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Acta Neuropathol (2013) 125:901–910DOI 10.1007/s00401-013-1120-y

ORIGINAL PAPER

BRAFV600E mutation is a negative prognosticator in pediatric ganglioglioma

Sonika Dahiya · Devon H. Haydon · David Alvarado · Christina A. Gurnett · David H. Gutmann · Jeffrey R. Leonard

Received: 19 November 2012 / Revised: 11 April 2013 / Accepted: 12 April 2013 / Published online: 23 April 2013 © Springer-Verlag Berlin Heidelberg 2013

tumors (38.3 %) had positive BRAFV600E staining, which was associated with shorter recurrence-free survival. Col-lectively, the combined use of histopathologic and molecu-lar features to stratify grade I gangliogliomas into low and high-risk groups provides important information relevant to the management of children and young adults with these rare tumors.

Keywords Ganglioglioma · BRAFV600E · Recurrence-free survival · Children

Introduction

Gangliogliomas are rare, well-differentiated, neuroepithe-lial tumors that most commonly affect children and young adults. Gangliogliomas are considered indolent tumors with excellent long-term survival, such that surgical resec-tion is potentially curative if an entire tumor can be suc-cessfully removed. A complete resection is not always possible, reflecting the proximity of the tumor to eloquent structures in the brain. Additionally, the literature contains several examples of ganglioglioma recurrence after imag-ing-confirmed complete resection [6, 12].

Factors predictive of ganglioglioma recurrence are largely unknown, possibly due to the rarity of the tumor. The current WHO grading classification only distinguishes benign grade I tumors from malignant grade III tumors. While several atypical histologic features have been pro-posed for grade II lesions, acceptance of a uniform standard for these atypical characteristics has not occurred, reflect-ing the subjective nature of these descriptive findings [2].

Recent work identified isocitrate dehydrogenase 1 (IDH1) mutations as a diagnostic tool to distinguish gan-gliogliomas from infiltrative gliomas (in patients older than

Abstract Gangliogliomas are typically low-grade neu-roepithelial tumors seen in the pediatric and young adult populations. Despite their often bland histologic appear-ance, these tumors recur with varying frequencies; how-ever, little data exist that adequately predict ganglioglioma recurrence in children. To identify potential histopathologic features predictive of recurrence-free survival, a series of 53 patients with World Health Organization (WHO) grade I gangliogliomas were evaluated, representing the larg-est cohort of pediatric gangliogliomas with accompany-ing histopathologic and survival data. Fifteen patients (28 %) exhibited disease recurrence during the study period. BRAFV600E immunohistochemistry was performed on 47 of these tumors. Histopathologic features associ-ated with shorter recurrence-free survival included an absence of oligodendroglial morphology, higher glial cell density, microvascular proliferation, and the presence of a high lymphoplasmacytic inflammatory infiltrate. Eighteen

S. Dahiya and D. Haydon made equal contributions to this study.

Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1120-y) contains supplementary material, which is available to authorized users.

S. Dahiya Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA

D. H. Haydon · J. R. Leonard (*) Department of Neurological Surgery, Washington University School of Medicine, 1 Children’s Place, Suite 4S20, St. Louis, MO 63110, USAe-mail: leonardj@wudosis.wustl.edu

D. Alvarado · C. A. Gurnett · D. H. Gutmann Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA

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20 years) and as a potential risk factor for ganglioglioma recurrence [11]. Immunohistochemical studies with IDH1 mutation-specific monoclonal antibodies are feasible, and provide an objective interpretation of positive findings. However, while IDH1 mutations are known to occur in ado-lescent malignant gliomas [25], to our knowledge, an IDH1 mutation has been found in only a single ganglioglioma patient less than 20 years of age [8]. Similarly, IDH1 muta-tions are rarely found in other pediatric low-grade tumors, such as grade II diffuse astrocytomas [14]. This low muta-tional frequency limits the clinical application of IDH1 testing for gangliogliomas in the pediatric population.

