6
ORIGINAL ARTICLE BRAF Duplications and MAPK Pathway Activation Are Frequent in Gliomas of the Optic Nerve Proper Fausto J. Rodriguez, MD, Azra H. Ligon, PhD, Iren Horkayne-Szakaly, MD, Elisabeth J. Rushing, MD, Keith L. Ligon, MD, PhD, Natalie Vena, MS, Denise I. Garcia, BS, J. Douglas Cameron, MD, and Charles G. Eberhart, MD, PhD Abstract Optic pathway gliomas represent a specific subtype of astrocy- toma with unique clinicopathologic and biologic properties, but studies of tumors in the optic nerve proper have been hampered by limited tissue availability. We analyzed optic nerve gliomas of 59 patients (median age, 9 years; range, 3 monthsY66 years; 33 female, 26 male) using formalin-fixed paraffin-embedded material in tissue microarrays. Seven patients had the clinical diagnosis of neurofibromatosis type 1 (NF1). Fluorescence in situ hybridization studies were performed for BRAF, PTEN, CDKN2A (p16), and NF1. Immunohistochemistry was performed for glial fibrillary acidic protein, phospho-ERK, and mutant IDH1 R132H protein. The BRAF duplication was present in 11 (73%) of 15 evaluable tumors, including 1 NF1 patient (1 of 4 tested; 25%). The single tumor lacking BRAF duplication or NF1 associ- ation had histologic features of a ganglioglioma. Conversely, het- erozygous PTEN deletions were present in 2 (8%) of 25 evaluable cases, one of which was BRAF duplicated and the other was NF1 associated. CDKN2A and NF1 deletions were absent in all tumors tested. Phospho-ERK immunoreactivity was present in 55 (96%) of 57 tumors and was mostly strong and diffuse (80%). Only 1 case of 53 expressed IDH1 R132H . Thus, optic nerve gliomas demon- strated molecular alterations typical of pilocytic astrocytomas, including the universal presence of either BRAF duplication or NF1 association and common mitogen-activated protein kinase pathway activation but very rare mutant IDH1 expression. Key Words: BRAF, Fluorescence in situ hybridization, Glioma, MAPK, Neurofibromatosis, Optic nerve, Pilocytic astrocytoma. INTRODUCTION Gliomas involving the optic nerve and other optic path- way structures (i.e. optic pathway gliomas) represent a specific subtype of astrocytoma with unique clinicopathologic and bio- logic properties. Optic nerve gliomas may occur in the setting of neurofibromatosis type 1 (NF1) or sporadically and affect approximately 15% of children with NF1 (1). At the patho- logical level, NF1-associated and sporadic optic nerve glio- mas are predominantly World Health Organization grade I pilocytic astrocytomas (PAs) (2Y4). Whereas optic pathway gliomas are often considered as a group, some clinical data suggest that the site within the optic pathways at which they arise can affect their biology. Tumors in the hypothalamic region can behave aggressively, and these are often of the pilomyxoid variant (5). In contrast, it has long been recognized that tumors involving the optic nerve proper, particularly in the setting of NF1, are typically indolent and, in some instances, regress spontaneously (6). Because of the benign behavior and typical radiological ap- pearance of optic nerve gliomas, in recent decades, they have rarely been biopsied, hampering investigations into their mo- lecular basis. Recent insights into the molecular mechanisms respon- sible for PAs have centered on the importance of mitogen- activated protein kinase (MAPK) pathway signaling. In NF1-associated tumors, the mechanism is biallelic NF1 gene inactivation (7). In sporadic PAs, the most frequent molec- ular alteration is a tandem duplication at chromosomal re- gion 7q34 involving the BRAF kinase domain, which leads to a novel KIAA1549:BRAF fusion (8Y11). Other alterations less commonly reported include BRAF (V600E) point mutation (12), K-RAS mutations (13), SRGAP3:RAF1 fusions (13, 14), small BRAF insertions (BRAF insT ) (14Y16), and the recently described FAM131B:BRAF fusion mediated by an interstitial deletion (17). The common biologic effect of all these alter- ations is MAPK pathway activation, which is an almost uni- versal feature of PAs. Conversely, IDH1/2 mutations are absent in almost all instances, in contrast to diffuse gliomas where they are common (18Y20). 789 J Neuropathol Exp Neurol Volume 71, Number 9, September 2012 J Neuropathol Exp Neurol Copyright Ó 2012 by the American Association of Neuropathologists, Inc. Vol. 71, No. 9 September 2012 pp. 789Y794 From the Department of Pathology, Johns Hopkins Hospital, Baltimore, Maryland (FJR, CGE); Brigham and Women’s Hospital (AHL, KLL); Children’s Hospital Boston (KLL); and Department of Medical Oncology and Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute (AHL, KLL, NV, DIG); and Harvard Medical School (AHL, KLL), Boston, and Massachusetts; Joint Pathology Center, Silver Spring, Maryland (IH-S); University Hospital Zurich, Institute of Neuropathology, Zurich, Switzerland (EJR); and Departments of Ophthalmology and Labora- tory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota (JDC). Send correspondence and reprint requests to: Fausto J. Rodriguez, MD, Department of Pathology, Division of Neuropathology, Johns Hopkins University, 720 Rutland Ave, Ross Bldg 558, Baltimore, MD 21205; E-mail: [email protected] This work was funded in part by the Children’s Cancer Foundation and con- tributors to the Pilocytic/Pilomyxoid Astrocytoma Fund, including Lauren’s First and Goal (Charles G. Eberhart), the Childhood Brain Tumor Founda- tion (Fausto J. Rodriguez), the DFCI Pediatric Low-Grade Astrocytoma Foundation (Keith L. Ligon, Azra H. Ligon), NCI P01CA142536, and an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology of the University of Minnesota (J. Douglas Cameron). Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.

