PI3K/AKT pathway alterations are associated with clinically aggressive and histologically anaplastic...

PI3K/AKT pathway alterations are associated with clinicallyaggressive and histologically anaplastic subsets of pilocyticastrocytoma

Erika F. RodriguezDepartment of Anatomic Pathology and Laboratory Medicine, Mayo Clinic, 200 First Street, SE,Rochester, MN 55905, USA

Bernd W. ScheithauerDepartment of Anatomic Pathology and Laboratory Medicine, Mayo Clinic, 200 First Street, SE,Rochester, MN 55905, USA

Caterina GianniniDepartment of Anatomic Pathology and Laboratory Medicine, Mayo Clinic, 200 First Street, SE,Rochester, MN 55905, USA

Amanda RynearsonDepartment of Anatomic Pathology and Laboratory Medicine, Mayo Clinic, 200 First Street, SE,Rochester, MN 55905, USA

Ling CenDepartment of Radiation Oncology, Mayo Clinic, Rochester, MN, USA

Bridget HoesleyAdvanced Genomics Technology Center, Mayo Clinic, Rochester, MN, USA

Heather Gilmer-FlynnAdvanced Genomics Technology Center, Mayo Clinic, Rochester, MN, USA

Jann N. SarkariaDepartment of Radiation Oncology, Mayo Clinic, Rochester, MN, USA

Sarah JenkinsBiomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, USA

Jin LongDepartment of Anatomic Pathology and Laboratory Medicine, Mayo Clinic, 200 First Street, SE,Rochester, MN 55905, USA

Fausto J. RodriguezDepartment of Anatomic Pathology and Laboratory Medicine, Mayo Clinic, 200 First Street, SE,Rochester, MN 55905, USA

Division of Neuropathology, Department of Pathology, Johns Hopkins University, 720 RutlandAvenue, Ross Building 512B, Baltimore, MN 21205, USA

AbstractPilocytic astrocytomas (PA) are well-differentiated gliomas having a favorable prognosis whencompared with other diffuse or infiltrative astrocytomas. Molecular genetic abnormalities and

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[email protected]; [email protected].

NIH Public AccessAuthor ManuscriptActa Neuropathol. Author manuscript; available in PMC 2012 August 11.

Published in final edited form as:Acta Neuropathol. 2011 March ; 121(3): 407–420. doi:10.1007/s00401-010-0784-9.

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activation of signaling pathways associated with clinically aggressive PA and histologicallyanaplastic PA have not been adequately studied. We performed molecular genetic, geneexpression, and immunohistochemical studies using three PA subsets, including conventional PA(n = 43), clinically aggressive/recurrent PA (n = 24), and histologically anaplastic PA (n = 25). Aclinical diagnosis of NF1 was present in 28% of anaplastic PA. Molecular cytogenetic studiesdemonstrated heterozygous PTEN/10q and homozygous p16 deletions in 6/19 (32%) and 3/15(20%) cases of anaplastic PA, respectively, but in neither of the two other groups. BRAFduplication was identified in 33% of sporadic anaplastic PA and 63% of cerebellar examples.BRAFV600E mutation was absent in four (of 4) sporadic cases lacking duplication. IDH1R132H

immunohistochemistry was negative in 16 (of 16) cases. Neither PDGFRA nor EGFRamplifications were present. pERK staining levels were similar among the three PA subsets, but astepwise increase in cytoplasmic pAKT and to a lesser extent pS6 immunoreactivity was noted byimmunohistochemistry in aggressive PA groups. This was particularly true in histologicallyanaplastic PA when compared with conventional PA (p<0.001 and p = 0.005, respectively). Inaddition, PTEN expression at the mRNA level was decreased in histologically anaplastic PA whencompared to the other groups (p = 0.05). In summary, activation of the PI3K/AKT in addition toMAPK/ERK signaling pathways may underlie biological aggressiveness in PA. Specifically, itmay mediate the increased proliferative activity observed in histologically anaplastic PA.

KeywordsPilocytic astrocytoma; Brain; Neurofibromatosis; Glioma; PTEN; AKT

IntroductionPilocytic astrocytoma (PA) is a well-differentiated astrocytoma occurring most frequently inchildren and young adults. Compared to diffuse or infiltrative astrocytomas, PA isassociated with a favorable prognosis, 5-year survival rates being >95% after gross totalresection [19]. Histologically, it is characterized by the presence of a biphasic patternincluding compact, fibrillary tissue featuring Rosenthal fibers and loosely arranged,microcyst-rich tissue containing eosinophilic granular bodies.

Despite its favorable prognosis, approximately 20% of PA patients experience tumorrecurrence or clinical progression [5]. A subset may result in significant morbidity ormortality, despite lack of atypical histologic features. Conversely, histologically anaplasticPAs are less frequent. These were defined in our previous study as PA demonstratinghypercellularity, moderate to severe cytologic atypia, and brisk mitotic activity with orwithout necrosis [25]. So defined, anaplasia was associated with decreased survival,corresponding to those of infiltrative astrocytomas of grades II and III, depending if necrosiswas absent or present, respectively.

In recent years, our molecular understanding of gliomas has greatly increased. Activation ofthe MAPK/ERK and PI3K/AKT pathways are hallmarks of a variety of malignancies,including high-grade astrocytomas [18]. Genetic alterations frequently involved, includeamplification of genes encoding for receptor tyrosine kinases (EGFR, PDGFRA) anddeletions/mutations in tumor suppressor genes (TP53, p16, PTEN) [18].

Recently, genetic alterations involved in the development of low grade astrocytomas such asPA have also been uncovered by high resolution genomic studies. MAPK/ERK pathwayactivation through aberrations of BRAF and RAF1 in sporadic PA or the NF1 gene inneurofibromatosis type 1 (NF1)-associated PA are most frequent [1, 6, 8, 13, 15, 22, 31].

Rodriguez et al.

Acta Neuropathol. Author manuscript; available in PMC 2012 August 11.

