Pilocytic astrocytoma (PA) is the most frequently encountered glial tumor (glioma or astrocytoma) in children. Recent studies have identified alterations in the BRAF serine/threonine kinase gene as the likely causative mutation in these childhood brain tumors. The majority of these genetic changes involve chromosome 7q34 tandem duplication, resulting in aberrant BRAF fusion transcripts. In this paper, we describe a novel KIAA1549:BRAF fusion transcript in a sporadic PA tumor associated with increased ERK activation and review the spectrum of BRAF genetic alterations in this common pediatric low-grade central nervous system neoplasm

Dahiya, Sonika

Gutmann, David H.

Kaul, Aparna

Leonard, Jeffrey R.

Yu, Jinsheng

English

PubMed

Pilocytic astrocytoma (PA) is the most frequently encountered glial tumor (glioma or astrocytoma) in children. Recent studies have identified alterations in theBRAFserine/threonine kinase gene as the likely causative mutation in these childhood brain tumors. The majority of these genetic changes involve chromosome 7q34 tandem duplication, resulting in aberrantBRAFfusion transcripts. In this paper, we describe a novelKIAA1549:BRAFfusion transcript in a sporadic PA tumor associated with increased ERK activation and review the spectrum ofBRAFgenetic alterations in this common pediatric low-grade central nervous system neoplasm.</jats:p