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Page 1: BRAF Duplications and MAPK Pathway Activation Are Frequent ...neuro.pathology.pitt.edu/conferences/jclub/JClub3-7-13MCFADDEN.pdf · BRAF Duplications and MAPK Pathway Activation Are

ORIGINAL ARTICLE

BRAF Duplications and MAPK Pathway Activation Are Frequentin Gliomas of the Optic Nerve Proper

Fausto J. Rodriguez, MD, Azra H. Ligon, PhD, Iren Horkayne-Szakaly, MD, Elisabeth J. Rushing, MD,Keith L. Ligon, MD, PhD, Natalie Vena, MS, Denise I. Garcia, BS, J. Douglas Cameron, MD,

and Charles G. Eberhart, MD, PhD

AbstractOptic pathway gliomas represent a specific subtype of astrocy-

toma with unique clinicopathologic and biologic properties, but studiesof tumors in the optic nerve proper have been hampered by limitedtissue availability. We analyzed optic nerve gliomas of 59 patients(median age, 9 years; range, 3 monthsY66 years; 33 female, 26 male)using formalin-fixed paraffin-embedded material in tissue microarrays.Seven patients had the clinical diagnosis of neurofibromatosis type 1(NF1). Fluorescence in situ hybridization studies were performed forBRAF, PTEN, CDKN2A (p16), and NF1. Immunohistochemistry wasperformed for glial fibrillary acidic protein, phospho-ERK, and mutantIDH1R132H protein. The BRAF duplication was present in 11 (73%)of 15 evaluable tumors, including 1 NF1 patient (1 of 4 tested;25%). The single tumor lacking BRAF duplication or NF1 associ-ation had histologic features of a ganglioglioma. Conversely, het-erozygous PTEN deletions were present in 2 (8%) of 25 evaluablecases, one of which was BRAF duplicated and the other was NF1associated. CDKN2A and NF1 deletions were absent in all tumorstested. Phospho-ERK immunoreactivity was present in 55 (96%) of57 tumors and was mostly strong and diffuse (80%). Only 1 caseof 53 expressed IDH1R132H. Thus, optic nerve gliomas demon-strated molecular alterations typical of pilocytic astrocytomas,including the universal presence of either BRAF duplication orNF1 association and common mitogen-activated protein kinasepathway activation but very rare mutant IDH1 expression.