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https://www.researchgate.net/publication/23183700_Frequent_Gains_at_Chromosome_7q34_Involving_BRAF_in_Pilocytic_Astrocytoma?el=1_x_8&enrichId=rgreq-fe8817449d1ea7f0d0f59c1408ddd178-XXX&enrichSource=Y292ZXJQYWdlOzQ5NjQxNjA5O0FTOjIxMzQzNzQwMzQ1OTU5MkAxNDI3ODk4ODUwNDY5

Our present study evaluates the status of MAPK/ERK and PI3K/AKT pathways in PAsubsets and explores their association with clinical and histological aggressiveness.

Materials and methodsPatients and tissue microarrays

Primary tumors were studied using primarily two tissue microarrays, including conventionalPA (n = 43), clinically aggressive/recurrent PA (n = 24), and histologically anaplastic PA (n= 25). Median patient age and gender were 13 years (M 20; F 23), 23 years (M 14; F 10),and 29 years (M 17; F 8) for the three groups, respectively. Clinical criteria for NF1 werepresent in 11 (26%), 1 (4%), and 7 (28%) cases, respectively. Anatomical sites forconventional, aggressive and histologically anaplastic PA, respectively, included opticpathway/hypothalamus (21, 13, 0%), supratentorial CNS (37, 42, 36%) and infratentorialCNS (42, 46, 64%).

Aggressive/recurrent PA was defined as PA progressing within a year of surgery despiteadequate therapy or requiring additional surgical intervention, but lacking histologic featuresof anaplasia. Histologically anaplastic PAs were obtained predominantly from theconsultation files from one of us (BWS); their clinicopathologic features were the subject ofour previous publication [25]. Their morphologic characteristics were summarized above.The tissue microarrays were constructed using 0.6 mm diameter cores per tumor intriplicate. All studies were approved by the Institutional Review Board and Biospecimencommittee at Mayo Clinic.

Fluorescence in situ hybridizationAll fluorescence in situ hybridization (FISH) studies were performed upon formalin-fixed,paraffin-embedded tissue microarray or whole tissue sections using previously describedmethods [26]. Locus-specific probes targeted the following regions: EGFR (7p12, AbbottMolecular/Vysis), P16 (9p21, Abbott Molecular/Vysis), PTEN (10q23, Abbott Molecular/Vysis), PDGFRA (Custom Made) and BRAF (Custom Made with clones RP4-592P3,RP4-726N20, RP5-839B19, RP4-813F11) (7q34). All target probes were labeled withSpectrumOrange™ (Abbott Molecular/Vysis, Des Plaines, Ill). Respective reference probes(CEP 4, 7, 9, 10) were labeled with SpectrumGreen™. A minimum of 100 non-overlappingnuclei were assessed per case. Chromosomal loses and gains were evaluated usingpreviously established cutoffs [26]. The identity of the BACs used to construct the custom-made BRAF probe was confirmed with PCR analysis (Supplementary Fig. 1).

BRAFV600E mutation analysisFive cases with sufficient material for DNA extraction were used for BRAFV600E mutationanalysis. Four to six 10-μm paraffin sections were used for genomic DNA extraction by thephenol:chloroform:isopropyl alcohol (Invitrogen) method. A 224-bp fragment of BRAFgene exon 15 was amplified using the following PCR primers: 5′-TCATAATGCTTGCTCTGATAGGA (forward) and 5′-GGCCAAA AAT TTAATCAGTGGA(reverse), as previously reported [17]. PCR products were separated on a 2% agarose gel andvisualized by ethidium bromide staining. Aliquoted products were used for bidirectionalDNA sequencing on an ABI Prism 377 DNA Sequencer (PE Applied Biosystems, FosterCity, CA, USA). NPA (thyroid tumor) cell lines with a known BRAFV600E mutation andPTC1 cells with a known wild type BRAF genotype were used for controls.

Real-time RT-PCRTotal RNA was extracted from 5 to 10 sections of formalinfixed paraffin-embedded tissue,each 10 μm thick, using the RecoverALL™ Total Nucleic Acid Isolation Kit (Applied

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Biosystems). Quantitation of gene expression was performed using Taqman gene expressionassays (Applied Biosystems), including PTEN (Hs02621230_s1) and BRAF kinase domain(Hs01052468_g1), GAPDH serving as an endogenous control. A total of 100–1,000 ng oftotal RNA were converted to cDNA, 10 ng loaded per well. Tumor and control brainsamples were run in triplicate on the ABI 7900HT Fast real-time PCR system (AppliedBiosystems) using 96-well plates. Data analysis was performed using ΔΔCt method withSDS software according to the manufactures recommendations(http://www.appliedbiosystems.com).

ImmunohistochemistryImmunohistochemical studies were performed using a Dako autostainer and the Dual LinkEnvision+ or ADVANCE detection systems on 5-μm thick formalinfixed, paraffin-embedded tissue microarray sections. Antibodies applied were directed against AKT (total)(Rabbit polyclonal, 1:500 dilution, cell signaling technology), pAKT (clone736E11recognizing phospho-ser473 site, 1:800; cell signaling technology), pERK (Rabbitpolyclonal Erk1/2) (Thr202/Tyr204; 1:250 cell signaling technology); pS6 (Rabbitpolyclonal p-S6 (Ser235/236) 1:200, cell signaling technology); EGFR (2-18C9, prediluted;Dako), p53 protein (clone DO7, 1:2,000; Dako), IDH1R132H (Clone H09, 1:50, Dianova),and ki67 (clone MIB-1, monoclonal, 1:300; Dako). Cytoplasmic staining for EGFR, AKT,pAKT, and pERK were scored on a four-tier semi-quantitative scale by two observers aspreviously described [26]. Nuclear stains (p53, ki67) were quantitatively estimated using theHamamatsu NanoZoomer Digital Pathology scanning and IHCScore software for computer-assisted analyses.

