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Embryonal Central Nervous System Neoplasms Arising inInfants and Young Children

A Pediatric Brain Tumor Consortium Study

Roger E. McLendon, MD; Adesina Adekunle, MD, PhD; Veena Rajaram, MD; Mehmet Kocak, MSc; Susan M. Blaney, MD

N Context.— Medulloblastomas (MBs) and atypical tera-toid/rhabdoid tumors (AT/RTs) arising in infants andchildren can be difficult to distinguish; however, histologiccharacterization is prognostically important.

Objective.— To determine histologic and phenotypicmarkers associated with utility with progression-freesurvival (PFS) and overall survival (OS) in children youngerthan 3 years with MBs and AT/RTs.

Design.— We undertook a histologic and immunopheno-typic study of MBs and AT/RTs arising in infants andchildren younger than 3 years treated in a Pediatric BrainTumor Consortium study. The 41 girls and 55 boys ranged inage from 2 to 36 months at enrollment. These infants andchildren exhibited 51 MBs, 26 AT/RTs, and 24 other tumors(not further studied). Median follow-up of the patients was17.2 months from diagnosis (range: 1.4–93 months).

Results.— Infants and children with AT/RT exhibited a

statistically significant shorter PFS and OS when comparedto infants and children with MBs (both P , .001). A lack of

nuclear BAF47 immunohistochemical reactivity provedreliable in identifying AT/RTs. Among MBs, our datasuggest an association of nodularity and prolonged PFSand OS, which must be independently confirmed. Anapla-

sia correlated with OTX2 reactivity and both OTX2 andmoderate to severe anaplasia correlated with PFS but notwith OS.

Conclusion.— Distinguishing AT/RT from MBs is clini-cally important. For expert neuropathologists, the diagno-ses of AT/RT and MB can be reliably made fromhematoxylin-eosin stains in the vast majority of cases.However certain rare small cell variants of AT/RT can beconfused with MB. We also found that immunohistochem-ical reactivity for BAF47 is clinically useful in distinguish-ing MBs from AT/RTs and for identifying certain small cellAT/RTs. Among MBs, nodularity may be an importantprognostic factor for improved PFS and OS in infants and

children.(Arch Pathol Lab Med. 2011;135:984–993)

Embryonal neoplasms of the central nervous system(CNS) represent 33% of tumors arising in the brains of

infants and children younger than 3 years,1 and thusrepresent a significant source of morbidity and mortalityin this age group. These tumors are largely comprised of medulloblastomas (MBs), atypical teratoid/rhabdoid tu-mors (AT/RTs), and CNS primitive neuroectodermaltumors (PNETs). In contrast to earlier literature,2 recentstudies suggest that accurate subclassification of theseembryonal tumors is important for prognostic and

therapeutic purposes.3 To better understand these neo-plasms, an optimum approach would be to evaluate

progression-free survival (PFS) and overall survival (OS)among a group of patients studied in a uniform manner,preferably under the same treatment protocol.

The Pediatric Brain Tumor Consortium (PBTC) wasformed by the National Cancer Institute in 1999 toimprove the treatment of primary brain tumors inchildren. As described on their Web site, ‘‘The PBTC’sprimary objective is to rapidly conduct novel phase I andII clinical evaluations of new therapeutic drugs, new biological therapies, treatment delivery technologies, and

radiation treatment strategies in children from infancy to21 years with primary central nervous system (CNS)tumors.’’ 4 They also have as a secondary objective to‘‘characterize reliable markers and predictors (direct orsurrogate) of brain tumors’ responses to new therapies.’’ 4

A major goal of the consortium is to further theunderstanding behind the biology and treatment of pediatric brain tumors by performing both basic andtranslational research on brain tumor specimens duringdrug trials. In phase 2, the PBTC supports centralpathology reviews of the patients’ biopsies and, whendeemed appropriate, an analysis of markers of potentialdiagnostic, prognostic, and therapeutic benefit. The

present study was undertaken during PBTC protocol 001(clinicaltrials.gov Identifier: NCT00042367), a trial to

Accepted for publication December 28, 2010.From the Department of Pathology, Duke University Medical Center,

Durham, North Carolina (Dr McLendon); the Department of Pathology,Baylor College of Medicine, Texas Children’s Hospital, Houston (DrAdekunle); the Department of Pathology, University of Chicago,Chicago, Illinois (Dr Rajaram); the Department of Biostatistics, St JudeChildren’s Research Hospital, Memphis, Tennessee (Mr Kocak); and theDepartment of Pathology, Texas Children’s Cancer Center, BaylorCollege of Medicine, Houston (Dr Blaney).