High frequency BRAFV600E mutations have been recently identified in gangliogliomas from a large screen of 1,320 central nervous system tumors [28]. BRAF altera-tions are increasingly recognized as critical components in the pathogenesis of other sporadic grade I gliomas in chil-dren [1, 13, 24, 32]. However, the histologic characteristics and prognostic significance of BRAFV600E mutations have not been previously explored in gangliogliomas. The pri-mary objective of this study was to identify molecular and histopathologic features that predict recurrence-free sur-vival in children with ganglioglioma.

Materials and methods

Patients

With prior approval by the Washington University School of Medicine Institutional Review Board, patients diagnosed with a WHO grade I ganglioglioma before 21 years of age and receiving treatment at either St. Louis Children’s Hos-pital or Barnes-Jewish Hospital in St. Louis were identi-fied. Fifty-nine consecutive patients treated between 1990 and 2011 originally met these inclusion criteria. Four patients were excluded, since they were lost to follow-up immediately following their diagnosis. Two patients were excluded after central pathological review revealed a con-flicting diagnosis.

Medical charts for the remaining 53 patients were ret-rospectively reviewed. Collected clinical data included demographic features, symptomatology, and treatments. Recurrence-free survival was the primary endpoint for this study, while overall survival was included as a secondary aim. Recurrence was defined as any new tumor visible on surveillance MRI, a ≥25 % increase in known residual tumor volume, and/or neurologic deterioration despite increased steroid use. This definition was adopted from the validated Macdonald criteria frequently used for other brain tumors [21]. Of note, 87 % of events were classified as recurrent disease according to the radiologic parameters alone.

Histopathology

Existing histopathologic slides for all cases were centrally reviewed by two authors (S.D. and D.H.). Tumor tissue was formalin-fixed and paraffin-embedded at the time of sur-gery according to standard protocol. All samples were pre-viously diagnosed as grade I gangliogliomas and confirmed with an immunohistochemical panel consisting of glial fibrillary acidic protein, synaptophysin, chromogranin, neurofilament, and CD34. p53 and Ki-67 immunostaining was performed on a case by case basis. Tumors were classi-fied using the 2007 WHO criteria [19].

Hematoxylin and eosin-stained slides from archival files were examined for the presence of eosinophilic granu-lar bodies (EGBs), Rosenthal fibers, calcifications, suba-rachnoid spread, microvascular proliferation, mitoses, and necrosis. The predominant histologic pattern was classified as “glial” or “neuronal”, while cases with an admixture of both components were classified as “mixed”. The glial component was also evaluated for the presence of cellular atypia or pleomorphism, and was stratified as minimal to mild (1+), mild-to-moderate (2+), or moderate (3+). None of our cases had marked atypia. Further, tumor cellularity was categorized as mild, moderate, or hypercellular. The presence of a chronic inflammatory infiltrate, as expected in these neoplasms, was noted, and the degree of this inflammatory infiltrate was scored as low (absent to mild) or high (moderate to marked).

BRAFV600E immunohistochemistry

Immunohistochemical staining was performed using the mouse monoclonal anti-human BRAFV600E antibody (clone VE1; Spring Bioscience; dilution 1:100), EDTA (pH 8) antigen retrieval with appropriate positive and negative controls. The stained sections were then evaluated. Posi-tive cases were further assessed for the degree of reactiv-ity (focal or widespread), intensity (mild, moderate, or marked) and localization (glial or neuronal).

Statistical analyses

Chi square or Fisher’s exact tests were used to determine the association of categorical variables. The Jonckheere–Terpstra test was used to evaluate the relationship between BRAFV600E status and tumor cellularity. The Cox propor-tional hazard model was used to obtain hazard ratios (HR) and 95 % confidence intervals (CI). Recurrence-free sur-vival differences were assessed using the Kaplan–Meier method with calculated p values from the log-rank test. Sta-tistical significance was defined as a p value <0.05. Anal-yses were conducted using SAS statistical software (SAS Inc., Cary, NC, USA).

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Results

Fifty-three children (33 male, 20 female) underwent treat-ment for a WHO grade I ganglioglioma for whom follow-up data were available. The median age at diagnosis was 12 years (range 0.9–20). The demographic and clinical fea-tures are listed in Table 1.

