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BRAF V600E expression and distribution in desmoplastic infantile astrocytoma / ganglioglioma1

Christian Koelsche1,2, Felix Sahm1,2, Werner Paulus3, Michel Mittelbronn4, Felice Giangaspero5, Manila Antonelli5, Jochen Meyer2, Felix Lasitschka6, Andreas von Deimling1,2 and David Reuss1,2
Department of Neuropathology, Ruprecht-Karls-Universitt Heidelberg, Heidelberg, Germany

Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, Germany Institute of Neuropathology, University Hospital Mnster, Mnster, Germany Institute of Neurology (Edinger Institute), University of Frankfurt, Frankfurt, Germany Department of Radiological Oncological Sciences and Pathology, Universit Sapienza, Roma, Italy Institute of Pathology, Ruprecht-Karls-Universitt Heidelberg, Heidelberg, Germany

Corresponding Author David Reuss, MD Ruprecht-Karls-Universitt Heidelberg Department of Neuropathology Im Neuenheimer Feld 224 D-69120 Heidelberg Fon: Fax: +49 (0)6221 56 4651 +49 (0)6221 56 4566

Email: david.reuss@med.uni-heidelberg.de

Running title: BRAF V600E mutation in DIA/DIG

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/nan.12072

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Key words: desmoplastic infantile astrocytoma, desmoplastic infantile ganglioglioma, BRAF, BRAF V600E, glioma, tumour, VE1, immunohistochemistry

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Abstract Aims: Desmoplastic infantile astrocytoma / ganglioglioma (DIA/DIG) is a rare primary neuroepithelial brain tumour typically affecting paediatric patients younger than 24 months. Knowledge about genetic alterations in DIA/DIG is limited. However, a previous study on BRAF V600E mutation in paediatric glioma revealed a BRAF mutation in one of two tested DIAs/DIGs. The limited number of cases in that study did not allow any conclusion about mutation frequency of BRAF in this tumour entity. Methods: We collected a series of 18 DIAs/DIGs for testing BRAF V600E mutational status by BRAF V600E immunohistochemistry (clone VE1). Cases with sufficient DNA were tested for BRAF V600E mutation by pyrosequencing. Results: Three out of 18 DIAs/DIGs presented with VE1 binding. A considerable proportion of BRAF V600E mutated tumour cells was detected in the cortical tumour whereas the pronounced leptomeningeal tumoral stroma was

component,

predominantly negative for VE1 binding. Pyrosequencing confirmed BRAF V600E

mutation in two of three VE1 positive cases. Conclusion: BRAF V600E mutation affects a subset of DIAs/DIGs and offers new

therapeutic opportunities.