The authors have no relevant financial interest in the products orcompanies described in this article.

Reprints: Roger E. McLendon, MD, Department of Pathology, Duke

University Medical Center, Box 3712, DUMC, Durham NC 27710(e-mail: [email protected]).

984 Arch Pathol Lab Med—Vol 135, August 2011 CNS Embryonal Tumors Arising in Infants and Young Children— McLendon et al



investigate the feasibility of the addition of regionaltherapy with intrathecal mafosfamide to an intensiveregimen of systemic chemotherapy in children youngerthan 3 years at diagnosis with MB, PNETs, ependymomawith metastatic disease, or other primary intracranialembryonal tumors.

Medulloblastoma is designated grade IV by the WorldHealth Organization and is one of the most commonmalignant solid tumors of infancy, but exhibits widelyvarying responses to radiation therapy and chemothera-py,3 responses that may vary by age, genetic abnormali-ties, and histologic features.5 The tumor has been thetarget of a large number of histologic and molecularstudies, with 3 major variants commonly recognized: anodular variant, a diffuse (or classic) variant, and a largecell/anaplastic variant, the last of which may or may not be a subset of diffuse tumors.6–11 Studies seem to indicatethat therapeutic responses show a correlation with themorphologic variants, with nodular tumors having asignificantly better than average prognosis12 and anaplas-tic variants having a much worse than average progno-sis.5,13,14 Recent studies have suggested that OTX2 is

overexpressed in MBs with anaplastic features and mayrepresent an objective means of identifying anaplasia.15

Not clear is whether or not degrees of anaplasia or focalregions of anaplasia in a small biopsy sample are of prognostic significance in this age group. Tumors withnodularity frequently also demonstrate desmoplasia asidentified by diffuse pericellular reticulin,16 however, notall cases with diffuse pericellular reticulin exhibit palenodules.5 Also controversial in this age group is whetherthe nodular variant known as MB with extensivenodularity (MBEN) deserves a grade IV designation.17

The AT/RT is an aggressive, malignant, embryonaltumor most commonly composed of epithelioid cells withround nuclei and prominent nucleoli with frequent

mitotic figures. Often the tumor displays a spectrum of rhabdoid cells, epithelioid cells, and spindle cells.18

Genetically, it exhibits silencing mutations affecting thehSNF5/INI1 complex,19 also known as the BRG1-associ-ated factors and called BAF47. Recently, it has been shownthat the immunohistochemical loss of BAF47 reactivityidentifies the vast majority of AT/RTs.20 Arising in thecerebrum and cerebellum, the tumor is often radiograph-ically mistaken for CNS PNET or MB, based on location.18

The advent of the immunohistochemical detection of BAF47 has helped not only to clarify the morphologicspectrum of these tumors, but also to reveal that certaintumors may lack the epithelioid cytoplasm and exhibitsmall round cells with high nuclear to cytoplasmic

ratios.20,21 Accordingly, recent studies have recommendedthat all cerebral and cerebellar tumors with embryonalfeatures be studied immunohistochemically for BAF47loss.21 Not only has this practice identified a widenedspectrum of tumors now recognized to be AT/RT, but alsothere has been a recognition of a worse than averagesurvival among these cryptic small round cell AT/RTsversus MBs, supporting the value of a pathologicdistinction.21,22

The present study undertook an analysis of histologic,histochemical, and immunohistochemical features of these infantile embryonal tumors. The goal was toinvestigate whether or not a set of histologic markers are

of diagnostic and prognostic utility. In order to accom-plish this, we analyzed the interobserver reproducibility

of the diagnoses issued by 3 neuropathologists withexpertise in pediatric brain tumors and compared thesediagnoses against the diagnoses issued by the originatinginstitutional pathologists. We also assessed the utility of reticulin staining and immunohistochemical reactions forOTX2 and BAF47 as objective standards for predicting PFSand OS.