Sonika Dahiya

Jinsheng Yu

Aparna Kaul

Jeffrey R. Leonard

David H. Gutmann

Crossref

Hindawi Publishing CorporationCase Reports in MedicineVolume 2012, Article ID 418672, 4 pagesdoi:10.1155/2012/418672Case ReportNovel BRAF Alteration in a Sporadic Pilocytic AstrocytomaSonika Dahiya,1 Jinsheng Yu,1 Aparna Kaul,2 Jeffrey R. Leonard,3 and David H. Gutmann21 Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA2 Department of Neurology, Washington University School of Medicine, P.O. Box 8111, 660 South Euclid Avenue,St. Louis, MO 63110, USA3 Department of Neurosurgery, Washington University School of Medicine, St. Louis, MO 63110, USACorrespondence should be addressed to David H. Gutmann, gutmannd@neuro.wustl.eduReceived 17 December 2011; Accepted 7 February 2012Academic Editor: Mark E. ShaﬀreyCopyright © 2012 Sonika Dahiya et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Pilocytic astrocytoma (PA) is the most frequently encountered glial tumor (glioma or astrocytoma) in children. Recent studieshave identified alterations in the BRAF serine/threonine kinase gene as the likely causative mutation in these childhood braintumors. The majority of these genetic changes involve chromosome 7q34 tandem duplication, resulting in aberrant BRAF fusiontranscripts. In this paper, we describe a novel KIAA1549:BRAF fusion transcript in a sporadic PA tumor associated with increasedERK activation and review the spectrum of BRAF genetic alterations in this common pediatric low-grade central nervous systemneoplasm.1. IntroductionPilocytic astrocytomas are the most common nonmalignantbrain tumor in the pediatric population. Children with theNeurofibromatosis type 1 (NF1) inherited cancer predispo-sition syndrome are prone to the development of these glialcell neoplasms, such that 15–20% of aﬀected individuals willdevelop gliomas involving the optic pathway, hypothalamus,and brainstem [1]. Molecular analysis of these tumors hasrevealed biallelic inactivation of the NF1 tumor suppressorgene, resulting in loss of NF1 protein (neurofibromin)expression. However, sporadic PA tumors do not exhibitmutational inactivation of the NF1 gene, suggesting thatother genetic mutations are responsible for the genesis ofthese histologically-identical low-grade brain tumors in thegeneral population [2].Over the past several years, the molecular basis for thesenonsyndromic pediatric brain cancers has been elucidatedwith the identification of signature molecular changesinvolving the BRAF serine/threonine kinase gene. The mostfrequently encountered genetic alteration is a tandem dupli-cation of the BRAF gene on chromosome 7q34, leadingto fusion of the KIAA1549 gene to the carboxyl terminalregion of the BRAF gene containing the kinase domain. Thismolecular change has been reported in 50–65% of sporadicpilocytic astrocytoma and is more frequent in cerebellar(∼80%) tumors. The majority of these alterations involvefusions between KIAA1549 exon 16 and BRAF exon 9,KIAA1549 exon 15 and BRAF exon 9, and KIAA1549 exon16 and BRAF exon 11 [3–9], while less common alterationsinclude tandem duplications involving SRGAP3 and RAF1 orFAM131B and BRAF [10, 11]. In this paper, we describe anovel KIAA1549-BRAF fusion event in a sporadic pediatricpilocytic astrocytoma.2. Case PresentationThe patient was a 14-year-old boy who presented with a6-month history of headache that progressed to a two-dayperiod of nausea, vomiting, and ataxia. Magnetic resonanceimaging (MRI) at that time showed a cystic mass in thecerebellum compressing the fourth ventricle (Figure 1(a)).He was taken to the operating room where a gross totalresection was performed. Neuropathological review revealeda classic pilocytic astrocytoma with alternating areas ofcompact and loose tissue architecture (Figure 1(b)). Thecompact areas were composed of piloid neoplastic cellscontaining numerous Rosenthal fibers and few eosinophilicgranular bodies (Figure 1(c)), while the paucicellular areas2 Case Reports in Medicine(g)(f)(d)(a)(b) (c)(e)(h)KIAA1549BRAFe18 e11 e10 e16 e15 e1Ladder1 kb600 bp200 bp100 bpNB PApERKERKFigure 1: Molecular characterization of a novel KIAA1549:BRAF fusion alteration in a sporadic pediatric pilocytic astrocytoma. (a) AxialT1-weighted 1.5-Tesla gadolinium-enhanced MRI scan reveals a cystic lesion in the cerebellum with a peripheral enhancing nodule (arrow).Hematoxylin and eosin staining demonstrates a classic pilocytic astrocytoma with compact and loose areas (b), including Rosenthal fibers(arrow) and eosinophilic granular bodies (arrowhead) (c). The tumor is composed of cells with strong GFAP expression (d) and rare Ki-67 immunoreactivity (arrowhead; (e)). Direct amplification of RNA from this tumor demonstrates a 599 bp fragment, which creates anovel fusion KIAA1549:BRAF transcript in which exon 16 of the KIAA1549 gene is joined to BRAF sequences in the middle of exon 10.The bars below the predicted amino acid sequence correspond to BRAF exon 10 (red), BRAF exon 11 (green), and KIAA1549 exon 16(blue) (f). Immunostaining with phospho-ERK-Thr202/Tyr204 antibodies