The clinicopathological features of ganglioglioma with CD34 expression and BRAF mutation in patients with epilepsy

Objective The aim of the study was to evaluate the clinicopathological features, as well as the surgical prognosis, of epilepsy-associated gangliogliomas (GG) with CD34 expression and BRAFV600E mutation. Methods Clinical data of patients who underwent epilepsy surgery for GG were retrospectively studied. Univariate and multivariate analyses were performed to evaluate the correlations of clinical and pathological factors with molecular markers of CD34 expression and BRAFV600E mutation in GG. Results A total of 208 patients with GG had immunohistochemical detection of CD34 expression (positive/negative: 184/24), and among them, 89 patients had immunohistochemical detection of BRAFV600E mutation (positive/negative: 54/35). By univariate and multivariate analyses, seizure aura (p = 0.025), concordance of ictal electroencephalogram (EEG) findings (p = 0.045) and medial temporal tumor (p = 0.030) were found to be related to CD34 expression, but only hospitalization time (p = 0.042) was different for BRAF-mutated status. In addition, drug-resistant epilepsy (p = 0.040) and concordance of interictal EEG findings (p = 0.009) were found to be associated with tumor progression-free survival (PFS) in univariate analysis, but only concordance of interictal EEG findings was with significance in multivariate analysis. However, CD34 expression or BRAFV600E mutation in GG was not found to be associated with surgical outcomes of seizure control and tumor PFS. Conclusion The CD34 expression or BRAFV600E mutation in GG may partly influence the distribution of clinicopathological features of patients with epilepsy, but they may be not able to predict the surgical prognosis of seizure outcome and tumor recurrence.


Introduction
Brain tumors are frequently met in patients with epilepsy surgery, and among them, gangliogliomas (GG) are the most common tumor entities (Blumcke et al., 2017;Slegers and Blumcke, 2020). Recently, the molecular markers of CD34 and BRAF mutation are interestingly found to be associated with brain tumors with epilepsy, especially with epilepsy-associated GG (Blümcke et al., 1999;Deb et al., 2006;Schindler et al., 2011;Giulioni et al., 2019;Xing et al., 2021).
CD34 is a transmembrane phosphoglycoprotein that was first identified on hematopoietic progenitor cells and subsequently vascular endothelial progenitors, multipotent mesenchymal stromal cells, and epithelial progenitors, etc., thus being regarded as a general marker of progenitor cells (Guo et al., 2003;Sidney et al., 2014). CD34 is transiently expressed in the central nervous system during early neurulation and cannot be detected in mature neuroectodermal cell progenies in the normal brain (Blümcke et al., 1999). Although CD34positive cells have been reported in gliosarcoma and giant cell variant of glioblastoma, or non-neoplastic glioneuronal hamartias or hamartomas, they are particularly represented in low-grade or developmental brain tumors, such as GG, pleomorphic xanthoastrocytoma (PXA), dysembroplastic neuroepithelial tumor (DNT) and pilocytic astrocytoma (PA), all of which are quite associated with chronic epilepsy (Blümcke et al., 1999;Reifenberger et al., 2003;Deb et al., 2006;Giulioni et al., 2019).
BRAF (v-raf murine sarcoma viral oncogene homolog B1) is a member of the RAF family of serine/threonine protein kinases, playing a critical role in transducing signals from membrane-bound, GTP-loaded RAS proteins to MEK and ERK kinases (RAS/RAF/ MEK/ERK pathway; Dougherty et al., 2010;Drosten and Barbacid, 2020). The vast majority of BRAF mutations (>90%) affect a mutational hot spot at amino acid position 600 and are characterized by the exchange of Valine by Glutamate, thus referred to as BRAF Val600Glu (or BRAF V600E ), which generates a constitutively active monomeric protein with high kinase activity that does not require RAS signaling (Davies et al., 2002;Dougherty et al., 2010). BRAF is mutated in about 8% of all human cancers, and these mutations primarily occur in melanomas and at much lower frequency in thyroid, lung, and colorectal cancer (Davies et al., 2002;Drosten and Barbacid, 2020). Recently, BRAF V600E mutations are found in brain tumors, mainly affecting glial or glioneuronal tumors, such as PXA, GG, DNT, and PA, as well as pediatric astrocytoma (Davies et al., 2002;Schindler et al., 2011;Xing et al., 2021), but glioblastoma and other gliomas were with low frequency or absence of mutations, and none of them in non-glial tumors Xing et al., 2021). More recently, the monoclonal BRAF V600E mutation-specific antibody via immunohistochemistry (IHC) detection has been found and widely used to screen for BRAF V600E mutation in the diagnostic work-up (Blümcke et al., 2016;Slegers and Blumcke, 2020), since the first BRAF V600E specific antibody was reported in 2011 (clone VE1; Capper et al., 2011).
Although the molecular expression of CD34 and BRAF V600E mutation could frequently and exclusively occur in GG with epilepsy, their clinical and pathological features were not yet well defined, as well as the prediction of long-term seizure outcome and tumor recurrence or progression (Blümcke et al., 1999;Dahiya et al., 2013;Zhang et al., 2017;Giulioni et al., 2019;Xing et al., 2021). Thus, we aimed to evaluate the associations of clinicopathological features, as well as surgical prognosis, with molecular expression of CD34 and BRAF V600E mutations in GG with epilepsy.