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Introduction Desmoplastic infantile astrocytoma / ganglioglioma (DIA/DIG) is a meningocerebral neuroepithelial tumour with a pronounced desmoplastic leptomeningeal tumour component [1]. The overall incidence of DIAs/DIGs has been estimated at less than 0.3 %, but when limited to infancy age, DIAs/DIGs accounts for approximately 16 % of intracranial tumours [2, 3]. Most DIAs/DIGs have been described in paediatric patients younger than 24 months. However, rare cases have been reported in paediatric patients exceeding this age-range [4]. DIAs/DIGs almost always grow supratentorial favouring the fronto-temporal region [4]. Upon neuroimaging DIAs/DIGs typically present with a cystic mass of deep localization and a peripheral solid tumour portion [5]. Histologically, the neuroepithelial tumour can present with purely astrocytic differentiation (DIA) or be composed of tumour cells with astrocytic and neuronal differentiation (DIG) [1]. Due to their very close histomorphological relationship, DIAs and DIGs have been categorized together in the WHO classification of tumours of the central nervous system [1]. DIA/DIG corresponds to WHO grade I because of their benign biological behaviuor with a favorable clinical course even after subtotal resection, [1, 2, 6]. Nevertheless, single cases have been reported with signs of malignant transformation or multifocal intracranial growth which then behaved aggressively [7, 8]. Knowledge of the genetic background in DIA/DIG is very limited and cytogenetic data are only available of a small number of cases. TP53 mutation, which is often found in diffuse astrocytoma, was not found in DIA/DIG and suggests no close genetic relation of these entities [9]. Furthermore, an array CGH based study of 3 DIAs/DIGs revealed no consistent chromosomal gains or losses [10]. Recently, point mutation of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) at codon position 600 has been shown in roughly 60 % of ganglioglioma and 10 % of pilocytic astrocytoma [11-13]. Due to their close relation regarding clinical presentation and histological features, a similar genetic background has been assumed [14]. Data about BRAF V600E mutation in DIA/DIG are very limited. One sequencing-based study revealed BRAF V600E mutation in a single DIG case [15]. However, the small number of investigated DIGs did not allow any extrapolation in terms of incidence of BRAF V600E mutation.
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The present study was conducted to investigate the frequency of BRAF V600E mutation in 18 DIAs/DIGs by applying the BRAF V600E mutation specific monoclonal antibody clone VE1. Materials and methods Tissue samples and patient characteristics and histology In total, 16 DIGs and 2 DIAs WHO grade I were included in this study. Tissue samples were retrieved from the archives of the Department of Neuropathology of the University Heidelberg, of the Department of Neuropathology of the University Bonn, of the Department of Radiological Oncological Sciences and Pathology of the University of Roma (Italy), of the Institute for Neuropathology of the University Mnster, of the Institute of Neurology (Edinger-Institute) Frankfurt. The median age at surgery was 10.5 months (ranging from 1 to 60 months) and the female/male gender ratio was 0.8. Tumour growth was located supratentorial for 17 cases and in the posterior fossa for one case. All cases with available neuroimaging report presented with a large cystic neocortical lesion (Table 1). All included DIAs/DIGs were reviewed by members of the Department of Neuropathology Heidelberg (CK, FS, DR) and diagnosed according to the revised WHO 2007 classification of brain tumours [1]. The study was performed in accordance with the guidelines of the ethical policies of the involved institutions. IHC, assessment and microscopy To ensure proper antigenicity for IHC we used fresh-cut slides from formalin-fixed paraffin embedded tissue which was not previously frozen and was free of coagulation artifacts. Sections cut to 4 m were dried at 80C for 15 min and stained with BRAF V600E specific clone VE1 on a Ventana BenchMark XT immuno stainer (Ventana Medical Systems, Tucson, USA). The Ventana staining procedure included pretreatment with cell conditioner 1 (pH 8) for 64 min, followed by incubation with VE1 hybridoma supernatant (monoclonal, dilution 1:5) at 37C for 32 min. Antibody incubation was followed by OptiView HQ Universal Linker for 12 min, incubation with OptiView HRP Multimer for 12 minutes, signal amplification including the Ventana OptiView Amplification Kit (Ventana, catalogue number 760-099), counterstaining with one drop of haematoxylin for 4 min and one drop of bluing reagent for 4 min. As
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positive and negative controls a tissue micro array consisting of 4 melanoma samples with known BRAF V600 status (2 BRAF wild type, 2 BRAF V600E) were included in every staining run. VE1 was scored as either positive or negative. Macro-, Laser-capture microdissection and DNA extraction Macro-dissection was performed of corresponding VE1 positive areas on unstained slides of 10 m thickness and collected in 1.5 ml Eppendorf Safe-Lock tubes (Eppendorf AG). For LASER-assisted microdissection VE1 stained slides of 10 m thickness were washed in ethanol and incubated for 5 min in xylene. After air-drying the slides were dissected by the Microbeam LMPC System (Carl Zeiss MicroImaging) using the RoboLPC method. Dissected material was captured and collected in 1.5 ml AdhesiveCaps opaque (Carl Zeiss MicroImaging). DNA was extracted applying the NucleoSpin Tissue XS kit (Machery-Nagel) according to the manufacturers instructions. Sequencing DNA was available from 4 DIGs and 1 DIA. Pyrosequencing for codon 600 of BRAF was performed as previously described [16]. The sequence was compared with GenBank sequence NM_004333 for BRAF as reference. Results A total of 16 DIGs and two DIAs were screened for BRAF V600E mutated protein by applying VE1 IHC (Table 1). Three cases, two DIGs and one DIA, presented with VE1 immunoreactivity (Figure 1). Expression and distribution of BRAF V600E mutated protein in DIA/DIG A case of an 6 month old infant with a cystic lesion localized in the temporal lobe (ID 60212) was diagnosed as DIG and exhibited the predominant BRAF V600E positive tumour cell proportion in the cortical tumour component, which presented as a subpial ribbon of cells (Figure 1a,b). BRAF V600E positive tumour cells were also found accentuated around cortical vessels (Figure 1c). In this region, spaceoccupying subpial cystic changes and distinctive regions with a spongy, microcystic appearance were apparent. Both the
6