Histopathologic features

All tumor specimens displayed two cellular populations, characterized by a neuronal component made up of dys-plastic and dysmorphic neuronal forms and a glial compo-nent without overt signs of anaplasia. Each specimen met the diagnostic criteria for a WHO grade I ganglioglioma. Most, but not all cases, had immunohistochemical studies performed to support glial (glial fibrillary acidic protein) and neuronal (synaptophysin, chromogranin and NeuN; former two to identify the dysplastic population and the

latter as a negative stain to highlight mature neurons) dif-ferentiation. Furthermore, a subset had neurofilament stain-ing to reveal a solid growth pattern in many of the cases. Ki-67 staining (MIB1 labeling indices; ranging from less than 1 % to as high as 6 % in focal areas) was evaluated, and was found to be variable in these cases. In some instances, this was reflected in scattered inflammatory cells. In addi-tion, CD34 immunostaining was available for 25 cases, and was positive in 18 cases with focal to widespread expres-sion. Table 2 lists all histopathologic features examined and

Table 1 Patient demographics and clinical variables

Variable Total n = 53 (%)

Gender

Male 33 (62)

Female 20 (38)

Presentation

Seizure 37 (69)

Headache 8 (15)

Focal weakness 3 (6)

Other 3 (6)

Incidental 2 (4)

Location

Temporal 29 (55)

Frontal 5 (9)

Parietal 5 (9)

Occipital 3 (6)

Deep nuclei 5 (9)

Infratentorial 6 (12)

Cystic component

Yes 29 (55)

No 24 (45)

Gadolinium enhancement

Yes 41 (77)

No 12 (23)

Extent of resection

Complete 34 (64)

Incomplete 19 (36)

Recurrence

Yes 15 (28)

No 38 (72)

Table 2 Histopathologic variables and their effect on recurrence-free survival

Variable Total n = 53 (%)

p value Hazard ratio (95 % confidence interval)

Predominant histology

Glial 33 (62)

Mixed 14 (27) 0.998 NA (0–∞)

Neuronal 6 (11) 0.998 NA (0–∞)

Oligodendroglial component

Yes 20 (38) 0.030 0.3 (0.1–0.9)

No 33 (62)

Microvascular proliferation

Yes 16 (30) 0.025 3.6 (1.2–11.0)

No 37 (70)

Hypercellularity

Yes 8 (15) 0.037 3.3 (1.1–10.1)

No 45 (85)

Glial atypia

Mild 42 (79)

Mild-to-moderate 3 (6) 0.334 2.2 (0.5–10.7)

Moderate 8 (15) 0.548 1.5 (0.4–5.8)

Mitoses (10HPF)

0 47 (89)

1 4 (7.5) 0.998 NA (0–∞)

>1 2 (3.8) 0.206 2.7 (0.6–12.2)

Inflammatory infiltrate

Low 37 (70) 0.006 4.8 (1.6–14.8)

High 16 (30)

Subarachnoid spread

Yes 5 (15) 0.998 NA (0–∞)

No 48 (85)

Rosenthal fibers

Yes 6 (11) 0.458 0.5 (0.1–3.6)

No 47 (89)

Eosinophilic granular bodies

Yes 34 (64) 0.250 2.2 (0.6–8.1)

No 19 (36)

Calcifications

Yes 20 (38) 0.755 0.8 (0.3–2.6)

No 33 (62)

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their effect on recurrence-free survival. A more detailed description of pertinent findings is provided below.

Tumor samples were categorized according to their predominant histologic pattern as “glial predominant” (33) (Fig. 1a), “neuronal-rich” (6) (Fig. 1b), or “mixed” with an equal ratio of the two components (14). The pres-ence of any oligodendroglioma-like region, regardless of extent, was noted in 20 cases (Fig. 1c); majority of these cases were predominantly oligodendroglial (7 cases) or had mixed glial phenotypes (6 cases), and a few were pre-dominantly piloid or fibrillary (4 and 3 cases, respectively) glial phenotypes. Sixteen tissue samples contained areas of glomeruloid type microvascular proliferation (Fig. 1d), but were otherwise scored as WHO grade I tumors.