demonstrates increased ERK activation in the PA tumor (bottompanel). Normal adult human frontal lobe (NB) from an autopsy specimen was included as reference tissue in the top panel (g). Western blotdemonstrates 282-fold increase in ERK activation (phospho-ERK-Thr202/Tyr204; p-ERK; Cell Signaling Technologies, catalog no. 4370S) inthe tumor (PA) relative to normal human brain (NB). Total ERK is included as internal control for protein loading (h).Case Reports in Medicine 3Table 1: Summary of reported BRAF fusion transcripts.Fusion partner BRAF Number of cases % casesKIAA1549 exon 16 exon 9 136 62.4KIAA1549 exon 15 exon 9 47 22.6KIAA1549 exon 11 exon 11 29 12.3KIAA1549 exon 18 exon 10 1 <1KIAA1549 exon 19 exon 9 1 <1KIAA1549 exon 16 exon 10∗ 1 <1FAM131B exon 9 3 1.4∗Current paper.were largely myxoid with scattered pleomorphic tumor cells,often containing multiple nuclei. Consistent with the glialnature of this tumor, there was diﬀuse and strong glialfibrillary acidic protein (GFAP) expression in the neoplasticcells (Figure 1(d)). The Ki67 labeling (proliferative) indexwas <1% (Figure 1(e)), and mitotic figures were not iden-tified. Upon two-year followup, there was no evidence ofrecurrent tumor onMRI. To identify themolecular alterationin this pilocytic astrocytoma, RNA was extracted from asnap-frozen tumor specimen using the RNeasy mini-kit(QIAGEN), reverse transcribed, and amplified by PCR usingBRAF and KIAA1549 primers as previously reported [8].Both strands of the resulting novel 599 base pair (bp)product were directly sequenced on an ABI 3730xl DNAAnalyzer. In contrast to previously reported KIAA1549:BRAFalterations, this tumor harbored a novel fusion transcript inwhich exon 16 of the KIAA1549 gene was fused to sequenceswithin exon 10 of the BRAF gene (Figure 1(f)), generatinga protein product in which the BRAF kinase domain isintact. This would result in a molecule in which the carboxylterminal kinase domain is not bound by the amino terminalBRAF regulatory domain and is thus “constitutively” active,leading to downstream MEK and ERK activation. Consistentwith this prediction, we found increased ERK activationusing activation-specific (phospho-Thr202/Tyr204) antibodiesin the tumor by both immunohistochemistry (Figure 1(g))and Western immunoblotting (Figure 1(h)).3. DiscussionThe vast majority of previously reported molecular alter-ations in sporadic involve BRAF exons 9 (85% of reportedKIAA1549:BRAF fusion transcripts) and 11 (12% of reportedKIAA1549:BRAF transcripts) (Table 1). Similarly, all of theFAM131B:BRAF fusion products also included BRAF exon 9sequences [11]. The current paper describes only the secondKIAA1549:BRAF fusion event involving exon 10 [7] and isthe first in which the alteration eliminates nearly half of theexon 10-encoded BRAF protein sequence. The inclusion ofthis specific genetic alteration to the growing list of BRAFmolecular changes supports a model in which fusion eventsthat maintain the BRAF open reading frame and includethe BRAF protein sequences encoded by exons 11–18 (BRAFkinase domain) are potentially tumorigenic.This proposed tumorigenicity is attributed to constitu-tive activation of the BRAF kinase domain as a result of theremoval of the amino terminal inhibitory domain, leading toincreased signaling to its immediate downstream eﬀectors,MEK and ERK. Similar to other BRAF mutations, thisnovel KIAA1549:BRAF molecular alteration is also associatedwith increased ERK activity. However, the exact mechanismby which deregulated MEK/ERK activation resulting fromKIAA1549:BRAF leads to pilocytic astrocytoma developmentis unclear. In this regard, several groups have shown thatthe expression of constitutively active (oncogenic) BRAF(BRAFV600; V600E mutation within the BRAF activationdomain) in human astrocytes and glial progenitor cells leadsto cellular senescence in vitro [12], and neither oncogenicBRAFV600 nor RAF1 expression in mice results in gliomaformation in vivo [13, 14]. However, forced expression ofthe kinase domain of BRAFV600, but not of wild-type BRAF(as exists in KIAA1549:BRAF fusion protein products), istransforming in primary human astrocytes in vitro and caninduce tumors in mice in vivo [13].NF1-associated pilocytic astrocytomas also exhibitincreased ERK activation as a result of mutation loss of theNF1 tumor suppressor protein, neurofibromin. In primarymouse astrocytes, loss of neurofibromin Ras GTPase ac-tivating protein (GAP) activity leads to high levels of Raseﬀector (ERK, AKT) activation. However, Nf1 geneticallyengineered mouse optic glioma growth is attenuated byinhibiting AKT pathway signaling [15]. In these studies,inhibition of AKT-mediatedmammalian target of rapamycin(mTOR) activity using the macrolide rapamycin resultedin reduced optic glioma volume and proliferation. In lightof these observations, the molecular mechanism shared byBRAF activation and neurofibromin loss will require furtherexperimental investigation.In this regard, future studies will likewise be requiredto determine precisely how BRAF activation leads to gliomaformation either