Key Words: BRAF, Fluorescence in situ hybridization, Glioma,MAPK, Neurofibromatosis, Optic nerve, Pilocytic astrocytoma.

INTRODUCTIONGliomas involving the optic nerve and other optic path-

way structures (i.e. optic pathway gliomas) represent a specificsubtype of astrocytoma with unique clinicopathologic and bio-logic properties. Optic nerve gliomas may occur in the settingof neurofibromatosis type 1 (NF1) or sporadically and affectapproximately 15% of children with NF1 (1). At the patho-logical level, NF1-associated and sporadic optic nerve glio-mas are predominantly World Health Organization grade Ipilocytic astrocytomas (PAs) (2Y4).

Whereas optic pathway gliomas are often considered asa group, some clinical data suggest that the site within theoptic pathways at which they arise can affect their biology.Tumors in the hypothalamic region can behave aggressively,and these are often of the pilomyxoid variant (5). In contrast,it has long been recognized that tumors involving the opticnerve proper, particularly in the setting of NF1, are typicallyindolent and, in some instances, regress spontaneously (6).Because of the benign behavior and typical radiological ap-pearance of optic nerve gliomas, in recent decades, they haverarely been biopsied, hampering investigations into their mo-lecular basis.

Recent insights into the molecular mechanisms respon-sible for PAs have centered on the importance of mitogen-activated protein kinase (MAPK) pathway signaling. InNF1-associated tumors, the mechanism is biallelic NF1 geneinactivation (7). In sporadic PAs, the most frequent molec-ular alteration is a tandem duplication at chromosomal re-gion 7q34 involving the BRAF kinase domain, which leadsto a novel KIAA1549:BRAF fusion (8Y11). Other alterationsless commonly reported include BRAF (V600E) point mutation(12), K-RAS mutations (13), SRGAP3:RAF1 fusions (13, 14),small BRAF insertions (BRAF insT ) (14Y16), and the recentlydescribed FAM131B:BRAF fusion mediated by an interstitialdeletion (17). The common biologic effect of all these alter-ations is MAPK pathway activation, which is an almost uni-versal feature of PAs. Conversely, IDH1/2 mutations areabsent in almost all instances, in contrast to diffuse gliomaswhere they are common (18Y20).

789J Neuropathol Exp Neurol � Volume 71, Number 9, September 2012

J Neuropathol Exp NeurolCopyright � 2012 by the American Association of Neuropathologists, Inc.

Vol. 71, No. 9September 2012

pp. 789Y794

From the Department of Pathology, Johns Hopkins Hospital, Baltimore,Maryland (FJR, CGE); Brigham and Women’s Hospital (AHL, KLL);Children’s Hospital Boston (KLL); and Department of Medical Oncologyand Center for Molecular Oncologic Pathology, Dana-Farber CancerInstitute (AHL, KLL, NV, DIG); and Harvard Medical School (AHL, KLL),Boston, andMassachusetts; Joint Pathology Center, Silver Spring, Maryland(IH-S); University Hospital Zurich, Institute of Neuropathology, Zurich,Switzerland (EJR); and Departments of Ophthalmology and Labora-tory Medicine and Pathology, University of Minnesota, Minneapolis,Minnesota (JDC).

Send correspondence and reprint requests to: Fausto J. Rodriguez, MD,Department of Pathology, Division of Neuropathology, Johns HopkinsUniversity, 720 Rutland Ave, Ross Bldg 558, Baltimore, MD 21205;E-mail: [email protected]

This work was funded in part by the Children’s Cancer Foundation and con-tributors to the Pilocytic/Pilomyxoid Astrocytoma Fund, including Lauren’sFirst and Goal (Charles G. Eberhart), the Childhood Brain Tumor Founda-tion (Fausto J. Rodriguez), the DFCI Pediatric Low-Grade AstrocytomaFoundation (Keith L. Ligon, Azra H. Ligon), NCI P01CA142536, and anunrestricted grant from Research to Prevent Blindness to the Department ofOphthalmology of the University of Minnesota (J. Douglas Cameron).

Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.

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Of interest, molecular alterations in PA, including opticpathway gliomas, appear to be site dependent. For example,global gene expression profiles in PA vary according to CNSsite of origin (21, 22). In addition, KIAA1549:BRAF fusionsare most frequent in cerebellar PAs in many studies, with in-cidences ranging from 72% to 94% (8, 9, 11, 13, 17, 23, 24),whereas BRAF (V600E) is more typical of hemispheric PA (12).Although BRAF alterations are also frequent in optic pathwaygliomas, with reported rates ranging from 43% to 69% (9, 17,24Y26), most of the tumors previously profiled were locatedin the hypothalamic region. The prevalence of BRAF alter-ations and MAPK pathway activation in gliomas of the opticnerve itself are therefore unclear. In this study, we tookadvantage of a unique historical archive containing a largenumber of optic nerve gliomas resected en bloc to per-form targeted immunohistochemical and molecular analysisof tumors at a site from which tissue is rarely removed incurrent clinical practice.

MATERIALS AND METHODS

Patients and Tumor SamplesTumors obtained from 59 patients were retrieved from

the archives of the former Armed Forces Institute of Pathol-ogy (Washington, DC). In most of these cases, the optic nervewas resected en bloc, often with enucleation of the globe.Initial histopathologic evaluation was consistent with PA/low-grade astrocytoma in all cases (Fig. 1). Based on thepresence of neoplastic ganglion cells, 2 cases were reclassi-fied after central rereview by several neuropathologists asgangliogliomas. Median age at surgery was 9 years old(range, 3 monthsY66 years), and there were 33 females and26 males. Five tumors demonstrated intraocular extension.Storage time for archived tissue ranged from 15 to 78 years(mean, 47 years). A tissue microarray was constructed fromformalin-fixed paraffin-embedded material using 2 to 5 cores,1 mm in diameter per tumor, with sampling of different neo-plastic regions when possible. Clinical evidence of NF1 waspresent in 7 (19%) of 37 patients for which detailed clinicalrecords were available. All studies were approved by institu-tional review boards at the participating institutions.

ImmunohistochemistryImmunohistochemical studies were performed using

antibodies recognizing phospho-ERK (1:400; rabbit mono-clonal antibody [D13.14.4E] XP, Cell Signaling Technology,Danvers, MA), glial fibrillary acidic protein ([GFAP] pre-diluted, rabbit monoclonal; Ventana, Tucson, AZ), and mutantIDH1R132H protein (clone H09, 1:50; Dianova, Hamburg,Germany). Scoring for pERK and GFAP was performed usinga 4-tiered semiquantitative scale (0 to +++) by a single neuro-pathologist (Fausto J. Rodriguez). The median value of mul-tiple scores was used. IDH1 mutant protein was scored aspositive or negative.

Fluorescence In Situ HybridizationBRAF

Fluorescence in situ hybridization (FISH) studies wereperformed using home brew probes targeting 3¶BRAF and

5¶BRAF and a commercial CEP7 probe (Abbott Molecular,Des Plaines, IL) and were scored as previously described (27).

P16, NF1, and PTENCommercially available probes that target CDKN2A/

p16 (9p21) and PTEN (10q23) were used, with respectivecontrol probes CEP9 and CEP10 (Abbott Molecular/Vysis).A custom-made probe targeting NF1 (17q11.2) with asso-ciated CEP17 was obtained from Empire Genomics (Buffalo,NY). Appropriate target hybridization was confirmed by hy-bridization with spread human metaphases. Interpretation ofFISH signals was performed using previously establishedcutoffs (23). In brief, at least 50 nonoverlapping nuclei wereenumerated per tumor, and only cases with clear probe hy-bridization were scored using a fluorescence microscope.Cutoffs for heterozygous deletion were defined as a target/control ratio of less than 0.80.