Patient selection
A retrospective chart review was conducted for all patients with epilepsy who underwent surgical treatment for GG between 2008 and 2021 at Sanbo Brain Hospital, Capital Medical University. This study was approved by the Capital Medical University Sanbo Brain Hospital Ethics Committee.
Patients who had epilepsy caused by brain tumors that were histopathologically confirmed as low-grade GG were enrolled in the study. The clinical data of patients with tumors that were detected by IHC with molecular markers of CD34 and BRAF V600E mutation was analyzed. Finally, a total of 208 consecutive patients who had tumors with IHC examination of CD34 expression, including 89 patients with IHC examination of BRAF V600E mutation, were enrolled in the study.

Preoperative evaluation and surgery
All patients underwent an individualized preoperative evaluation, including detailed medical history and physical examination, seizure semiology, video electroencephalogram (EEG) and brain magnetic resonance imaging (MRI). The lesion size was represented by the mean tumor diameter of T1-weighted MRI scans. The video EEG monitoring was performed in all patients for at least 16 h, and the concordant EEG findings of interictal epileptiform discharges and ictal seizure rhythms were defined as epileptiform discharge sources localized in the same tumor-invading brain hemisphere.
After detailed preoperative evaluations by neurologists, neurosurgeons, neuroradiologists and electrophysiologists, surgical plans were made. The aim of the operation was to remove the tumor and relevant epileptogenic zone (EZ). The EZ was determined by the findings of the detailed preoperative evaluation and/or intraoperative electrocorticography (ECoG). Intraoperatively, neurological electrophysiological monitoring and neuronavigation were also performed for safe tumor resection. In particular, according to the resection extent of brain tissue (determined by operative records and postoperative MRI), extensive tumor resection was defined as resection of both tumor and peritumoral cortex (or hippocampus), while simple tumor resection was those with simply resection of the tumor.

Immunohistochemical staining and pathological diagnosis
The surgically removed brain tissue specimens were fixed with 3.7% neutral formaldehyde, embedded in conventional paraffin, sliced into 5-μm thick sections, and then stained with hematoxylin and eosin (H&E). Immunohistochemical staining was performed with the following primary antibodies: anti-BRAF V600E (Spring Bioscience, monoclonal, clone VE1, 1:50), glial fibrillary acidic protein (GFAP; Dako, 1:1000), neuronal nuclear antigen (NeuN; Chemicon, 1:2000), Histopathological records were systematically reviewed by two experienced neuropathologists according to the WHO classification scheme from 2016, including a panel of immunohistochemical markers. Ki-67 proliferation index was defined by the percentage of Ki-67 positive cells in the total cell population at 40 magnifications for 10 microscopic fields. In particular, tumors with IHC detection of CD34 expression and BRAF V600E mutation were analyzed in terms of their associations with clinical and pathological features.

Follow-up examination
Patients were periodically followed up at the 3rd month and 6th month postoperatively and yearly thereafter. Follow-up evaluations of seizure and tumor recurrence or progression, as well as neurological statuses, were performed by neurosurgeons at the clinic and/or by telephone interview in each patient. Favorable seizure outcomes with Engel classification were defined as Engel class I, and unfavorable seizure outcomes were Engel class II-IV at the last follow-up evaluation.