alone or in concert with other genetic orstromal (microenvironment) changes. Despite these seem-ingly contradictory experimental observations, the identifi-cation of BRAF as a seminal genetic alteration in pilocyticastrocytoma sets the stage for therapeutic trials aimed atrestoring deregulated BRAF/RAF signaling in this commonpediatric brain tumor.AcknowledgmentsThe authors appreciate the technical assistance of Mr. RyanEmnett. The authors have no disclosures to report. Se-quencing core support was provided by a Grant from theNational Institutes of Health (UL RR024992).References[1] R. Listernick, J. Charrow, M. J. Greenwald, and N. B. Esterly,“Optic gliomas in children with neurofibromatosis type 1,”Journal of Pediatrics, vol. 114, no. 5, pp. 788–792, 1989.[2] L. Kluwe, C. Hagel, M. Tatagiba et al., “Loss of NF1alleles distinguish sporadic from NF1-associated pilocytic as-trocytomas,” Journal of Neuropathology and ExperimentalNeurology, vol. 60, no. 9, pp. 917–920, 2001.4 Case Reports in Medicine[3] S. Pfister, W. G. Janzarik, M. Remke et al., “BRAF gene dupli-cation constitutes a mechanism of MAPK pathway activationin low-grade astrocytomas,” Journal of Clinical Investigation,vol. 118, no. 5, pp. 1739–1749, 2008.[4] E. E. Bar, A. Lin, T. Tihan, P. C. Burger, and C. G. Eberhart,“Frequent gains at chromosome 7q34 involving BRAF inpilocytic astrocytoma,” Journal of Neuropathology and Exper-imental Neurology, vol. 67, no. 9, pp. 878–887, 2008.[5] D. T. W. Jones, S. Kocialkowski, L. Liu et al., “Tandem dupli-cation producing a novel oncogenic BRAF fusion gene definesthe majority of pilocytic astrocytomas,” Cancer Research, vol.68, no. 21, pp. 8673–8677, 2008.[6] A. J. Sievert, E. M. Jackson, X. Gai et al., “Duplication of7q34 in pediatric low-grade astrocytomas detected byhigh-density single-nucleotide polymorphism-based geno-type arrays results in a novel BRAF fusion gene,” Brain Pa-thology, vol. 19, pp. 449–458, 2009.[7] T. Forshew, R. G. Tatevossian, A. R. J. Lawson et al., “Activationof the ERK/MAPK pathway: a signature genetic defect inposterior fossa pilocytic astrocytomas,” Journal of Pathology,vol. 218, no. 2, pp. 172–181, 2009.[8] J. Yu, H. Deshmukh, R. J. Gutmann et al., “Alterations ofBRAF and HIPK2 loci predominate in sporadic pilocyticastrocytoma,” Neurology, vol. 73, no. 19, pp. 1526–1531, 2009.[9] K. Jacob, S. Albrecht, C. Sollier et al., “Duplication of 7q34is specific to juvenile pilocytic astrocytomas and a hallmarkof cerebellar and optic pathway tumours,” British Journal ofCancer, vol. 101, no. 4, pp. 722–733, 2009.[10] D. T. W. Jones, S. Kocialkowski, L. Liu, D. M. Pearson, K.Ichimura, and V. P. Collins, “Oncogenic RAF1 rearrangementand a novel BRAF mutation as alternatives to KIAA1549:BRAFfusion in activating the MAPK pathway in pilocytic astrocy-toma,” Oncogene, vol. 28, no. 20, pp. 2119–2123, 2009.[11] H. Cin, C. Meyer, R. Herr et al., “Oncogenic FAM131B-BRAFfusion resulting from 7q34 deletion comprises an alternativemechanism of MAPK pathway activation in pilocytic astro-cytoma,” Acta Neuropathologica, vol. 121, no. 6, pp. 763–774,2011.[12] E. H. Raabe, K. S. Lim, J. M. Kim et al., “BRAF activationinduces transformation and then senescence in human neuralstem cells: a pilocytic astrocytoma model,” Clinical CancerResearch, vol. 17, no. 11, pp. 3590–3599, 2011.[13] J. Gronych, A. Korshunov, J. Bageritz et al., “An activatedmutant BRAF kinase domain is suﬃcient to induce pilocyticastrocytoma inmice,” Journal of Clinical Investigation, vol. 121,no. 4, pp. 1344–1348, 2011.[14] Y. Lyustikman, H. Momota, W. Pao, and E. C. Holland,“Constitutive activation of raf-1 induces glioma formation inmice,” Neoplasia, vol. 10, no. 5, pp. 501–510, 2008.[15] B. Hegedus, D. Banerjee, T. H. Yeh et al., “Preclinical cancertherapy in a mouse model of neurofibromatosis-1 optic gli-oma,” Cancer Research, vol. 68, no. 5, pp. 1520–1528, 2008.Submit your manuscripts athttp://www.hindawi.comStem CellsInternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014MEDIATORSINFLAMMATIONofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Behavioural NeurologyEndocrinologyInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Disease MarkersHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014BioMed Research InternationalOncologyJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Oxidative Medicine and Cellular LongevityHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014PPAR ResearchThe Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Journal ofObesityJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014 Computational and  Mathematical Methods in MedicineOphthalmologyJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Diabetes ResearchJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Research and TreatmentAIDSHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Gastroenterology Research and PracticeHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Parkinson’s DiseaseEvidence-Based Complementary and Alternative MedicineVolume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