The cellularity of each tumor was stratified as “mild” (15) (Fig. 2a), “moderate” (30) (Fig. 2b), or “hypercellu-lar” (8) (Fig. 2c). The degree of cellular atypia in the glial subset of tumor cells was similarly evaluated. No specimen displayed marked glial atypia. Forty-two samples showed mild atypia (Fig. 2d), while 3 and 8 samples showed mild-to-moderate (Fig. 2e) and moderate atypia (Fig. 2f), respectively.

Mitotic figures were difficult to find in 47 tissue sam-ples, and only a rare mitotic figure was noted in 4 cases. In the remaining 2 cases, mitoses were relatively frequent, with an estimated count of 5 per 10 high-power fields (HPF) for both cases; these mitoses were found in glial cells (Supplementary Fig. 1). However, none of these had

areas of necrosis, microvascular proliferation, or a high degree of glial cellularity or atypia.

Lastly, the presence and degree of chronic inflamma-tory infiltrates were reviewed. These infiltrates were com-posed predominantly of mature lymphocytes with scattered plasma cells and histiocytes. A low degree of inflammatory cell infiltration was noted in 37 samples (Fig. 3a), while a higher degree of inflammatory cell infiltration was found in the remaining 16 specimens (Fig. 3b).

BRAFV600E immunohistochemistry

Sufficient diagnostic material was available for BRAFV600E immunohistochemistry in 47 of 53 samples. Positive BRAFV600E staining was observed in 18 of 47 specimens (38.3 %). Thirteen of the immunopositive tumors displayed widespread positivity (Fig. 4a), while 5 of the samples showed focal staining (Fig. 4b). Widespread staining corre-sponded to >90 % tumor cell positivity, while focal stain-ing demonstrated <50 % positivity by tumor volume. The majority of focally positive samples exhibited 10–25 % staining of overall tumor volume. Both the glial and neu-ronal cellular populations were BRAFV600E positive in 13 samples (Fig. 4a). BRAFV600E positivity was exclusive to the glial population in 5 cases (Fig. 4b), whereas no tumor exhibited exclusive neuronal positivity. With a single excep-tion, the staining intensity of the neuronal cellular popula-tion was either equal to or greater than the glial population

Fig. 1 Histopathologic char-acteristics of gangliogliomas. Hematoxylin and eosin-stained sections from different gangli-oglioma cases show piloid (a) and neuronal predominance (b). Some ganglioglioma samples exhibit areas of oligodendroglial morphology (c). An otherwise WHO grade I ganglioglioma with glomeruloid type micro-vascular proliferation (d)

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(Fig. 4a). Sequencing was performed on four cases, of which three had mutations, two of which were positive for the BRAFV600 mutation by both immunohistochemistry and sequencing (Fig. 5), and the remaining case was nega-tive by both immunohistochemistry and sequencing.

Recurrence-free survival and overall survival

Fifteen children (28 %) experienced a recurrence dur-ing the study period. The median time-to-recurrence was 8.8 months (range 2.6–107.1 months). The 5-year recurrence-free survival was 70 %. A single patient died 7 months following the diagnosis of a brainstem gan-glioglioma. The overall survival at last follow-up was 98 %. Mean duration of follow-up was 4.2 years (range 3.1–162.3 months).

Prognostic features

The effect of each histopathologic feature on clinical out-come was assessed. Improved recurrence-free survival was observed for the presence of oligodendroglial morphol-ogy (p = 0.030), even when such areas were only focally present (Fig. 6a). In contrast, the presence of piloid histol-ogy showed a trend toward worse survival, but failed to reach statistical significance (p = 0.052). The presence of microvascular proliferation (p = 0.025), even if incipient,

marked tumor cellularity (p = 0.037), and a high chronic inflammatory infiltrate (p = 0.006) were also negatively associated with recurrence-free survival (Fig. 6b–d). Micro-vascular proliferation was often associated with piloid his-tology, but was not limited to the predominance of this glial component.