Study variables and statistical analysis
Clinical variables of interest were evaluated between two groups of patients with or without CD34 expression and BRAF V600E mutation, including patient or demographic characteristics, tumor or pathological characteristics, seizure semiology and electrophysiological findings, surgical and follow-up variables.
Continuous variables were described with medians and interquartile ranges (IQR), while categorical variables were described with absolute and relative (%) frequencies. Descriptive statistics between compared groups were analyzed by t tests and χ 2 tests for continuous and categorical variables, respectively. When necessary, Fisher's exact test and the Kruskal-Wallis rank-sum test were used. Variables showing a p < 0.05 in the univariate analysis were then entered into the multivariate binary logistic or Cox regression model. Statistical tests were considered significant if p < 0.05. Odds ratios (OR) and hazard ratios (HR) were presented with 95% confidence intervals (CI). All data were analyzed using the software package SPSS, version 21.
Regarding video EEG findings, concordant interictal EEG findings were found in 148 patients (71.1%), while discordant findings were in 43 patients (20.7%); 17 patients (8.2%) were with unknown results due to lack of significant epileptiform discharges or being in a normal EEG setting. Concordant EEG findings of ictal seizure rhythms were found in 107 patients (51.4%), and discordant findings were found in 43 patients (20.7%), but 58 patients (27.9%) were with unknown results due to no significant ictal seizures (Table 1).

Follow-up outcomes
All patients were followed up, except for 12 patients (5.8%) lost, with the median follow-up time of 54 months (IQR: 25-79 months). Of 192 patients who were followed up for at least 12 months, 165 patients (85.9%) were seizure-free and had a favorable seizure outcome (Engel class I), while 27 patients (14.1%) had an unfavorable seizure outcome (Engel class II/9, III/13 and IV/5; Table 1). In total, 146 patients (70.2%) had anti-epileptic drugs reduced (40) or discontinued (106). During the whole follow-up period, 5 (2.6%) patients had tumor recurrence, including one with subtotal tumor resection, and the accumulated 10-year tumor progression-free survival (PFS) was 96%. Among them, 3/5 of cases had seizure recurrence, and 2 cases of GG had malignant progression.

Univariate and multivariate analyses
Clinical and pathological factors in 208 patients were compared between two groups [tumor with CD34 (+) vs. CD34 (−)] (Table 1). Significant differences were found in age of seizure onset (p = 0.017), seizure aura (p = 0.032), concordance of ictal EEG findings (p = 0.024) and medial temporal tumor (p = 0.035) by univariate analysis. In particular, surgical outcomes of seizure control (p = 0.791) and tumor recurrence (p = 0.913) were not found with differences between two groups. Multivariate binary logistic regression analysis finally included the seizure aura (p = 0.025, OR = 2.94), the concordance of ictal EEG findings (discordant vs. concordant; p = 0.045, OR = 0.35) and the medial temporal tumor (p = 0.030, OR = 0.37) into the predicting model of GG with CD34 positive expression ( Table 2).
The clinical and pathological features of 89 patients who had IHC detection of BRAF V600E mutation were also compared between two groups [tumor with BRAF (+) vs. BRAF (−)] (Table 3). Significant differences were found in hospitalization time (p = 0.042), and statistic tendency (0.05 < p < 0.1) was found in concordance of ictal EEG findings (concordant vs. discordant; p = 0.078) and age at surgery  Frontiers in Molecular Neuroscience 07 frontiersin.org (p = 0.064), but none in surgical outcomes of seizure control (p = 0.895) and tumor recurrence (p = 1.000).

The CD34 expression and BRAF V600E mutation in gangliogliomas
As a molecular marker of progenitor cells, CD34 often expresses in low-grade or developmental brain tumors, which are usually accompanied by chronic epilepsy, such as the so-called "low-grade epilepsy-associated neuroepithelial tumors (LEAT)," but none of Frontiers in Molecular Neuroscience 08 frontiersin.org normal adult or developing human brain and tumors without epilepsy are CD34 positive (Blümcke et al., 1999;Deb et al., 2006;Chappé et al., 2013;Sidney et al., 2014;Giulioni et al., 2019). The GG are the most common tumor type in patients with epilepsy and also frequently reported with CD34 expression, ranging from 60 to 90% (Deb et al., 2006;Giulioni et al., 2019). In our study, we reviewed the CD34 expression in GG with epilepsy and found 88.5% of tumors were CD34 positive, which was line with the previous studies (Deb et al., 2006;Chappé et al., 2013;Giulioni et al., 2019).
Frontiers in Molecular Neuroscience 09 frontiersin.org specified), but no difference of BRAF V600E mutation was found between GG and mixed GG (p = 0.903).