Case Reports in Medicine

Novel<i>BRAF</i>Alteration in a Sporadic Pilocytic Astrocytoma

Leonard, Jeffrey R

Gutmann, David H

Digital Commons@Becker

Washington University School of MedicineDigital Commons@BeckerOpen Access Publications2012Novel BRAF alteration in a sporadic pilocyticastrocytomaSonika DahiyaWashington University School of Medicine in St. LouisJinsheng YuWashington University School of Medicine in St. LouisAparna KaulWashington University School of Medicine in St. LouisJeffrey R. LeonardWashington University School of Medicine in St. LouisDavid H. GutmannWashington University School of Medicine in St. LouisFollow this and additional works at: http://digitalcommons.wustl.edu/open_access_pubsPart of the Medicine and Health Sciences CommonsThis Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in OpenAccess Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact engeszer@wustl.edu.Recommended CitationDahiya, Sonika; Yu, Jinsheng; Kaul, Aparna; Leonard, Jeffrey R.; and Gutmann, David H., ,"Novel BRAF alteration in a sporadicpilocytic astrocytoma." Case Reports in Medicine.2012,. Article ID 418672. (2012).http://digitalcommons.wustl.edu/open_access_pubs/1102Hindawi Publishing CorporationCase Reports in MedicineVolume 2012, Article ID 418672, 4 pagesdoi:10.1155/2012/418672Case ReportNovel BRAF Alteration in a Sporadic Pilocytic AstrocytomaSonika Dahiya,1 Jinsheng Yu,1 Aparna Kaul,2 Jeffrey R. Leonard,3 and David H. Gutmann21 Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA2 Department of Neurology, Washington University School of Medicine, P.O. Box 8111, 660 South Euclid Avenue,St. Louis, MO 63110, USA3 Department of Neurosurgery, Washington University School of Medicine, St. Louis, MO 63110, USACorrespondence should be addressed to David H. Gutmann, gutmannd@neuro.wustl.eduReceived 17 December 2011; Accepted 7 February 2012Academic Editor: Mark E. ShaﬀreyCopyright © 2012 Sonika Dahiya et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Pilocytic astrocytoma (PA) is the most frequently encountered glial tumor (glioma or astrocytoma) in children. Recent studieshave identified alterations in the BRAF serine/threonine kinase gene as the likely causative mutation in these childhood braintumors. The majority of these genetic changes involve chromosome 7q34 tandem duplication, resulting in aberrant BRAF fusiontranscripts. In this paper, we describe a novel KIAA1549:BRAF fusion transcript in a sporadic PA tumor associated with increasedERK activation and review the spectrum of BRAF genetic alterations in this common pediatric low-grade central nervous systemneoplasm.1. IntroductionPilocytic astrocytomas are the most common nonmalignantbrain tumor in the pediatric population. Children with theNeurofibromatosis type 1 (NF1) inherited cancer predispo-sition syndrome are prone to the development of these glialcell neoplasms, such that 15–20% of aﬀected individuals willdevelop gliomas involving the optic pathway, hypothalamus,and brainstem [1]. Molecular analysis of these tumors hasrevealed biallelic inactivation of the NF1 tumor suppressorgene, resulting in loss of NF1 protein (neurofibromin)expression. However, sporadic PA tumors do not exhibitmutational inactivation of the NF1 gene, suggesting thatother genetic mutations are responsible for the genesis ofthese histologically-identical low-grade brain tumors in thegeneral population [2].Over the past several years, the molecular basis for thesenonsyndromic pediatric brain cancers has been elucidatedwith the identification of signature molecular changesinvolving the BRAF serine/threonine kinase gene. The mostfrequently encountered genetic alteration is a tandem dupli-cation of the BRAF gene on chromosome 7q34, leadingto fusion of the KIAA1549 gene to the carboxyl terminalregion of the BRAF gene containing the kinase domain. Thismolecular change has been reported in 50–65% of sporadicpilocytic astrocytoma and is more frequent in cerebellar(∼80%) tumors. The majority of these alterations involvefusions between KIAA1549 exon 16 and BRAF exon 9,KIAA1549 exon 15 and BRAF exon 9, and KIAA1549 exon16 and BRAF exon 11 [3–9], while less common alterationsinclude tandem duplications involving SRGAP3 and RAF1 orFAM131B and BRAF [10, 11]. In this paper, we describe anovel KIAA1549-BRAF fusion event in a sporadic pediatricpilocytic astrocytoma.2. Case PresentationThe patient was a 14-year-old boy who presented with a6-month history of headache that progressed to a two-dayperiod of nausea, vomiting, and ataxia. Magnetic resonanceimaging (MRI) at that time showed a cystic mass in thecerebellum compressing the fourth ventricle (Figure 1(a)).He was taken to the operating room where a gross totalresection was performed. Neuropathological review revealeda classic pilocytic astrocytoma with alternating areas ofcompact and loose tissue architecture (Figure 1(b)). Thecompact areas were composed of piloid neoplastic cellscontaining numerous Rosenthal fibers and few eosinophilicgranular bodies (Figure 1(c)), while the paucicellular