Statistical analysisSurvival rates were described using Kaplan–Meier curves and analyzed with Coxproportional hazard regression. The time to event was defined as time from surgery to death(or last follow-up if censored). Fisher's exact tests were used to compare proportions, andthe Student's t, Wilcoxon rank sum, or Kruskal–Wallis tests were used to comparecontinuous variables between groups of interest. Statistical analyses were performed usingSAS version 9 or JMP version 8 software (SAS Institute, Inc., Cary, NC). P values <0.05were considered statistically significant.

ResultsMolecular genetic abnormalities of histologically anaplastic PA

The molecular genetic abnormalities observed in this subset of tumors are summarized inTable 1 and are illustrated in Fig. 1.

PTEN and p16—Molecular cytogenetic analysis demonstrated heterozygous PTEN (10q)deletions in 6/19 (32%) histologically anaplastic PA but in none of 39 conventional orclinically aggressive PA (p<0.001). Whole chromosome 10 loss was not observed.Homozygous deletions of p16 were noted in 3/15 (20%) histologically anaplastic PA but innone of 16 conventional or clinically aggressive PA. These alterations were present both inthe histologically anaplastic component and PA precursor when tested (2 cases with PTENdeletion and one with p16 deletion). All p16 homozygous deleted tumors had PTENdeletions. However, three PTEN-deleted tumors had intact p16 loci (Table 2).

BRAF—Duplication of BRAF was present in 5/15 (33%) sporadic histologically anaplasticPA (mean BRAF/CEP7 ratio 1.38), but in none of two NF1 associated histologicallyanaplastic PA tested. The frequency of BRAF duplication was highest in cerebellarexamples (5/8) (63%) cases, and limited to this anatomic location. Conversely, proportional

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gains/polysomies of chromosome 7 were more frequent (15/21 cases (71%). In addition,BRAF duplication was present in (16/40) (40%) sporadic PA without anaplasia. Re-examination of histologically anaplastic PA lacking BRAF duplication demonstratedconvincing classical histological features of conventional PA in these tumors (Fig. 2),arguing against the alternative diagnosis of infiltrating glioma.

Because of the increased frequency of polysomies in histologically anaplastic PA whencompared with the other groups, we performed qRT-PCR analysis using primers targetingRNA coding for the BRAF kinase domain to see if increasing copy number was associatedwith gene overexpression. There were no differences in BRAF expression levels notedamong the groups (p>0.05).

Testing for BRAFV600E mutation by DNA sequencing was successfully performed in foursporadic histologically anaplastic PA lacking BRAF duplication. This demonstrated wildtype BRAF (Supplementary Fig. 2).

PDGFRA and EGFR—PDGFRA or EGFR, two cell surface receptor tyrosine kinases, areoften amplified in pediatric and adult high-grade astrocytomas, respectively. Neither waspresent in the histologically anaplastic PA tested (0/15 and 0/18, respectively). Nonetheless,increased EGFR protein immunoreactivity was noted in aggressive PA and histologicallyanaplastic PA as compared to conventional PA (p = 0.05 and 0.02, respectively).

IDH1R132H—To exclude the possibility of histologically anaplastic PA representingprogression from a diffuse glioma, immunohistochemistry using an antibody recognizing themost frequent mutant protein, IDH1R132H, was performed and was negative in 16 (of 16)cases tested.

Activation of the PI3K/AKT signaling pathway is associated with an aggressive PAphenotype

PTEN gene expression—Given the increased frequency of PTEN heterozygousdeletions present in histologically anaplastic PA, we hypothesized that the PI3K/AKTpathway may be differentially activated in this subset of tumors as compared to conventionalPA. Upon evaluating tumors for PTEN gene expression, PTEN mRNA levels weredecreased in histologically anaplastic PA relative to PA without anaplasia (p = 0.05)(Wilcoxon Rank Sum) (Fig. 3). As a group, PTEN mRNA levels were approximatelytwofold lower in histologically anaplastic when compared with non-anaplastic PA. With thecaveat that subgroups within the histologically anaplastic PA group were small, we found nodifferences in PTEN gene expression levels between PTEN-deleted and non-deleted tumors,or between pAKT/pS6 immunohistochemistry. However, there was an increase in pS6staining in PTEN-deleted versus non-deleted tumors.

PI3K/AKT pathway—Using phospho-specific antibodies reflecting activation of theMAPK/ERK and PI3K/AKT pathways, immunohistochemical studies were undertaken (Fig.4). A stepwise increase in pAKT staining in clinically aggressive/recurrent PA andhistologically anaplastic PA was noted when compared with conventional PA (respectively,p = 0.02 and p = 0.006). Although the staining for total AKT was weak, this was uniform inall tumor groups (p>0.05). This finding could be related to differences in antibodysensitivity between AKT and pAKT, but support that the increases in the latter are due tophosphorylation rather than a simple increase in total levels. pS6 staining intensity was alsoincreased in histologically anaplastic PA when compared with conventional PA (p = 0.005),but there was only a non-significant trend for increased staining when compared withaggressive/recurrent PA (p = 0.09). In contrast, pERK staining intensity was similarly

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increased in all three PA groups, a reflection of consistent activation of MAPK/ERKsignaling in astrocytomas of all types and grades. When comparing pERK/pS6/pAKTstaining between conventional and anaplastic components of the same tumor (in the sameresection or prior precursor) or between the precursor and recurrence in recurrent/clinicallyaggressive PA (available in 13, 10, and 12 cases, respectively), we found that in mostinstances, increased staining was present in the precursors. These findings suggest thatactivation of these pathways is an early feature of clinical aggressiveness/anaplasia in thesetumors.

Cellular proliferation and p53—Quantification of MIB1-labeling indices showedincreased cell proliferation in histologically anaplastic PA when compared withconventional as well as aggressive/recurrent PA (p<0.001). No difference was notedbetween conventional and aggressive/recurrent PA (p = 0.31) (Fig. 5). In addition, there wasan association between increased MIB1-labeling indices and pAKT immunostaining (p =0.008). There was a trend for increased p53 nuclear staining in histologically anaplastic PA,but no stepwise increase was seen between the three groups (p>0.05).