ganglionic

and

the

undifferentiated

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neuroepithelial tumour cell component were positive for synapthophysin and VE1. The desmoplastic leptomenigeal component contributed the largest tumour part and presented with small nests of BRAF V600E expressing tumour cells. VE1 negative spindle cells surrounded these nests. Nevertheless, even if most cortical tumour cells were VE1 positive, they made only the minor tumour cell fraction in the desmoplastic leptomeningeal tumour region (Figure 1c). The second VE1 positive DIG (ID 60192) developed in the occipital lobe of a 5 year old child. The tissue solely comprised of the desmoplastic leptomeningeal tumour component. Here, small nests of tumour cells faintly bound VE1 (Figure 1d,e). The BRAF V600E mutated DIA (ID 56972) occurred in the suprasellar region of a 4 months young infant. The tumour bulk completely consisted of desmoplastic tumour tissue with VE1 positive tumour cells arranged in nests surrounded by predominantly VE1 negative spindle-shaped cells (Figure S1). Confirmation of BRAF V600E mutation by DNA-based method VE1 specificity was verified by pyrosequencing of BRAF codon 600 in 5 cases (Table 1). All VE1 negative cases with DNA available were confirmed as BRAF wt. Pyrosequencing revealed BRAF V600E mutation in case 57094 and 60212. For the latter one macro-dissection of VE1 positive cells was performed to enrich tumour DNA. The cortical and desmoplastic leptomeningeal tumour components were separately dissected. BRAF V600E mutated allele was detected in both fractions, however the allelic frequency of BRAF V600E mutation was somewhat lower in the desmoplastic region (Figure 1b,c inlet). The small amount of VE1 positive cells in case 60192 impeded the confirmation of BRAF mutation by sequencing. Preceding laser-capture microdissection was performed to enrich the proportion of VE1 positive tumour cell DNA. However, BRAF V600E mutant alleles were not detected. Discussion BRAF mutation has been shown to play a pivotal role in glioma tumorigenesis, especially in paediatric patients [12, 15, 17]. The close relationship of DIA/DIG with pilocytic astrocytoma and ganglioglioma, the latter harboring BRAF V600E mutation in the majority of cases, and the previously described detection of BRAF V600E
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mutation in a single DIG case raised the hypothesis that BRAF mutation might also be a common genetic alteration contributing to DIA/DIG tumorigenesis [12-14]. BRAF V600E (VE1) binding was detected in 3 of 18 DIA/DIG and confirmation by sequencing was yielded in two cases (IDs 57094 and 60212). VE1 binding was confined to the non-spindle cell component whereas the desmoplastic spindleshaped cells were predominantly VE1 negative. Of note, almost all DIAs/DIGs reported here presented with a pronounced leptomeningeal desmoplastic tumour component, whereas the cortical component was lacking or represented a minor fraction of samples. Since spindle-shaped cells in the leptomeningeal component were predominantly VE1 negative, tumour cell dilution by these VE1 negative cells possibly reduced the probability of finding mutation by DNA-based methods and might explain the failure of BRAF V600E confirmation by sequencing for case 60192. However, BRAF V600E mutation specific antibody staining is a useful tool in particular for tumour regions with low tumour-cell density resulting from prominent desmoplasia, for instance. VE1 IHC allows the detection of BRAF V600E mutated protein close to the single cell level. Previous studies have confirmed the high reliability, sensitivity and feasibility of BRAF V600E (VE1) antibody application in such tumour entities [12, 18-23]. Furthermore, VE1 IHC has the advantage of being able to evaluate the distribution of BRAF V600E mutated protein in tissue. Case 60212 was suitable for a comprehensive examination of the expression and distribution of BRAF V600E mutated protein in both the cortical and leptomeningeal tumour component. Interestingly, the cortical tumour region presented with a prominent subpial ribbon and angiocentric pattern of VE1 positive tumour cells, whereas the leptomeningeal tumour part had intermingled groups of VE1 binding cells. Previous studies had assumed that somatic mutations of common neoplastic precursor cells like specialized subpial glial cells might lead to brain tumours with neocortical localization and a close relation to the meninges [9]. The cortical ribbon of VE1 positive cells might reflect the subpial origin of DIA/DIG. Case 60192 solely covered a small part of the leptomeningeal desmoplastic tumour component. Thus, this case was ineligible for a comprehensive analysis of tumour cell distribution. Nevertheless, the VE1 positive cells arranged in nests and were surrounded by VE1 negative spindle shaped cells, a similar pattern as seen in case
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60212 and 57094. The distribution of VE1 positive cells in these cases may argue for the cortical tumour cells as the true neoplastic component whereas the prominent desmoplastic component may be purely reactive in nature. Recently, we revealed the expression of BRAF V600E mutated protein in the ganglionic tumour cell component of ganglioglioma [12]. Corresponding to the finding in ganglioglioma, we also found an overlap of synaptophysin- and BRAF V600E (VE1) staining in DIG case ID 60212 (Fig. S2). VE1 positive ganglionic cells were detected in both the cortical and the desmoplastic leptomenigeal tumour part. Interestingly, the frequency of BRAF V600E mutation in DIAs/DIGs is considerably lower than in ganglioglioma (60%) [12, 13]. This may point to a different cell of origin in the neuronal lineage which has a different susceptibility to BRAF V600E -induced tumor initiation. Further studies are required to reveal more details about the molecular relation between DIA/DIG and ganglioglioma including DNA-methylome analysis. The low rate of BRAF V600E mutation in DIA/DIG argues for additional molecular