areas2 Case Reports in Medicine(g)(f)(d)(a)(b) (c)(e)(h)KIAA1549BRAFe18 e11 e10 e16 e15 e1Ladder1 kb600 bp200 bp100 bpNB PApERKERKFigure 1: Molecular characterization of a novel KIAA1549:BRAF fusion alteration in a sporadic pediatric pilocytic astrocytoma. (a) AxialT1-weighted 1.5-Tesla gadolinium-enhanced MRI scan reveals a cystic lesion in the cerebellum with a peripheral enhancing nodule (arrow).Hematoxylin and eosin staining demonstrates a classic pilocytic astrocytoma with compact and loose areas (b), including Rosenthal fibers(arrow) and eosinophilic granular bodies (arrowhead) (c). The tumor is composed of cells with strong GFAP expression (d) and rare Ki-67 immunoreactivity (arrowhead; (e)). Direct amplification of RNA from this tumor demonstrates a 599 bp fragment, which creates anovel fusion KIAA1549:BRAF transcript in which exon 16 of the KIAA1549 gene is joined to BRAF sequences in the middle of exon 10.The bars below the predicted amino acid sequence correspond to BRAF exon 10 (red), BRAF exon 11 (green), and KIAA1549 exon 16(blue) (f). Immunostaining with phospho-ERK-Thr202/Tyr204 antibodies demonstrates increased ERK activation in the PA tumor (bottompanel). Normal adult human frontal lobe (NB) from an autopsy specimen was included as reference tissue in the top panel (g). Western blotdemonstrates 282-fold increase in ERK activation (phospho-ERK-Thr202/Tyr204; p-ERK; Cell Signaling Technologies, catalog no. 4370S) inthe tumor (PA) relative to normal human brain (NB). Total ERK is included as internal control for protein loading (h).Case Reports in Medicine 3Table 1: Summary of reported BRAF fusion transcripts.Fusion partner BRAF Number of cases % casesKIAA1549 exon 16 exon 9 136 62.4KIAA1549 exon 15 exon 9 47 22.6KIAA1549 exon 11 exon 11 29 12.3KIAA1549 exon 18 exon 10 1 <1KIAA1549 exon 19 exon 9 1 <1KIAA1549 exon 16 exon 10∗ 1 <1FAM131B exon 9 3 1.4∗Current paper.were largely myxoid with scattered pleomorphic tumor cells,often containing multiple nuclei. Consistent with the glialnature of this tumor, there was diﬀuse and strong glialfibrillary acidic protein (GFAP) expression in the neoplasticcells (Figure 1(d)). The Ki67 labeling (proliferative) indexwas <1% (Figure 1(e)), and mitotic figures were not iden-tified. Upon two-year followup, there was no evidence ofrecurrent tumor onMRI. To identify themolecular alterationin this pilocytic astrocytoma, RNA was extracted from asnap-frozen tumor specimen using the RNeasy mini-kit(QIAGEN), reverse transcribed, and amplified by PCR usingBRAF and KIAA1549 primers as previously reported [8].Both strands of the resulting novel 599 base pair (bp)product were directly sequenced on an ABI 3730xl DNAAnalyzer. In contrast to previously reported KIAA1549:BRAFalterations, this tumor harbored a novel fusion transcript inwhich exon 16 of the KIAA1549 gene was fused to sequenceswithin exon 10 of the BRAF gene (Figure 1(f)), generatinga protein product in which the BRAF kinase domain isintact. This would result in a molecule in which the carboxylterminal kinase domain is not bound by the amino terminalBRAF regulatory domain and is thus “constitutively” active,leading to downstream MEK and ERK activation. Consistentwith this prediction, we found increased ERK activationusing activation-specific (phospho-Thr202/Tyr204) antibodiesin the tumor by both immunohistochemistry (Figure 1(g))and Western immunoblotting (Figure 1(h)).3. DiscussionThe vast majority of previously reported molecular alter-ations in sporadic involve BRAF exons 9 (85% of reportedKIAA1549:BRAF fusion transcripts) and 11 (12% of reportedKIAA1549:BRAF transcripts) (Table 1). Similarly, all of theFAM131B:BRAF fusion products also included BRAF exon 9sequences [11]. The current paper describes only the secondKIAA1549:BRAF fusion event involving exon 10 [7] and isthe first in which the alteration eliminates nearly half of theexon 10-encoded BRAF protein sequence. The inclusion ofthis specific genetic alteration to the growing list of BRAFmolecular changes supports a model in which fusion eventsthat maintain the BRAF open reading frame and includethe BRAF protein sequences encoded by exons 11–18 (BRAFkinase domain) are potentially tumorigenic.This proposed tumorigenicity is attributed to constitu-tive activation of the BRAF kinase domain as a result of theremoval of the amino terminal inhibitory domain, leading toincreased signaling to its immediate downstream eﬀectors,MEK and ERK. Similar to other BRAF mutations, thisnovel KIAA1549:BRAF molecular alteration is also associatedwith increased ERK activity. However, the exact mechanismby which deregulated MEK/ERK activation resulting fromKIAA1549:BRAF leads to pilocytic astrocytoma developmentis unclear. In this regard, several groups have shown thatthe expression of constitutively active (oncogenic) BRAF(BRAFV600; V600E mutation within the BRAF activationdomain) in human astrocytes and glial progenitor cells leadsto cellular senescence in vitro [12], and neither oncogenicBRAFV600 nor RAF1 expression in mice results