Survival analysesOverall survival decreased from conventional through clinically aggressive/recurrent PA tohistologically anaplastic PA, with anaplasia remaining the strongest adverse prognosticfactor (p<0.001) (Fig. 6a). pAKT staining was also associated with decreased survival in allgroups combined after adjusting by extent of resection, age and location, but not whenadjusting for clinical and histologic aggressiveness (Fig. 6b). Other features showing aninverse association with overall survival on univariate analyses included increasing age,MIB1-labeling index, and extent of resection (gross total vs. subtotal/biopsy only) (p<0.05).Only age and extent of resection remained statistically significant after adjusting byhistologic anaplasia (p<0.05).

DiscussionActivation of the PI3K/AKT and MAPK/ERK pathways by various molecular mechanismsis ubiquitous in a variety of malignancies, including high grade astrocytomas [18]. Our studyevaluated the association of alterations of these pathways with various PA subsets. In recentyears, several studies have highlighted the importance of MAPK/ERK activation in thepathobiology of PA. Activation occurs most frequently by alterations in the BRAF gene,particularly a tandem duplication of its kinase domain resulting in a BRAF:KIAA1549 genefusion [15]. More recently, a similar duplication–fusion event was reported in a minority ofPA involving RAF1 and SRGAP3 genes [16]. Other less specific alterations reported in PAinclude the BRAFV600E point mutation, which leads to constitutive activation of BRAF.BRAFV600E may more often be found in pediatric diffuse gliomas [27] and gangliogliomas[6]. The BRAFV600E mutation also occurs in melanoma and papillary thyroid carcinoma.

In PA developing in the context of NF1 disease, homozygous inactivation of the NF1 geneis an alternative mechanism for MAPK/ERK activation and seems to be a mutuallyexclusive alternative to BRAF mutation [34]. Although NF1-associated PA seems to beassociated with a better outcome when compared with its sporadic counterpart, associationsof BRAF aberrations with prognosis have been less consistent [8, 13, 15, 22]. Of note, 28%of the histologically anaplastic PA in our series were NF1-associated and not related to theoptic pathways. Although NF1-associated PA tend to have a better outcome when comparedwith their sporadic counterparts, this is highlighted more in optic pathway tumors, whichmay even spontaneously regress, and are usually not resected in most instances at the currenttime [20, 24]. The optic pathway, at the biologic level, represents a unique niche in NF1

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[33], with a low potential for clinical and histological progression, explaining in part theabsence of these tumors in our series. However, infiltrating and high grade astrocytomasmay occur throughout the neuraxis in NF1, especially in older patients, and be associatedwith similar molecular abnormalities and adverse outcome as their sporadic counterparts[11, 24]. Another possible explanation for the increased prevalence of NF1 cases in ourseries is that they are predominantly consultation derived, and NF1-associated tumors otherthan PA, especially with unusual morphologies, are more likely to be shared in consultation.

It is of interest that BRAF duplications were identified in 63% of the cerebellar anaplasticPA, but in none of seven non-cerebellar examples. This may suggest that non-cerebellaranaplastic PA may activate the ERK pathway by alternative mechanisms, in spite of similarhistology, which may involve, among other alterations, point mutations (e.g. RAS) or RAF1rearrangements as previously described in a minority of PA [16, 30]. In addition, only 40%of the sporadic non-anaplastic PA in our series showed BRAF duplication, although thisalteration was present in almost half (47%) of posterior fossa examples. Variation in BRAFaberration frequency according to anatomic site has been reported, with prior studiesshowing a decreased frequency of BRAF rearrangements in non-cerebellar examples [34],down to 38% in one study [12]. In addition, BRAF rearrangements may also be associatedwith lower patient age [12], which can in part explain the lower prevalence in our relativelyolder patient population.

Although clinical observations and prior studies both noted aggressive clinical behavior in asubset of PA showing otherwise conventional histology [5], molecular abnormalitiesassociated with the worse outcome remained elusive, comprehensive gene expressionstudies demonstrating no global changes reliably distinguishing conventional from clinicallyaggressive PA [28]. However, alterations in specific genes and proteins associated with aclinically aggressive phenotype were noted including increased levels of matrilin-2 [29] anddecreased levels of ALDH1L1 [23].

Recent studies have also explored the relationship of various molecular alterations andoutcome in PA. Tibbetts et al. [32] did not find an association with recurrence free survivalin PA with respect to ERK or mTOR activation. However, their study did not includehistologically anaplastic PA, which had the highest degree of pS6 staining in our study, andAKT activation (pAKT) was not tested. At difference with our study, Horbinski et al. [12]found homozygous p16 deletions by FISH in 6% of PA and LOH at the PTEN locus in 50%by PCR-based microsatellite LOH analysis, findings not associated with adverse outcome.Their study also did not include histologically anaplastic PA, and the prevalence ofhomozygous p16 deletions was higher in this cohort (20%) in our study. With respect toPTEN, we did not find heterozygous deletions by FISH in non-anaplastic PA, but in 32% ofhistologically anaplastic PA. These findings suggest that even though PTEN LOH may befound in conventional PA, large deletions identified by FISH may be more closelyassociated with anaplastic histology.

Amplifications of receptor tyrosine kinases are important genetic events in the developmentof high-grade astrocytomas. EGFR amplification is most frequently seen in primaryglioblastomas of adults, while PDGFRA amplification has been identified in a subset ofpediatric high-grade astrocytomas [21]. In our study, we did not encounter these aberrations.

In the current study, we demonstrate that, in addition to MAPK/ERK pathway activation,increased PI3K/AKT activity also seems to be a feature of clinically aggressive PA andparticularly histologically anaplastic PA. A possible model summarizing these findings isillustrated in Fig. 7. Notably, however, NF1-associated tumors, including PA, are associatedwith increased mTOR pathway signaling [4, 14]. In addition to aberrations of MAPK/ERK,

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histologically anaplastic PA develop alterations characteristic of high grade astrocytomas,including heterozygous PTEN/10q and homozygous p16 deletions. Frequent alterations inthe PTEN/PI3K/AKT pathway were also noted in a prior study of malignant transformationin pediatric low grade gliomas [3], although only one example progressed from a PA.