BRAFV600E positive staining was associated with worse recurrence-free survival (Fig. 7). BRAFV600E staining was not related to oligodendroglial morphology (p = 0.999), astrocytic morphology (p = 0.349) including piloid pheno-type (p = 0.37), microvascular proliferation (p = 0.524), or tumor cellularity (p = 0.403). BRAFV600E immunostaining positively correlated with increased inflammatory cell infil-tration (p = 0.007). BRAFV600E staining did not correlate with other clinical characteristics, including the extent of surgical resection (p = 0.110), tumor location (p = 0.150), or seizure history (p = 0.493).

Discussion

Gangliogliomas represent a significant clinical problem in the fields of pediatric epilepsy and neuro-oncology. While seizure outcomes are well described for gangliogliomas in children, comprehensive information regarding tumor behavior is lacking [12, 15, 23]. Existing reports address-ing tumor behavior often include multiple histologic grades

Fig. 2 Additional histopathologic features of gangliogliomas. Glial cellularity may vary from mild (a), to moderate (b), to frankly hyper-cellular (c). Similarly, the degree of atypia/pleomorphism in glial ele-

ments may vary from minimal to “mild” (d) to “mild-to-moderate” (e), and “moderate” (f)

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and diagnoses, which can be misleading if different crite-ria are used [6, 12, 20, 30]. Ganglioglioma recurrence is relatively common, but the histological factors predictive of recurrence have not been extensively studied. In the cur-rent series of WHO grade I gangliogliomas, 15 of the 53 children exhibited tumor recurrence, with a 5-year event-free survival rate of 70 % and a mean follow-up period over 4 years. To our knowledge, the present study repre-sents the largest cohort of WHO grade I gangliogliomas in children with accompanying histopathologic and survival data. Importantly, our study also demonstrates that hyper-cellularity, the lack of an oligodendroglial component, the presence of a moderate to marked (high) chronic inflam-matory cell infiltrate, microvascular proliferation (even if

incipient), and BRAFV600E mutation correlate with shorter recurrence-free survival. These findings support the crea-tion of an “atypical” designation for this particular subset with high-risk findings, and raise the possibility that these gangliogliomas should be classified as WHO grade II tumors.

On comparison with Luyken et al.’s work [20], which is so far the largest study pertaining to gangliogliomas, the common parameters for tumor recurrence included increased cellularity, conspicuous nuclear pleomorphism in glial cell elements and microvascular proliferation. Presence of microvascular proliferation had a modifier of ‘prominent’ in their work which is in contrast to our find-ings whereby even ‘incipient’ microvascular proliferation

Fig. 3 The presence of a chronic lymphoplasmacytic infiltrate is common in gangli-oglioma samples. Representa-tive images demonstrate a low (a) and high (b) degree of infiltration

Fig. 4 Ganglioglioma BRAFV600E immunohistochemi-cal staining. Widespread (a) compared to patchy (b) staining is depicted. Both glial and neu-ronal cellular populations reveal BRAFV600E immunopositivity in the majority of samples (arrow indicates positive neuronal staining). Staining was exclu-sive to the glial population for a subset of specimens and nearly all of these cases had only patchy mutant protein expres-sion (arrows indicate negative neuronal staining). Double labeling highlights reactivity in glial (GFAP: brown; BRAFv600e: red; positive cells: arrows) and neuronal cells (NeuN: red; BRAFv600e: green; positive cells: arrows)

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has a bearing on clinical outcome. It is though worth men-tioning that this series was limited to supratentorial gangli-ogliomas only and had adult patients too.