in gliomaformation in vivo [13, 14]. However, forced expression ofthe kinase domain of BRAFV600, but not of wild-type BRAF(as exists in KIAA1549:BRAF fusion protein products), istransforming in primary human astrocytes in vitro and caninduce tumors in mice in vivo [13].NF1-associated pilocytic astrocytomas also exhibitincreased ERK activation as a result of mutation loss of theNF1 tumor suppressor protein, neurofibromin. In primarymouse astrocytes, loss of neurofibromin Ras GTPase ac-tivating protein (GAP) activity leads to high levels of Raseﬀector (ERK, AKT) activation. However, Nf1 geneticallyengineered mouse optic glioma growth is attenuated byinhibiting AKT pathway signaling [15]. In these studies,inhibition of AKT-mediatedmammalian target of rapamycin(mTOR) activity using the macrolide rapamycin resultedin reduced optic glioma volume and proliferation. In lightof these observations, the molecular mechanism shared byBRAF activation and neurofibromin loss will require furtherexperimental investigation.In this regard, future studies will likewise be requiredto determine precisely how BRAF activation leads to gliomaformation either alone or in concert with other genetic orstromal (microenvironment) changes. Despite these seem-ingly contradictory experimental observations, the identifi-cation of BRAF as a seminal genetic alteration in pilocyticastrocytoma sets the stage for therapeutic trials aimed atrestoring deregulated BRAF/RAF signaling in this commonpediatric brain tumor.AcknowledgmentsThe authors appreciate the technical assistance of Mr. RyanEmnett. The authors have no disclosures to report. Se-quencing core support was provided by a Grant from theNational Institutes of Health (UL RR024992).References[1] R. Listernick, J. Charrow, M. J. Greenwald, and N. B. Esterly,“Optic gliomas in children with neurofibromatosis type 1,”Journal of Pediatrics, vol. 114, no. 5, pp. 788–792, 1989.[2] L. Kluwe, C. Hagel, M. Tatagiba et al., “Loss of NF1alleles distinguish sporadic from NF1-associated pilocytic as-trocytomas,” Journal of Neuropathology and ExperimentalNeurology, vol. 60, no. 9, pp. 917–920, 2001.4 Case Reports in Medicine[3] S. Pfister, W. G. Janzarik, M. Remke et al., “BRAF gene dupli-cation constitutes a mechanism of MAPK pathway activationin low-grade astrocytomas,” Journal of Clinical Investigation,vol. 118, no. 5, pp. 1739–1749, 2008.[4] E. E. Bar, A. Lin, T. Tihan, P. C. Burger, and C. G. Eberhart,“Frequent gains at chromosome 7q34 involving BRAF inpilocytic astrocytoma,” Journal of Neuropathology and Exper-imental Neurology, vol. 67, no. 9, pp. 878–887, 2008.[5] D. T. W. Jones, S. Kocialkowski, L. Liu et al., “Tandem dupli-cation producing a novel oncogenic BRAF fusion gene definesthe majority of pilocytic astrocytomas,” Cancer Research, vol.68, no. 21, pp. 8673–8677, 2008.[6] A. J. Sievert, E. M. Jackson, X. Gai et al., “Duplication of7q34 in pediatric low-grade astrocytomas detected byhigh-density single-nucleotide polymorphism-based geno-type arrays results in a novel BRAF fusion gene,” Brain Pa-thology, vol. 19, pp. 449–458, 2009.[7] T. Forshew, R. G. Tatevossian, A. R. J. Lawson et al., “Activationof the ERK/MAPK pathway: a signature genetic defect inposterior fossa pilocytic astrocytomas,” Journal of Pathology,vol. 218, no. 2, pp. 172–181, 2009.[8] J. Yu, H. Deshmukh, R. J. Gutmann et al., “Alterations ofBRAF and HIPK2 loci predominate in sporadic pilocyticastrocytoma,” Neurology, vol. 73, no. 19, pp. 1526–1531, 2009.[9] K. Jacob, S. Albrecht, C. Sollier et al., “Duplication of 7q34is specific to juvenile pilocytic astrocytomas and a hallmarkof cerebellar and optic pathway tumours,” British Journal ofCancer, vol. 101, no. 4, pp. 722–733, 2009.[10] D. T. W. Jones, S. Kocialkowski, L. Liu, D. M. Pearson, K.Ichimura, and V. P. Collins, “Oncogenic RAF1 rearrangementand a novel BRAF mutation as alternatives to KIAA1549:BRAFfusion in activating the MAPK pathway in pilocytic astrocy-toma,” Oncogene, vol. 28, no. 20, pp. 2119–2123, 2009.[11] H. Cin, C. Meyer, R. Herr et al., “Oncogenic FAM131B-BRAFfusion resulting from 7q34 deletion comprises an alternativemechanism of MAPK pathway activation in pilocytic astro-cytoma,” Acta Neuropathologica, vol. 121, no. 6, pp. 763–774,2011.[12] E. H. Raabe, K. S. Lim, J. M. Kim et al., “BRAF activationinduces transformation and then senescence in human neuralstem cells: a pilocytic astrocytoma model,” Clinical CancerResearch, vol. 17, no. 11, pp. 3590–3599, 2011.[13] J. Gronych, A. Korshunov, J. Bageritz et al., “An activatedmutant BRAF kinase domain is suﬃcient to induce pilocyticastrocytoma inmice,” Journal of Clinical Investigation, vol. 121,no. 4, pp. 1344–1348, 2011.[14] Y. Lyustikman, H. Momota, W. Pao, and E. C. Holland,“Constitutive activation of raf-1 induces glioma formation inmice,” Neoplasia, vol. 10, no. 5, pp. 501–510, 2008.[15] B. Hegedus, D. Banerjee, T. H. Yeh et al., “Preclinical cancertherapy in a mouse model of neurofibromatosis-1 optic gli-oma,” Cancer Research, vol. 68, no. 5, pp. 1520–1528, 2008.