RESULTS

BRAF Duplication Is Frequent in Glioma of theOptic Nerve, Whereas CDKN2A, NF1, and PTENDeletions Are Rare to Absent

Only the subset of TMA cases with immunofluores-cent signals sufficient for evaluation was scored, and the factthat most cases were more than 4 decades old likely limited

FIGURE 1. Optic nerve glioma pathology. (A) Optic nervegliomas cause fusiform expansions of the optic nerve that inthe past often led to large resections. (B) Extension into thesubarachnoid space may be a prominent feature (hematox-ylin and eosin [H&E], original magnification: 100�). (C) Theoverwhelming majority of optic nerve gliomas demonstratepilocytic features, including biphasic architecture with alter-nating microcystic and compact areas (H&E, original magni-fication: 200�).

Rodriguez et al J Neuropathol Exp Neurol � Volume 71, Number 9, September 2012

� 2012 American Association of Neuropathologists, Inc.790

Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.

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the technical success rate. Of 59 cases, 26 (44%) lackedsuccessful hybridizations with any probe; 4 (7%) casesshowed successful hybridizations with 1 probe only; and 29(49%) were successful with 2 or more probes. These find-ings confirm that hybridization failures/weak hybridizationswere not uniform across samples. Duplication of the BRAFkinase domain (3¶ portion of the gene) was the most fre-quent molecular alteration, present in 11 (73%) of 15 ofthe evaluxable tumor samples (Fig. 2A). Conversely, heter-ozygous PTEN deletions were present in 2 (8%) of 25 cases,whereas CDKN2A and NF1 deletions were absent in all casessuccessfully tested (n = 29 and 23, respectively) (Fig. 2BYD).Both cases with PTEN deletions lacked p16 deletions; onehad a concurrent BRAF duplication and another was associ-ated with NF1. Polysomies involving chromosomes 7, 9, 10,and 17 were present in 2 (13%), 2 (7%), 1 (4%), and 2 (9%)cases, respectively. Of interest, 17 of 18 cases with suc-cessful BRAF FISH studies and/or known NF1 clinical statushad either BRAF duplication or NF1 syndrome, and the singlecase lacking either was histologically classified as a ganglio-glioma. Of 4 patients with NF1 syndrome, 1 had a concur-rent BRAF duplication (Tables 1, 2).

MAPK Activation Is Frequent in Optic NerveGlioma, Whereas Mutant IDH1 ProteinExpression Is Very Rare

Given the prolonged storage age of many of the sam-ples, immunohistochemistry for GFAP was performed to eval-uate for adequacy of immunoreactivity. Staining for GFAP waspreserved in 53 (95%) of 56 cases, suggesting intact immu-noreactivity in most of the tumors (Fig. 3A). Cases lackingimmunoreactivity for GFAP or the relevant marker were ex-cluded from interpretation.

Immunolabeling for pERK was present in 55 (97%) of57 cases. When present, pERK immunoreactivity was usuallystrong and diffuse (81%) (Fig. 3B), supporting MAPK path-way activation in the vast majority of gliomas of the opticnerve and similar to PAs in other anatomic compartments.The pERK was also positive in all cases with BRAF duplica-tion (11 of 11); in this group, 9 were +++ and 2 were +. Of7 NF1 cases, 6 demonstrated strong (+++) pERK immunor-eactivity and 1 was negative. Conversely, only 1 case of 53expressed IDH1R132H mutant protein (Fig. 3C, D), but FISHstudies failed in that case. Results are summarized in Table 1.

Optic Nerve Gliomas Are AssociatedWith a Relatively Favorable Outcome

Outcome data were available for 15 patients (25%). Tenpatients were alive and stable 1 to 12 years after surgery(median follow-up, 6.5 years). Two patients underwent sub-sequent surgeries for recurrence/progression 3 months and48 years after the first surgery. Two patients died of peri-operative complications, and 1 patient died with progressivedisease 5 years after surgery, which was confirmed at au-topsy. No firm associations between molecular findings andoutcome were identified, although the single patient who diedwith progressive disease had the clinical diagnosis of NF1as well as a heterozygous PTEN deletion.