The clinicopathological features of CD34 expression
The relationship between clinicopathology and CD34 expression in GG has been studied in some studies, but the results were always inconsistent or in different types of tumors (Blümcke et al., 1999;Deb et al., 2006;Vornetti et al., 2017;Giulioni et al., 2019).
With respect to demographic features, for example, Blümcke et al. (1999) found patients with brain tumors with epilepsy and CD34 expression had younger age at seizure onset or at surgery and Vornetti et al. (2017) found CD34 expression in LEAT was significantly associated with a longer duration of epilepsy, which was similarly reported by Giulioni et al. (2019) who also reported that CD34 expression in LEAT appeared to be significantly related to older age at surgery, higher AED intake, and female sex by univariate analysis. In present study, we only found GG with CD34 expression occurred more in patients with older age (p = 0.017) by univariate analysis.
The tumor or pathological characteristics were less reported for GG with CD34 expression, except that Lisievici et al. (2021) found CD34 expression in GG was more in temporal lobe. In our study, we found GG with CD34 expression were less occurring in medial temporal sites than other sites (OR = 0.37; p = 0.030).
Although CD34 expression tends to occur in lesions with epilepsy, the seizure semiology or EEG finding is seldom reported to be related to GG with CD34 expression (Giulioni et al., 2019;Lisievici et al., 2021). In present study, however, we found patients with CD34 expression in GG had more tendency of experiencing seizure aura (OR = 2.94, p = 0.025) and concordant ictal EEG findings (discordant vs. concordant; OR = 0.34, p = 0.045) than those without CD34 expression. The Kaplan Meier curves of significant clinical factors in univariate Cox regression analysis. Patients with drug-resistant epilepsy were with a lower rate of tumor recurrence than those without drug-resistant epilepsy (Left; Log Rank test: χ 2 = 5.551, p = 0.018), as well as in patients with concordant interictal video electroencephalogram (EEG) findings when compared to those with unknown EEG findings (Right; Log Rank test: χ 2 = 11.76, p = 0.003).

The clinicopathological features of BRAF V600E mutation
Although the BRAF V600E somatic mutation in neuronal linage cells (or glial lineage cells) was proved to play a key role in epileptogenic properties (or tumorigenic properties) of GG (Koh et al., 2018), the relationships between clinicopathological features and BRAF V600E mutations in GG were not well studied or with less data (Dahiya et al., 2013;Koelsche et al., 2013;Prabowo et al., 2014;Vornetti et al., 2017;Zhang et al., 2017;Xing et al., 2021).
Several demographic features were reported to be related to GG or brain tumor with BRAF V600E mutation, including younger age at surgery for GG (p = 0.005; Koelsche et al., 2013), younger age of seizure onset for epilepsy-associated brain tumors (p = 0.020; Xing et al., 2021), and female patients for glioneuronal tumors (GNT) with epilepsy (p = 0.022; Zhang et al., 2017). However, Schindler et al. (2011) noted no significant differences of patient age at surgery for GG with BRAF V600E mutation; Zhang et al. (2017) reported no significant correlation between the BRAF status in GNT and age at surgery, as well as age of seizure onset and duration of epilepsy; and Xing et al. (2021) also did not find brain tumors with epilepsy and BRAF V600E mutation were associated with gender and duration of epilepsy. In present study, we did not find any associations of BRAF V600E mutation in GG with age of seizure onset, duration of epilepsy and patient gender, except for the statistic tendency (0.05 < p < 0.1) in older age at surgery (p = 0.064).
For tumor or pathological characteristics, Schindler et al. (2011) found GG with BRAF V600E mutation were more in temporal lobe. Prabowo et al. (2014) found in both GG and DNT, the presence of BRAF V600E mutation was significantly associated with the expression of CD34. Vornetti et al. (2017) found BRAF mutation in LEAT was predominant in right-sided lesions. However, Koelsche et al. (2013) found CD34 was not differentially expressed in BRAF wild-type and-mutated tumors of GG, and Xing et al. (2021) found there was no statistical difference between BRAF V600E mutations and wild type for tumor site. Also, we did not find GG with BRAF V600E mutation were associated with CD34 expression, tumor locations, calcification or encystation, et al.
With respect to seizure semiology or EEG findings, BRAF V600Emutated LEAT (Vornetti et al., 2017), as well as GNT , were reported to be with more seizure types. However, we did not find GG with BRAF V600E mutation were associated seizure semiology, except that the statistic tendency existed in concordance of EEG findings was different (discordant vs. concordant; p = 0.078).