mechanisms that drive tumorigenesis. Candidate alterations include those who also activate the MAPK signaling pathway like Neurofibromin 1 (NF1) gene mutation or BRAF-fusion. There are indeed single case reports about neurofibromatosis type I associated DIG [24]. BRAF-fusion has almost exclusively been restricted to pilocytic astrocytoma [25]. Accordingly, we did not find BRAF-KIAA1549 fusion in two cases (ID 57094, 60210) analyzed by FISH analysis (data not shown). Nevertheless, a very recent whole-exome sequencing based study of different paediatric low-grad glioma revealed a new gene fusion involving Fragile X related protein 1 (FXR1) and BRAF in

one investigated DIG case. However, deregulation of BRAF activity by fusion or point mutation indicates the importance of MAPK signaling pathway in the development of DIA/DIG. A favorable clinical course of DIA/DIG after gross total resection has been reported for the great majority of cases, but malignant transformation and multifocal growth have been reported in some cases [7, 8]. The latter cases may benefit from a systemic therapeutic approach. New drugs targeting the MAPK signaling pathway, by selective inhibition of BRAF mutated protein for instance, offer new therapeutic options with promising results in BRAF V600E mutated melanoma metastases and glioma [26-28].
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Recently, a case with a prenatal diagnosis of DIG has been reported [29]. The young age of patients with DIA/DIG has raised the question whether DIA/DIG formation starts with the mutation of a progenitor cell conferring to a selective growth advantage. Some DIA/DIG cases might be caused by congenital mutations. Thus, further investigation of the temporal and spatial development of DIA/DIG might give new insights in the biology of this rare disease. In summary, we described the presence of BRAF V600E mutation in a subset of DIA/DIG and assigned the major proportion of BRAF V600E expressing tumour cells to the cortical tumour region. Figure 1: BRAF V600E (VE1) immunohistochemistry of case 60212 (a-c) and case 60192 (d,e). (a) Overview of a DIG with cortical and desmoplastic leptomeningeal tumour parts. (b) Corresponding magnification of the cortical tumour region with BRAF V600E positive ribbon-like staining pattern (arrow) and angiotropism of BRAF V600E mutated tumour cells (two-headed arrow). The inlet shows the BRAF codon 600 pyrogramm of this region with a T to A substitution in 16 % of alleles. (c) Corresponding leptomeningeal tumour part with nests of VE1 positive tumour cells surrounded by VE1 negative spindle-shaped cells. The inlet shows the BRAF codon 600 pyrogramm of this region with a T to A substitution in 10 % of alleles. (d) VE1 staining of a DIG with faint positive tumour cell nests surrounded by VE1 negative desmoplastic stroma. (e) Corresponding magnification of VE1 positive cell nests. Magnification: (a) 4-fold; (b,c,e) 200-fold; (d) 30-fold. Table 1: Clinical, immunohistochemical and molecular data of DIAs/DIGs ID Diagnosis (WHO grade) DIG (I) DIG (I) DIG (I) DIG (I) DIA (I) DIG (I) DIG (I) DIG (I) Age at surgery (month) 1 2 3 4 4 6 6 6 Sex m m f f m f m f
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Location temporal supratentorial posterior fossa temporal suprasellar temporo-parietal supratentorial temporal