Moreover, we sought to determine whether a predomi-nance of either a glial or neuronal cellular component had prognostic value. Histologic stratification by glial, mixed,

Fig. 5 Sequence analysis reveals the BRAFV600E mutation in one of the patients which was also positive by immunohistochemistry

Fig. 6 Kaplan–Meier curves of recurrence-free survival according to oligodendroglial morphology (a), microvascular proliferation (b), hypercel-lularity (c), and inflammatory infiltrates (d). Tumors with oligodendroglial morphol-ogy (solid black line) have significantly longer recurrence-free survival rates compared to those tumors without this trait (dashed blue line). Tumors with microvascular proliferation and hypercellularity (dashed blue lines) have significantly shorter recurrence-free survival rates compared to other samples without these features (solid black lines). A high degree of lymphocytic inflammatory infiltration (dashed blue line) is associated with worse recur-rence-free survival compared to a low degree of inflammatory cell infiltrate (solid black line)

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or ganglionic predominant samples revealed a negative association between increasing glial composition and recurrence-free survival; however, this trend did not reach statistical significance. Improved survival was similarly noted when tumor samples contained even focal oligo-dendroglioma-like areas, irrespective of the predominant histology. This particular relationship has been previously reported for both low [17] and high-grade [7] mixed oli-goastrocytomas, in which the oligodendroglial component is an independent prognostic factor for improved survival.

The presence of microvascular proliferation was also associated with reduced recurrence-free survival. This rela-tionship was true even for focal, incipient proliferation. The microvascular proliferation noted in our samples was similar to the glomeruloid type observed in pilocytic astro-cytomas [3]. Interestingly, the majority of cases of micro-vascular proliferation occurred in tumors with a piloid background.

Increased inflammatory cell infiltration is a common finding in ganglioglioma, as seen in pleomorphic xanthoas-trocytoma. The exact frequency of this histologic feature is unknown, with few reports attempting to quantify its presence [10, 29]. Lymphocytic infiltration was noted in 77 % of gan-glioglioma samples in our series. Classification according to a simple low versus high degree of infiltration demonstrated a statistically worse recurrence-free survival for tumors with increased inflammatory infiltrates. The prognostic importance of lymphocytic infiltrates has been documented for other sys-temic malignancies, including some forms of breast carci-noma [26]. In this regard, a significant survival advantage for patients with glioblastomas containing intermediate or exten-sive CD8+ T cell infiltrates has been described relative to those patients whose tumors had focal or rare infiltrates [31].

Atypical features have also been reported to varying degrees in gangliogliomas, including cytologic atypia, necrosis, increased mitotic activity, and increased cellular-ity. Russell and Rubinstein [27] first postulated that only the glial population is capable of malignant change and that outcomes are based on the degree of glial anaplasia. A semi-quantitative assessment of overall atypia in the glial tumor component (i.e. mild, mild-to-moderate, or moder-ate) was not associated with survival.

No areas of necrosis were seen in any tumor sample in our series. The mitotic activity of gangliogliomas is gener-ally low, and only 2 cases from our series contained ele-vated mitotic activity. Of note, while none of the patients with isolated mitotic figures experienced disease recur-rence, both patients with more than one mitotic figure expe-rienced tumor recurrence. The proliferative index of intrac-ranial tumors may be falsely elevated due to a predominant inflammatory infiltrate. However, it should be stressed that the mitotic figures observed in the present study were found in GFAP-positive glial cells, rather than in infiltrative lym-phoplasmacytic cells. Increased tumor cellularity was also associated with worse recurrence-free survival. However, the limitations of the anaplastic descriptors continue to pre-clude a more detailed ganglioglioma grading scheme, due to their inherent subjectivity and associated inter-observer variability.

BRAFV600E mutations have recently been identified in 18 % of ganglioglioma samples by traditional sequenc-ing methods [28]. More sensitive techniques may in fact determine that BRAF mutations are far more common in gangliogliomas. A pilot study at our institution employing next-generation sequencing techniques identified BRAF mutations in 3 of the 4 ganglioglioma specimens, with the BRAFV600E mutation observed in 2 of the 3 BRAF mutant samples. A mutation-specific BRAFV600E monoclonal antibody has recently become available, which accurately identifies the presence of this mutation in pathologic speci-mens [4]. Immunohistochemical studies using BRAFV600E-specific antibodies are 97 % sensitive and 98 % specific for detecting this mutation in other tumor types [18]. More recently, a 94 % concordance has been demonstrated between immunohistochemistry and sequencing in gan-gliogliomas; this report also showed a frequency of 58 % for the mutant protein using IHC [16]. However, the his-tologic characteristics and prognostic significance of this mutation have not been previously described in pediatric gangliogliomas.