Although the status of the remaining PTEN allele was not tested in our study, decreasedexpression of the gene at the mRNA level in histologically anaplastic PA suggests that thisaberration may have biological significance. Furthermore, it seems to occur early in theirevolution, being present in two (of 2) cases in which the PA precursors were tested. Ourfindings also reinforce recent findings highlighting the importance of PTEN loss inmalignant progression of other NF1-associated tumors, particularly neurofibromasprogressing to malignant peripheral nerve sheath tumors [10]. Now, in the current study, wewere unable to demonstrate a definite relationship between PTEN deletion, PTEN geneunderexpression and pAKT/pS6 immunohistochemistry, even though each of these variableswas more prevalent in histologically anaplastic PA. This suggests that alternativemechanisms to PTEN loss may also activate AKT signaling in histologically anaplastic PA,in a similar way to other astrocytomas that have mutations in other PI3K pathwaycomponents [18]. Future studies should be more illustrative in this regard.

PI3K/AKT activation underlies increased cellular proliferation characteristic of anaplasiaand an adverse histologic phenotype in PA. Indeed, an inverse relation between MIB1-labeling index and progression-free survival was noted in one prior study of PA [2].Interestingly, a worsening of prognosis with increased MIB1 labeling was not confirmed byother studies [5, 7, 9]. We did not identify a significant difference in MIB1-labeling indicesbetween clinically aggressive/recurrent PA and conventional PA, but did find a significantincrease in histologically anaplastic PA. These findings support the notion that increasedcellular proliferation is an important, defining trait of histologic anaplasia in PA and a factorunderlying the worse prognosis associated with these tumors.

In summary, this study assessed the prevalence of key molecular genetic abnormalities inclinically and histo-logically aggressive PA, and highlighted important differences in PAsubsets with respect to PI3K/AKT activation status. Further, more comprehensive studiesshould confirm these findings and identify additional molecular aberrations in aggressive PAsubsets, perhaps identifying therapeutic targets beneficial to astrocytoma patients in general.

AcknowledgmentsThis work was funded in part by grant P50 CA108961 from the Mayo SPORE in Brain Cancer (CG, FJR, JNS) andMayo Clinic CTSA (FJR) through grant number UL1 RR024150 from the National Center for Research Resources(NCRR), a component of the National Institutes of Health (NIH). The authors also thank the microarray, tissue, cellmolecular analysis and cytogenetic shared resources of the Mayo Clinic for excellent technical assistance.

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27. Schiffman JD, Hodgson JG, VandenBerg SR, et al. Oncogenic BRAF mutation with CDKN2Ainactivation is characteristic of a subset of pediatric malignant astrocytomas. Cancer Res. 2010;70:512–519. [PubMed: 20068183]

28. Sharma MK, Mansur DB, Reifenberger G, et al. Distinct genetic signatures among pilocyticastrocytomas relate to their brain region origin. Cancer Res. 2007; 67:890–900. [PubMed:17283119]

29. Sharma MK, Watson MA, Lyman M, et al. Matrilin-2 expression distinguishes clinically relevantsubsets of pilocytic astrocytoma. Neurology. 2006; 66:127–130. [PubMed: 16401863]

30. Sharma MK, Zehnbauer BA, Watson MA, Gutmann DH. RAS pathway activation and anoncogenic RAS mutation in sporadic pilocytic astrocytoma. Neurology. 2005; 65:1335–1336.[PubMed: 16247081]

31. Sievert AJ, Jackson EM, Gai X, et al. Duplication of 7q34 in pediatric low-grade astrocytomasdetected by high-density single-nucleotide polymorphism-based genotype arrays results in a novelBRAF fusion gene. Brain Pathol. 2009; 19:449–458. [PubMed: 19016743]

32. Tibbetts KM, Emnett RJ, Gao F, Perry A, Gutmann DH, Leonard JR. Histopathologic predictors ofpilocytic astrocytoma event-free survival. Acta Neuropathol. 2009; 117:657–665. [PubMed:19271226]

33. Warrington NM, Woerner BM, Daginakatte GC, et al. Spatiotemporal differences in CXCL12expression and cyclic AMP underlie the unique pattern of optic glioma growth inneurofibromatosis type 1. Cancer Res. 2007; 67:8588–8595. [PubMed: 17875698]

34. Yu J, Deshmukh H, Gutmann RJ, et al. Alterations of BRAF and HIPK2 loci predominate insporadic pilocytic astrocytoma. Neurology. 2009; 73:1526–1531. [PubMed: 19794125]

Rodriguez et al.


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Fig. 1.Molecular genetic alterations in histologically anaplastic PA. Case 8: Conventional PAcomponent characterized by astrocytes with degenerative atypia, hyalinized vasculature andnumerous eosinophilic granular bodies (a). Features of anaplasia in PA include coagulativenecrosis (b) and brisk mitotic activity (inset). Molecular alterations included heterozygousPTEN deletions (c), homozygous p16 deletions (arrow points to a non-neoplastic endothelialcell with preservation of both p16 loci) (d), and BRAF duplications (arrows) in associationwith polysomies (e). Amplifications of receptor tyrosine kinases were not identified,although proportional chromosomal gains/polysomies were frequent (f)

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Fig. 2.Histologically anaplastic PA lacking BRAF duplication have morphological componentsidentical to conventional PA. Case 15: Well circumscribed spinal cord neoplasm in a 51-year-old man (a); histological review demonstrates numerous Rosenthal fibers (arrows) in arelatively circumscribed astrocytoma with piloid cells (b); immunohistochemistry forIDH1R132H was negative (c). Case 4: Histologically anaplastic PA involving the frontal lobeof an 11-year-old male with prominent microcysts and hyalinized vessels (d); eosinophilicgranular bodies were also present (arrows) (e); neurofilament protein immunostainhighlights compressed axons at the periphery of the tumor consistent with a solidarchitecture (f); Case 6: Loose textured tumor with microcysts involving the cerebellum of a29-year-old with NF1 (g); Rosenthal fibers were present in other piloid areas (h);histologically malignant component with nuclear hyperchromasia, hypercellularity andmitoses (arrows) (i)