DISCUSSIONOur study supports the feasibility of using older archi-

val material for phenotypic and molecular studies, which maybe of particular use in rare tumors for which surgical ex-cess material is frequently not available. Based on GFAP andpERK immunostaining, we demonstrate a high frequency(È95%) of antigen preservation in formalin-fixed paraffin-embedded tumor tissues. Hybridization failures for FISHstudies were higher, but objective scoring was possible inalmost half of the cases. A particularly high extent of failurefor FISH studies was noted in cases stored more than 55 years,with only 1 case of 8 in this group providing interpretable re-sults with 2 FISH probe sets.

The BRAF duplications and strong pERK immunos-taining were frequent, whereas PTEN, CDKN2A (p16), andNF1 deletions were rare to absent. This confirms MAPKpathway activation through genetic alterations as an essentialbiologic feature of optic nerve gliomas, analogous to PAsarising in other anatomic sites such as the cerebellum. Themain difference is the greater proportion of NF1 syndromepatients with optic nerve gliomas (19%), whereas cerebellarPAs are almost always sporadic. It is of note that essentially

FIGURE 2. Molecular cytogenetic findings in optic nerve gli-oma. (A) Themain molecular cytogenetic finding in optic nervegliomas was duplication of the 3¶ region of the BRAF gene,leading to 3 red signals in neoplastic cells (overlapped withgreen yielding yellow signals in 2 cells). Arrow indicates thenormal cell pattern for reference. (B) NF1 deletions were absentin all cases, including those obtained from patients with theclinical diagnosis of NF1. (C) Heterozygous PTEN deletions werepresent in 2 cases only; 1 of these died with progressive disease.(D) Polysomies involving several chromosomes, including Chr 9recognized with a probe targeting p16/CEP9, were present in aminority of cases (original magnification: 1,000�).

J Neuropathol Exp Neurol � Volume 71, Number 9, September 2012 MAPK Pathway Alterations in Optic Nerve Glioma

� 2012 American Association of Neuropathologists, Inc. 791

Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.

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TABLE

1.Optic

Nerve

Glio

maCases

With

BRAF

Dup

licationan

dNF1

Clin

ical

Status

Data

Case

Age,

years

Sex

Clinical

Presentation

Site

Patholog

yChiasm

Involvem

ent

NF

Status-

Clinical

BRAF

FISH

PTEN

FISH

p16

FISH

NF1

FISH

GFAP

IHC

IDH1

IHC

pERK

IHC

12.5

FCough,drow

siness,vomiting,

andloss

ofappetite

Optic

nerve

Optic

glioma

VNF1

Polysom

yNormal

Normal

Normal

3Neg

3

25

FPainlessproptosisfor3months

Loptic

nerve

Optic

glioma

VNF1

Polysom

yPolysom

yPolysom

yNormal

3Neg

3

34

FRapidly

prog

ressiveproptosis

for9months,reducing

vision

Optic

nerve

Optic

glioma

VNF1

Normal

Normal

VNormal

2Neg

3

42

MGradual

blindnessright9left

Rop

tic

nerve/

chiasm

Optic

glioma

Yes

NF1

VHet

del

Normal

Normal

3Neg

0

56

FExo

phthalmos

Optic

nerve

Optic

glioma

Yes

NF1

VNormal

Normal

V-

Neg

3

62

FBilateral

loss

ofvision,

nystagmus,andopticatrophy

Bilateral

optic

nerve

Optic

glioma

-NF1

VV

VV

3Neg

3

77

M3-year

historyof

proptosis,

amaurosis,exophthalm

os,and

amass

Rop

tic

nerve

Optic

glioma

Absent

NF1

Dup

Normal

VNormal

3Neg

3

85

MV

Optic

nerve

Optic

glioma

VSporadic

Dup

Normal

Normal

Normal

3Neg

1

97

MV

Optic

nerve

Optic

glioma

VSporadic

Dup

Normal

Normal

Normal

-Neg

3

101

FSeizure,increasedhead

circum

ference

Chiasm

Optic

glioma

Yes

Sporadic

Dup

Normal

Normal

Normal

3Neg

3

118

FProptosisof

6mon

ths’