Novel BRAF alteration in a sporadic pilocytic astrocytoma

Directory of Open Access Journals

Novel BRAF Alteration in a Sporadic Pilocytic Astrocytoma

A .J .S i e v e r t ,E .M .J a c k s o n ,X .G a ie ta l . ,“ D u p l i c a t i o no f 7q34 in pediatric low-grade astrocytomas detected by high-density single-nucleotide polymorphism-based genotype arrays results in a novel BRAF fusion gene,”

An activated mutant BRAF kinase domain is suﬃcient to induce pilocytic

BRAF activation induces transformation and then senescence in human neural stem cells: a pilocytic astrocytoma model,”

BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas,”

Duplication of 7q34 is speciﬁc to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours,”

Frequent gains at chromosome 7q34 involving BRAF in pilocytic astrocytoma,”

Oncogenic FAM131B-BRAF fusion resulting from 7q34 deletion comprises an alternative mechanism of MAPK pathway activation in pilocytic astrocytoma,”

Optic gliomas in children with neuroﬁbromatosis type 1,”

orshew ,R.G.T atev ossian,A.R.J .La wsonetal.,“ A ctivation of the ERK/MAPK pathway: a signature genetic defect in posterior fossa pilocytic astrocytomas,”

Preclinical cancer therapy in a mouse model of neuroﬁbromatosis-1 optic glioma,”

Tandem duplication producing a novel oncogenic BRAF fusion gene deﬁnes the majority of pilocytic astrocytomas,”

Tatagiba et al., “Loss of NF1 alleles distinguish sporadic from NF1-associated pilocytic astrocytomas,”

y u s t i k m a n ,H .M o m o t a ,W .P a o ,a n dE .C .H o l l a n d , “Constitutive activation of raf-1 induces glioma formation in mice,”

http://digitalcommons.wustl.edu/cgi/viewcontent.cgi?article=2098&context=open_access_pubs

Novel BRAF Alteration in a Sporadic Pilocytic Astrocytoma

Abstract

Similar works

Full text

Available Versions

Crossref

Digital Commons@Becker

Directory of Open Access Journals