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Fig. 3.Decreased PTEN gene expression in histologically anaplastic PA (APA). Decreased levelsof PTEN mRNA were identified by qRT-PCR in a subset of APA tumors (n = 11) whencompared with conventional PA (n = 17) (a). In contrast, there was no difference in BRAFmRNA levels between the two groups (b) (Wilcoxon Rank-Sum; error bars SEM)

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Fig. 4.AKT pathway signaling is increased in histologically anaplastic PA (APA) when comparedwith conventional and clinically aggressive PA. Tissue microarray sections stained withimmunohistochemistry using total protein and phospho-specific antibodies (a).Semiquantification results corresponding to the immunohistochemistry tests are illustratedon the right (b) (white bars conventional PA; gray bars clinically aggressive PA; black barsAPA(B)(Wilcoxon rank sum, error bars SEM)

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Fig. 5.Increased cellular proliferation in histologically anaplastic PA (APA) when compared withaggressive and conventional PA. MIB1 and p53 nuclear immunostains in conventional PA,aggressive PA, and APA. Increased MIB1-labeling indices, in particular, are a feature ofAPA (a). Increased p53 staining was present in some histologically anaplastic PA, but thedifferences between the groups were not as evident as proliferation and pAKT staining. Re-review of the histology of this specific case demonstrating strong p53 staining (b) showedclassic pilocytic histology in part of the tumor (Case 6, Fig. 2) (white bars conventional PA,gray bars clinically aggressive PA, black bars APA (Wilcoxon rank sum, error bars SEM)

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Fig. 6.Histologic anaplasia and AKT activation are associated with decreased overall survival inPA. Kaplan–Meier curves illustrate decrease survival in APA (3) when compared withclinically aggressive (2) and conventional PA (1) (p<0.005, Log rank test) (a). Higher levelsof pAKT as assessed by immunohistochemistry are also associated with decreased survivalin PA (p<0.001) (b)

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Fig. 7.Model of molecular mechanisms underlying PA and histologically anaplastic PA. RAS/BRAF/MEK pathway activation by various mechanisms is a key feature of sporadic PA andNF1 PA. Histologically anaplastic PA develop in addition hyperactivation of the PI3K/PTEN/AKT pathway and inactivation of cell cycle inhibitors such as p16 leading toincreased proliferation

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Rodriguez et al.

Tabl

e 1

Mol

ecul

ar g

enet

ic f

eatu

res

in 2

2 ca

ses

of h

isto

logi

cally

ana

plas

tic P

A

Cas

eA

geSe

xL

ocat

ion

NF

1 cl

inic

al s

tatu

sH

isto

ry o

f ra

diat

ion

Nec

rosi

sP

TE

NB

RA

FB

RA

F V

600E

Chr

7P

16P

DG

FR

AE

GF

RID

HR

132H

IH

C

15

MC

ereb

ellu

mSp

orad

icN

oN

oN

AD

upN

AN

orm

alN

orm

alN

o A

mp

No

Am

pN

eg

270

MC

ereb

ellu

mSp

orad

icN

oY

esH

et d

elN

orm

alN

AN

orm

alH

DN

o A

mp

No

Am

pN

eg

328

MC

ereb

ellu

mSp

orad

icY

esY

esW

TN

AN

A+

7N

AN

AN

o A

mp

Neg

411

MR

fro

ntal

lobe

Spor

adic

No

Yes

WT

Nor

mal

WT

+7

Nor

mal

No

Am

pN

o A

mp

Neg

536

FC

ereb

ellu

mSp

orad

icN

oN

oH

et d

elD

upN

A+

7N

orm

alN

o A

mp

No

Am

pN

eg

629

MC

ereb

ellu

mN

F1N

oN

oH

et d

elN

orm

alN

AN

orm

alH

DN

o A

mp

No

Am

pN

eg

720

MC

ereb

ellu

mSp

orad

icN

oY

esW

TD

upN

A+

7N

orm

alN

o A

mp

NA

Neg

827

MC

ereb

ellu

mSp

orad

icY

esY

esH

et d

elD

upN

A+

7H

DN

o A

mp

NA

Neg

975

ML

tem

pora

l lob

eSp

orad

icN

oN

oW

TN

orm

alW

T+

7N

orm

alN

o A

mp

No

Am

pN

eg

1010

M3r

d ve

ntri

cle

Spor

adic

No

Yes

WT

Nor

mal

NA

+7

Nor

mal

No

Am

pN

o A

mp

Neg

1139

FC

ereb

ellu

mSp

orad

icY

esY

esN

AD

upN

A+

7N

orm

alN

o A

mp

No

Am

pN

eg

1246

ML

par

ieta

l lob

eN

F1N

oY

esW

TN

AN

AN

orm

alN

AN

AN

o A

mp

Neg

1346

FR

tem

pora

l lob

eN

F1N

oN

oW

TN

orm

alN

A+

7N

orm

alN

o A

mp

NA

Neg

1473

FT

ectu

mSp

orad

icN

oY

esW

TN

orm

alW

T+

7N

orm

alN

o A

mp

No

Am

pN

eg

1551

MSp

inal

cor

dSp

orad

icN

oN

oH

et d

elN

orm

alN

AN

orm

alN

orm

alN

AN

o A

mp

Neg

1619

MC

ereb

ellu

mN

F1N

oN

oW

TN

AN

AN

orm

alN

AN

AN

o A

mp

NA

1726

MC

ereb

ellu

mN

F1N

oY

esW

TN

AN

A+

7N

AN

AN

o A

mp

NA

1816

MSp

inal

cor

dSp

orad

icN

oY

esH

et d

elN

orm

alN

A+

7N

orm

alN

o A

mp

No

Am

pN

A

1949

FC

ereb

ellu

mSp

orad

icN

oY

esW

TN

orm

alN

A+

7N

AN

o A

mp

No

Am

pN

A

2021

FM

edul

laSp

orad

icN

oY

esW

TN

orm

alW

T+

7N

orm

alN

o A

mp

No

Am

pN

A

2133

ML

par

ieta

l lob

eN

F1N

oN

oW

TN

AN

A+

7N

AN

AN

o A

mp

NA

2255

FC

ereb

ellu

mSp

orad

icN

AN

oN

AN

orm

alN

A+

7N

AN

AN

AN

A

NF1

neu

rofi

brom

atos

is ty

pe 1

, Het

del

het

eroz

ygou

s de

letio

ns, H

D h

omoz

ygou

s de

letio

ns, W

T w

ild ty

pe, C

hr c

hrom

osom

e, N

o A

mp

no a

mpl

ific

atio

n, N

eg n

egat

ive,

NA

test

fai

led

or n

ot d

one


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Rodriguez et al.