duration

Lop

tic

nerve

Optic

glioma

Absent

Sporadic

Dup

Normal

Normal

Normal

3Neg

3

1230

FBlurred

vision,visual

loss,

prop

tosis

Rop

tic

nerve

Optic

glioma

Yes

Sporadic

Dup

Normal

Normal

V3

Neg

3

134

FUnilateralinterm

ittent

prop

tosis

Lop

tic

nerve

Optic

glioma

VSporadic

Dup

Normal

V3

Neg

3

146

FV

Optic

nerve

Optic

glioma

VV

Dup

Het

del

Normal

V3

Neg

3

1516

MV

Optic

nerve

Optic

glioma

VV

Dup

Normal

Normal

Normal

3Neg

3

1622

FV

Optic

nerve

Optic

glioma

VV

Dup

VV

V2

Neg

1

176

MV

Optic

nerve

Optic

glioma

VV

Dup

VV

V3

Neg

3

185

FProgressive

proptosis,recent

pain

Rop

tic

nerve

Ganglioglioma

Absent

Sporadic

Normal

VNormal

V3

Neg

3

1913

FGradual

unilateral

visual

loss

for3years,op

ticatroph

yRop

tic

nerve

Optic

glioma

VV

VV

VV

3Pos

3

Dup,duplication;

F,female;

FISH,fluorescence

insitu

hybridization;

GFAP,glialfibrillary

acidic

protein;

Hetdel,heterozygous

deletion;IH

C,im

munohistochem

istry;

L,left;M,male;

Neg,negative;NF1,

neurofi

brom

atosis

type

1;R,right.

Rodriguez et al J Neuropathol Exp Neurol � Volume 71, Number 9, September 2012

� 2012 American Association of Neuropathologists, Inc.792

Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.

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100% of optic nerve gliomas in this series had either BRAFduplication or NF1 association. The single case lacking BRAFduplication or NF1 association was more consistent with aganglioglioma than a PA, although it also had strong pERKstaining, supporting MAPK pathway activation.

The PTEN and CDKN2A (p16) deletions are frequentfindings in diffusely infiltrating gliomas, particularly glio-blastoma (28). Conversely, PTEN deletions were rare in ourcohort of optic nerve gliomas, and CDKN2A deletions wereabsent. This is in agreement with prior studies showing thesealterations to be relatively rare in conventional PAs (29, 30).Of note, these alterations occur at a higher rate in rare PAswith anaplasia (23), which almost never arise in the opticpathways. It is also of interest that the only tumor resultingin patient death with progressive disease in the subset ofpatients for whom outcome data are available had a hetero-zygous PTEN deletion.

NF1-associated PAs are characterized at the geneticlevel by homozygous inactivation of the NF1 gene, which isnot a feature of PAs that arise sporadically. In most instances,BRAF alterations and NF1 syndrome are mutually exclusive.Only a single NF1-associated tumor in our cohort had aBRAF alteration, an occurrence that is rare but has beendocumented (13, 15, 17). We did not identify NF1 deletionsby FISH in our cohort, which is consistent with small geneticalterations to be responsible for NF1 biallelic inactivation inthese tumors not detectable by FISH. In fact, constitutionalNF1 microdeletions occur only in a small subset of NF1patients (5%Y10%) (31).