VE1 + +

Seq NA NA NA ND V600E NA NA V600E

60194 60196 60214 60206 56972 60188 60198 60212

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60200 60216 60202 60218 60220 60222 60204 60190 60210 60192

DIG (I) DIG (I) DIG (I) DIA (I) DIG (I) DIG (I) DIG (I) DIG (I) DIG (I) DIG (I)

7 8 8 9 9 10 12 12 17 60

m m m f m m f f f m

parietal temporo-parietal frontal frontal frontal occipital supratentorial supratentorial fronto-parietal occipital

NA NA NA NA wt NA NA wt NA wt

ID ~ internal patient number; DIG ~ desmoplastic infantile ganglioglioma; DIA ~ desmoplastic infantile astrocytoma; m ~ male; f~ female; NA ~ not available; VE1 ~ antibody clone VE1; - ~ immunonegative; + ~ immunopositive; Seq ~ BRAF codon

600 pyrosequencing status; V600E ~ BRAF V600E mutation; wt ~ BRAF wild type;

Figure S1: Sagittal postgadolinium T1-weighted (a) and sagittal T2- (CISS) weighted (b) MR images of a 4 month old child showing a large primarily supratentorial solid-cystic tumour. (a) The solid component (white arrow) shows heterogeneous enhancement following gadolinium injection. (b) T2 (CISS) weighted images visualize a large anterior and small posterior cystic component (white arrows). Note the subdural hygroma surrounding the anterior cystic tumour mass. (c) H&E staining shows an astrocytic tumour with abundant desmoplasia. (d) BRAF V600E (VE1) immunohistochemistry shows positive tumor cells embedded in a desmoplastic matrix. Magnification: (c-d) 200-fold. (e) BRAF codon 600 pyrogramm with a T to A substitution in 20 % of alleles. Figure S2: (a) BRAF V600E (VE1) staining of tumour cells in the desmoplastic component of DIG case 60212. (b) Corresponding region stained against Synaptophysin (Syn) revealed a similar staining pattern compared to VE1 IHC. Magnification: 100-fold. Acknowledgments The authors thank Tanja Goeck and Jutta Scheuerer for excellent technical assistance, Philipp Kickingereder for MRI examination and images, and Andrey
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Korshunov for BRAF-KIAA1549 FISH analysis. This work was in part funded by the Deutsche Forschungsgemeinschaft, SFB 938/TP Z2 (F.L.). Conflict of interest AvD has applied for a patent on the diagnostic use of BRAF V600E mutant-specific antibody VE1. All terms are being managed by the German Cancer Research Center in accordance with its conflict of interest policies. Authors contribution CK histologic imaging, data analysis and manuscript preparation FS data analysis WP collection of cases, clinical data MM collection of cases, clinical data FG collection of cases, clinical data MA collection of cases JM molecular analysis FL molecular analysis AvD data analysis, manuscript preparation DR project conception, data analysis and manuscript preparation References

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