Tabl

e 2

Alte

ratio

ns o

f th

e M

APK

/ER

K a

nd P

I3K

/AK

T p

athw

ays

in h

isto

logi

cally

ana

plas

tic P

A (

n =

25)

, clin

ical

ly a

ggre

ssiv

e PA

(n

= 2

4), a

nd c

onve

ntio

nal P

A(n

= 4

3)

Cas

e #

Age

Sex

Loc

atio

nN

F1

clin

ical

sta

tus

Tum

or t

ype

PT

EN

FIS

HP

TE

N q

RT

-PC

R F

CF

INA

LpA

KT

IH

CpS

6 IH

CB

RA

F F

ISH

pER

K I

HC

P16

FIS

H

15

MC

ereb

ellu

mSp

orad

icA

PA1.

072

2D

up3

Nor

mal

270

MC

ereb

ellu

mSp

orad

icA

PAH

et D

el2

2N

orm

alH

D

328

MC

ereb

ellu

mSp

orad

icA

PAW

T2

32

411

MR

fro

ntal

lobe

Spor

adic

APA

WT

−13

33

+7

3N

orm

al

536

FC

ereb

ellu

mSp

orad

icA

PAH

et D

el−

191

3D

up+

73

Nor

mal

629

MC

ereb

ellu

mN

F1A

PAH

et D

el−

3.9

23

Nor

mal

3H

D

720

MC

ereb

ellu

mSp

orad

icA

PAW

T1

2D

up2

Nor

mal

827

MC

ereb

ellu

mSp

orad

icA

PAH

et D

el1.

92

3D

up2

HD

975

ML

tem

pora

l lob

eSp

orad

icA

PAW

T2

1+

73

Nor

mal

1010

M3r

d ve

ntri

cle

Spor

adic

APA

WT

32

+7

3N

orm

al

1139

FC

ereb

ellu

mSp

orad

icA

PA−

1.44

3D

upN

orm

al

1246

ML

par

ieta

l lob

eN

F1A

PAW

T−

62

0

1346

FR

tem

pora

l lob

eN

F1A

PAW

T−

3.6

00

+7

3N

orm

al

1473

FT

ectu

mSp

orad

icA

PAW

T−

72

+7

3N

orm

al

1551

MSp

inal

cor

dSp

orad

icA

PAH

et D

el−

1.75

23

Nor

mal

Nor

mal

1619

MC

ereb

ellu

mN

F1A

PAW

T

1726

MC

ereb

ellu

mN

F1A

PAW

T

1816

MSp

inal

cor

dSp

orad

icA

PAH

et D

el2

+7

Nor

mal

1949

FC

ereb

ellu

mSp

orad

icA

PAW

T0

2021

FM

edul

laSp

orad

icA

PAW

T2

+7

Nor

mal

2133

ML

par

ieta

l lob

eN

F1A

PAW

T

2255

FC

ereb

ellu

mSp

orad

icA

PA3

+7

2341

MPi

neal

reg

ion

Spor

adic

APA

−1.

9

2411

FR

occ

ipita

l lob

eN

F1A

PA3

3

2514

MPo

ster

ior

foss

aSp

orad

icA

PA0

Faile

d

2639

FC

ereb

ellu

mSp

orad

icA

gg P

AW

T1

0N

orm

al3

274

FSu

pras

ella

rSp

orad

icA

gg P

A2

0D

up3


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Rodriguez et al.

Cas

e #

Age

Sex

Loc

atio

nN

F1

clin

ical

sta

tus

Tum

or t

ype

PT

EN

FIS

HP

TE

N q

RT

-PC

R F

CF

INA

LpA

KT

IH

CpS

6 IH

CB

RA

F F

ISH

pER

K I

HC

P16

FIS

H

2828

M3r

d ve

ntri

cle

Spor

adic

Agg

PA

0N

orm

al2

2932

MR

lat v

entr

icle

Spor

adic

Agg

PA

WT

13

+7

3

3010

MR

par

ieto

occi

pita

lSp

orad

icA

gg P

AW

T0

0D

up

3123

F4t

h ve

ntri

cle

Spor

adic

Agg

PA

WT

11

+7

3

324

FR

thal

amus

NF1

Agg

PA

336

FL

fro

ntal

lobe

Spor

adic

Agg

PA

WT

02

Nor

mal

3N

orm

al

3426

MR

tem

poro

pari

etal

Spor

adic

Agg

PA

WT

20

+7

3N

orm

al

3550

MC

ereb

ellu

mSp

orad

icA

gg P

A

3639

FL

fro

ntal

lobe

Spor

adic

Agg

PA

00

3N

orm

al

371

FPo

ster

ior

foss

aSp

orad

icA

gg P

AW

T−

1.56

22

Dup

3N

orm

al

3842

FL

tem

pora

l lob

eSp

orad

icA

ggPA

WT

−1.

052

0D

up3

Nor

mal

3944

MR

fro

ntal

lobe

Spor

adic

Agg

PA

WT

2D

up3

Nor

mal

406

FPo

ster

ior

foss

aSp

orad

icA

ggPA

WT

23

Nor

mal

2N

orm

al

4118

MC

ereb

ellu

mSp

orad

icA

gg P

AW

T1

0D

up2

Nor

mal

4237

MC

ereb

ellu

mSp

orad

icA

gg P

A2

Nor

mal

3N

orm

al

4323

FL

thal

amus

Spor

adic

Agg

PA

WT

1.04

21

Nor

mal

3N

orm

al

4422

M3r

d ve

ntri

cle

Spor

adic

Agg

PA

WT

−2.