Why NF1-related gliomagenesis favors the optic nerveand pathways has always been an enigmatic feature of thesyndrome. Recent insights have been provided by murinemodels of NF1 and optic nerve glioma. For example, War-rington et al (32) studied the role of the tumor microenvi-ronment in optic gliomagenesis, where non-neoplastic cellssecrete factors that stimulate neoplastic glial cell growth atthis specific site. Little is known regarding initiation and tumorprogression in sporadic PA at this site. However, the high in-cidence of BRAF duplications in optic nerve PA as compared

with those arising in the cortex or deep gray matter suggestsdifferences in initiating factors possibly linked to cell of origin.

With respect to biologic behavior, recent studies havedescribed the phenomenon of oncogene-induced senescenceto limit growth in PAs. Through this mechanism, an initialoncogenic growth stimulus mediated by BRAF activation limitssubsequent growth by promoting growth arrest. This is sup-ported by the expression of markers of senescence such asp16 and activation of A-galactosidase in PA cultures and cellcultures after activated BRAF transfection (33, 34). Oncogene-induced senescence may also occur after NF1 loss (35). Thespecific mechanisms responsible for avoiding senescence inPA are unclear, although p16 protein loss was associated withworse overall survival in a prior study (34).

Few pathological features have been associated with out-come in tumors involving the optic pathways. The pilomyxoidastrocytoma is a unique PA variant that has a propensity toinvolve the hypothalamic region of young children and isassociated with a worse clinical outcome (5). However, pilo-myxoid astrocytomas centered in the optic nerve proper havenot been described. In the setting of NF1, increased mitoticactivity and infiltrative features do not seem to be associatedwith a worse outcome in optic nerve gliomas (2), providingfurther support for the concept that the optic nerve is a uniqueanatomic site associated with a distinct biology in gliomas.

TABLE 2. Summary of Molecular and ImmunohistochemicalMarkersMarker n (%)

BRAF* duplication 11 (73)

PTEN* deletion 2 (8)

CDKN2A (p16)* deletion 0

NF1* deletion 0

pERK†

+++ 46 (81)

++ 6 (11)

+ 3 (5)

0 2 (3)

IDH1R132H†

Negative 52 (98)

Positive 1 (2)

*Evaluated by fluorescence in situ hybridization.†Evaluated by immunohistochemistry using a 4-tiered semiquantitative scale

(0 to +++) by a single neuropathologist (Fausto J. Rodriguez).

FIGURE 3. Immunohistochemical findings in optic nerveglioma. (A) Preserved immunoreactivity was reflected bystrong glial fibrillary acidic protein staining in most of the casesstudied (original magnification: 40�). (B) Strong pERKimmunoreactivity, consistent with MAPK pathway activation,was present in most of the cases (original magnification: 200�)(inset: original magnification: 600�, with a negative internalvessel control). (C, D) Almost all cases were negative for mutantIDH1 protein expression ([C] original magnification: 200�),except for a single case with diffuse moderate reactivity ([D]original magnification: 600�).

J Neuropathol Exp Neurol � Volume 71, Number 9, September 2012 MAPK Pathway Alterations in Optic Nerve Glioma

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Outcome data in a subset of patients in our cohort alsoemphasize this relatively good prognosis of gliomas in theoptic nerve.

A major finding of our study is the evidence of MAPKactivation in essentially 100% of tumors with available data.This has therapeutic implications given that inhibitors ofBRAF and other pathway components are actively beingtested in clinical trials, including sorafenib and AZD6244(clinicaltrials.gov). In view of the increased morbidity asso-ciated with surgery and standard chemoradiation regimens atthis site, pharmacological blockade may be an attractive ap-proach for the optic pathway in specific cases.

In summary, our study supports an important role forBRAF duplication and MAPK pathway activation in gliomasof the optic nerve proper, similar to PAs in the cerebellum.Future studies should provide insight into the prognostic andbiologic features of tumors arising in this unique anatomiccompartment and hopefully will lead to much needed targetedtherapies for these patients.

ACKNOWLEDGMENTThe authors thank the Cytogenetic Shared Resource Core

at Mayo Clinic and Antoinette Price at Johns Hopkins Uni-versity for technical assistance.

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