In our series, we found positive BRAFV600E immu-nostaining in 18 of 47 tumors (38.3). Thirteen of 18 posi-tive samples (72 %) revealed staining of both the glial and neuronal cellular populations. These findings suggest that both cellular components may be derived from a common progenitor cell and that BRAF mutations might be an early

Fig. 7 Kaplan–Meier curve comparing recurrence-free survival rates of patients with BRAFV600E-negative (solid black line) and positive (dashed blue line) immunostaining. BRAFV600E-positive staining is associated with significantly shorter recurrence-free survival

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step in ganglioglioma pathogenesis. Indeed, BRAF altera-tions are considered important steps in the pathogenesis of sporadic pilocytic astrocytomas, another grade I brain tumor [1, 13, 24, 32], although BRAF alterations were not associated with astrocytic glial morphology in our series. An absence of neuronal staining in a subset of cases also raises the possibility that BRAF mutations occur with neo-plastic transformation of only astroglial elements. How-ever, we believe that this hypothesis is less likely, since only a small minority of cases revealed exclusive glial immunostaining. This hypothesis is also supported by a recent report of predominant expression of this mutant pro-tein in neuronal component [16].

Of particular interest was the fact that BRAFV600E immunopositivity was associated with worse recurrence-free survival. To exclude possible confounding effects, we examined the relationship between BRAFV600E immuno-positivity and other clinical parameters thought to affect ganglioglioma outcome. The extent of resection is widely regarded as affecting ganglioglioma outcome [12, 20, 22] as well as tumor location, which likely reflects the extent of surgical resection. Additional studies have also identi-fied seizure history as a potential prognostic indicator in gangliogliomas [9]. As seizures are far more common with cortically based tumors, seizure history is likely a surrogate for tumor location and, in turn, resectability. In our series, none of these clinical features were associated with positive BRAFV600E immunostaining.

We also examined the relationship between BRAFV600E status and other histopathologic features. Oligodendro-glial morphology, tumor cellularity, and microvascular proliferation were not associated with positive BRAFV600E immunostaining. Interestingly, BRAFV600E mutation status was associated with the presence and degree of inflam-matory cell infiltration. In this regard, it is possible that lymphocytic infiltration in gangliogliomas is a marker for more aggressive tumor biology, which then elicits a T-cell dependent immune response. Future investigation will be required to define the relationship between BRAFV600E mutation and immune cell infiltration.

The availability of an objective immunohistochemical test to stratify an otherwise homogenous group of WHO grade I tumors into low and high-risk groups has important clinical implications, especially in light of the recent suc-cesses of BRAF-targeted therapies for numerous other can-cers. Improved recurrence-free survival and overall survival have recently been reported in a phase III BRAF inhibitor (vemurafenib) clinical trial for patients with BRAFV600E-mutated metastatic melanoma [5]. The future use of BRAF-targeted therapies to treat BRAFV600E-mutated ganglioglio-mas might provide a similarly effective option for patients with tumors refractory to conventional surgical and/or adju-vant treatment.

Conclusion

The clinical behavior of WHO grade I gangliogliomas in children is quite diverse. Select histopathologic features are associated with clinical outcome, although their interpreta-tions are variably subjective. BRAFV600E-mutated gangli-ogliomas exhibit worse recurrence-free survival compared to BRAFV600E-negative tumors. The identification of this high-risk group may assist clinicians in determining future treatment and disease surveillance strategies, particularly now that BRAF-targeted therapies have been successfully employed for other malignancies.

Acknowledgments The authors gratefully acknowledge the work of Drs. Jingqin Luo and Kathryn Trinkaus (Division of Biostatistics, Washington University School of Medicine) who assisted with the statistical analyses. Support for this study was provided by the Chil-dren’s Discovery Institute (MC-LI-2009-03R) and the National Insti-tute of Health (T32: 3857-54941D).

Conflict of interest The authors declare that they have no conflict of interest.

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