32

3D

up2

Nor

mal

457

M3r

d ve

ntri

cle

Spor

adic

Agg

PA

WT

13

Nor

mal

2N

orm

al

463

MC

ereb

ellu

mSp

orad

icA

gg P

A0

0N

orm

al

4734

MPo

ster

ior

foss

aSp

orad

icA

gg P

A3

482

MC

ereb

ellu

mSp

orad

icA

gg P

A2

Nor

mal

4923

MR

tem

pora

l lob

eSp

orad

icA

gg P

A−

2.6

Nor

mal

507

FL

tem

pora

l lob

eSp

orad

icPA

10

Nor

mal

2

5117

ML

fro

ntal

lobe

NF1

PAW

T−

1.72

22

Nor

mal

3

5214

F3r

d ve

ntri

cle

Spor

adic

PAW

T2

2N

orm

al2

5313

MR

fro

ntal

lobe

NF1

PAW

T1.

642

2N

orm

al3

5433

MSp

inal

cor

dSp

orad

icPA

WT

0Fa

iled

2

5510

MC

ereb

ellu

mN

F1PA

−2.

560

3N

orm

al3

5622

MR

par

ieta

l lob

eSp

orad

icPA

WT

00

Nor

mal

2

579

FSu

pras

ella

rSp

orad

icPA

10

Nor

mal

2

5814

FR

tem

pora

l lob

eSp

orad

icPA

WT

00

Nor

mal


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Rodriguez et al.

Cas

e #

Age

Sex

Loc

atio

nN

F1

clin

ical

sta

tus

Tum

or t

ype

PT

EN

FIS

HP

TE

N q

RT

-PC

R F

CF

INA

LpA

KT

IH

CpS

6 IH

CB

RA

F F

ISH

pER

K I

HC

P16

FIS

H

5918

FC

ereb

ellu

mSp

orad

icPA

WT

00

Nor

mal

2

602

ML

opt

ic n

erve

NF1

PA−

1.78

6121

ML

par

ieto

occi

pita

lSp

orad

icPA

WT

10

Dup

2

625

MPo

ster

ior

foss

aSp

orad

icPA

WT

10

Nor

mal

2

6321

FT

empo

ral l

obe

NF1

PA1

3N

orm

al3

645

FL

opt

ic n

erve

NF1

PA0

2

652

FL

lat v

entr

icle

NF1

PA0

0N

orm

al2

6630

FR

lat v

entr

icle

NF1

PA0

3N

orm

al3

6718

FC

ervi

com

edul

lary

Spor

adic

PAW

T0

2D

up2

684

MPo

ster

ior

foss

aSp

orad

icPA

WT

00

Nor

mal

2

6916

MPo

ster

ior

foss

aSp

orad

icPA

WT

23

Nor

mal

3

7058

ML

fro

ntot

empo

ral

Spor

adic

PA−

2.6

+7

7111

FM

idbr

ain

Spor

adic

PA0

Dup

2

721

FPo

ster

ior

foss

aSp

orad

icPA

WT

00

Dup

2

7337

ML

fro

ntal

lobe

Spor

adic

PAW

T2

+7

3

7437

FL

thal

amus

Spor

adic

PA2

3

7513

FPo

ster

ior

foss

aSp

orad

icPA

WT

13

Nor

mal

3

765

FSu

pras

ella

rSp

orad

icPA

WT

01

Dup

2

7712

MR

thal

amus

Spor

adic

PA0

1N

orm

al3

7846

FC

ereb

ellu

mSp

orad

icPA

WT

10

Dup

2

7918

F3r

d ve

ntri

cle

Spor

adic

PA3

Nor

mal

3

8047

FH

ypot

hala

mus

Spor

adic

PA1

0+

73

814

MC

ereb

ellu

mSp

orad

icPA

WT

10

Dup

3

8219

M4t

h ve

ntri

cle

Spor

adic

PAW

T0

0D

up1

837

MSu

pras

ella

rSp

orad

icPA

WT

01

Nor

mal

3

8411

FPo

ster

ior

foss

aSp

orad

icPA

WT

10

Dup

3

853

FR

opt

ic n

erve

NF1

PA0

01

862

MSu

pras

ella

rSp

orad

icPA

2D

up3

873

FC

ereb

ellu

mSp

orad

icPA

WT

00

Dup

2N

orm

al

8813

MR

tem

poro

occi

pita

lN

F1PA

WT

13

+7

3N

orm

al

8945

FB

rain

stem

NF1

PA0

02


NIH


NIH


NIH


Rodriguez et al.

Cas

e #

Age

Sex

Loc

atio

nN

F1

clin

ical

sta

tus

Tum

or t

ype

PT

EN

FIS

HP

TE

N q

RT

-PC

R F

CF

INA

LpA

KT

IH

CpS

6 IH

CB

RA

F F

ISH

pER

K I

HC

P16

FIS

H

903

MC

ereb

ellu

mSp

orad

icPA

−1.

7N

orm

al

9119

M4t

h ve

ntri

cle

Spor

adic

PA3.

66D

up

9276

FC

ereb

ellu

mSp

orad

icPA

−2.

6N

orm

al

9340

ML

tem

pora

l lob

eSp

orad

icC

ontr

olW

T1

00

Nor

mal

0

9452

ML

tem

pora

l lob

eSp

orad

icC

ontr

olW

T6.

4286

040

0N

orm

al0

951

FC

ereb

ellu

mSp

orad

icC

ontr

olW

T0

1N

orm

al0

NF1

neu

rofi

brom

atos

is ty

pe 1

, Het

del

het

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s de

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D h

omoz

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ns, W

T w

ild ty

pe


PI3K/AKT pathway alterations are associated with clinically aggressive and histologically anaplastic...

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