622 aa in BRAF, respectively [64]. Most BRAF non-V600E mutations exist in the G-loop and the activation segment [16,64]; therefore, we selected the two cohorts as BRAF non-V600E & IDH1/2 WT with mutations in the G-loop and activation segment. We compared them with BRAF V600E & IDH1/2 WT , and no difference was found between the BRAF V600E & IDH1/ 2 WT cohorts and those of the BRAF non-V600E & IDH1/ 2 WT cohorts. Furthermore, we compared BRAF non-V600E & IDH1/2 WT with mutations in the G-loop with the remaining BRAF non-V600E & IDH1/2 WT patients and found no difference between them. Although there was no statistical significance, the estimated mean survival of BRAF non-V600E & IDH1/2 WT with mutations in the G-loop was the shortest in all cohorts. We propose that a larger sample is necessary for confirmation of this finding. Our data indicated that the BRAF non-V600E cohort had no survival advantage from co-occurrence with IDH mutations compared with the BRAF non-V600E cohort of adult patients with glioma.

Limitations
Because the BRAF V600E mutation is rare in adult glioma, there were few patients in both cohorts retrieved from the publicly available data (cBioPortal). In this study, while their apparent survival times were substantially different, they were not significantly different. To prove the mechanism by which BRAF mutations promote cancer growth via an enhanced antiapoptotic effect of Bcl-2, further study using appropriate clinical tissue samples or animal models are necessary.

Conclusions
In conclusion, we found that in adult patients with gliomas, BRAF non-V600E , rather than BRAF V600E , frequently co-occurs with TP53, IDH1/2, and ATRX mutations. Both BRAF non-V600E and BRAF V600E frequently overlapped with CDKN2A/2B HDs, whereas there were no significant differences between the two cohorts. Although there were significant differences in co-occurring gene mutations and copy number alterations, no difference was found in survival between cohorts of BRAFnon-V600E and BRAF V600E with and without IDH1/2 favorable effects on survival. We also found that the estimated mean survival of BRAF non-V600E & IDH1/2 WT with mutations in the G-loop was the shortest; however, no difference was observed between that cohort and other cohorts. Due to the poor available mRNA and protein data in the TCGA database we retrieved in this study, no expression data were evaluated. More clinical data or models are necessary to elucidate the mechanism involved in BRAF non-V600E -associated glioma in the future.