Seizure outcomes and tumor recurrence
The correlations between CD34 expression or BRAF V600E mutation in GG and postoperative seizure outcomes have been evaluated in previous studies (Prabowo et al., 2014;Shen et al., 2017;Wang et al., 2022), but they were always with negative results (Vornetti et al., 2017;Zhang et al., 2017;Xing et al., 2021). For example, Wang et al. (2022) found 9 patients with GG had postoperative seizure recurrence, and 8 of them were immunoreactive for CD34, and Prabowo et al. (2014) found the expression of BRAF V600E in GNT was associated with a worse postoperative seizure outcome. However, Vornetti et al. (2017) did not find LEAT with BRAF V600E mutation or CD34 expression were associated with seizure outcomes. Zhang et al. (2017) did not find any significant correlations between the BRAF status in GNT and postoperative seizure freedom. Also, Xing et al. (2021) reported there was no statistical difference of epilepsy-associated brain tumors between BRAF V600E mutations and wild type in Engel outcome comparison. Similarly, we defined no differences between CD34 expression (p = 0.807) or BRAF V600E mutation (p = 0.937) in GG and postoperative seizure outcomes (Figure 3).
The GG are benign, slow-growing tumors, and patients with GG rarely experience tumor progression or recurrence, although 5% (or less) of GG with anaplasia or malignant progression were reported in previous studies (Zaky et al., 2018;Slegers and Blumcke, 2020). During the whole follow-up period of 54 months (ranging from 6 months to160 months) in our study, 5 patients had tumor recurrence and 2 cases had malignant progression (Figure 3), with the accumulated 10-year tumor PFS reaching 96%. Through univariate Cox regression The pie graphs showing the percentages of prognostic results in seizure outcomes (left) and tumor recurrence (right) based on differential diagnosis of gangliogliomas with CD34 expression or with BRAF V600E mutation.
Frontiers in Molecular Neuroscience 11 frontiersin.org analysis, we found drug-resistant epilepsy (HR = 0.15) and concordant interictal EEG findings (unknown vs. concordant; HR = 14.75) were associated with longer PFS, but only the concordance of interictal EEG findings (p = 0.009) was significant in the multivariate Cox regression analysis. In particular, when compared the Kaplan Meier curves between two groups [tumor with CD34 (+) vs. CD34 (−)] or groups [tumor with BRAF (+) vs. BRAF (−)], no difference was found in patients with detection of CD34 expression or BRAF V600E mutation. The relationship of CD34 expression or BRAF V600E mutation in GG with tumor survival (PFS or overall survival) have been studied, previously (Chappé et al., 2013;Dahiya et al., 2013;Chen et al., 2017;Zaky et al., 2018;Lisievici et al., 2021). Although some of studies reported the significant correlation of CD34 expression or BRAF V600E mutation in GG with tumor recurrence or progression (Lisievici et al., 2021), the extent of the surgical resection (or tumor location), instead of CD34 expression and BRAF V600E mutation, may play an important role of the tumor prognosis of low-grade GG (Chappé et al., 2013;Dahiya et al., 2013;Zaky et al., 2018;Wang et al., 2022). However, when analyzing the association of tumor recurrence with resection extent or tumor locations, we did not find any statistic differences in resection extent (p = 0.417) and tumor locations (temporal vs. non-temporal, p = 0.761; or medial temporal vs. extra temporal, p = 0.174), which may be partly attributed to the high rate of complete tumor resection (99%) in our surgical cohort.

Limitations
The evidence from our study with GG cohort may compromise its retrospective nature, as well as its inhomogeneity, such as in patient gender or age and tumor locations. However, we did not find any significant impacts of different patient gender (female vs. male), age population (children vs. adults) and tumor locations (temporal vs. nontemporal) on either CD34 expression (see Table 1) or BRAF mutation (see Table 3), or even surgical prognosis (see Table 4), in GG with epilepsy in our cohort. In addition, the limited follow-up time for patients with low-grade GG might weaken the evidence of our study when evaluating tumor recurrence or progression after surgery. Even so, our results could partly complement the undefined domains of the clinicopathological features of molecular alterations (CD34 and BRAF mutation) in GG with epilepsy, as well as the long-term surgical outcomes.

Conclusion
CD34 expression and BRAF V600E mutation are closely associated with GG in patients with epilepsy, which may also partly influence the distribution of clinical and pathological features in patients with GG. However, CD34 expression or BRAF V600E mutation in GG may not impact the surgical prognosis of seizure outcome, as well as tumor PFS, if complete tumor resection could be achieved.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The studies involving human participants were reviewed and approved by Research Ethics Committee, Sanbo Brain Hospital. Written informed consent for participation was not provided by the participants' legal guardians/next of kin because: The study was retrospective and does not contain individual clinical data, and thus informed consent was not required.