The results indicate that the diagnoses of MB, AT/RT,and PNET are valid and reproducible as determined by a 2out of 3 consensus being at least 95%. The data alsosupport the routine use of BAF47 immunoreactivity inanalyzing small cell embryonal tumors, the use of OTX2 tocharacterize anaplasia in MBs, the identification of moderate to severe anaplasia in MBs, and the recognitionof nodularity in MBs, all of which proved significant incharacterizing prognosis in embryonal tumors of thepediatric CNS.

MATERIALS AND METHODS

Tumors in this study were embryonal neoplasms from infantsand children younger than 3 years entered into the PBTCProtocol 001 (clinicaltrials.gov Identifier: NCT00042367), a pilot

study that tested the utility of intrathecal mafosfamide tosupplement intensive chemotherapy and radiation therapy of embryonal tumors and metastatic ependymomas. This study wasperformed with multi-institutional institutional review boardapproval. The diagnosis of the originating institution wasconsidered the gold standard, as the institutional pathologistshad full access to the clinical histories, the radiographic studies,and any ancillary diagnostic studies required to establish thediagnosis. The primary goal of the 3 neuropathologists (R.E.M.,A.A., and V.R.) was to independently render a diagnosis basedon hematoxylin-eosin stains of representative sections of tumors.A secondary goal was to characterize and/or quantitate thepresence of a variety of histologic features often associated withpediatric embryonal neoplasms. For MBs, the presence of reticulin, OTX2 expression, and nodular, classic, anaplastic,

and large cell morphologies were identified and recordeddirectly into a central, online database, ProtoTrak, maintained by the PBTC Operations and Biostatistics Center. Cases in whichat least 2 of 3 reviewers identified a case as exhibiting anaplasiawere subsequently evaluated for degree of anaplasia and extentof anaplasia by 1 reviewer (R.E.M.). Cases identified by at least 2of 3 reviewers as being nodular were further analyzed for thediagnosis of MBEN by the same neuropathologist.

Medulloblastoma is a PNET defined by location in thecerebellum. The diffuse pattern of MB presents a histologicappearance of densely packed cells with round-to-oval or carrot-shaped hyperchromatic nuclei surrounded by scanty cytoplasm.3

The classic pattern is similarly characterized by a diffuse growthpattern populated by groups of monomorphic cells with round,regular nuclei in which the chromatin is less condensed;

neuroblastic or Homer-Wright rosettes are most commonlyencountered in this group. Although this definition is unrelatedto the presence of reticulin, reticulin is only rarely encountered inthese tumors.3

Nodular variants of MB exhibit many of the cytologic featuresof the classic variant; however, they vary by 2 distinctivefeatures: pericellular reticulin in the monotonous zones andaround the nodules (Figure 1, A and B). Nodules are composedof circumscribed collections of cells with neurocytic features,including round nuclei and relatively more cytoplasm thanfound in the surrounding tumor, and are occasionally associatedwith obvious streaming neuropil. Reticulin is absent in thesenodules and mitotic activity is infrequent.3 Some tumors exhibit aprofound collection of such nodules, many of which areassociated with sweeping lines of neuropil and rare compressed

regions of internodular cells. These MBENs3

are considered asubgroup of the nodular variant for this study.

Arch Pathol Lab Med—Vol 135, August 2011 CNS Embryonal Tumors Arising in Infants and Young Children— McLendon et al 985



Some MBs exhibit regions of pericellular reticulin, which mayoccur either focally or diffusely.23,24 Pericellular reticulin invari-ably occurs where tumor cells invade the leptomeninges.However, pericellular reticulin is more commonly a widespreadphenomenon among the internodular cells of nodular MBs. Insmall biopsies, it is often not possible to distinguish between the2 etiologies of pericellular reticulin. Therefore for the purposes of this study, reticulin was categorized as negative, multifocal, ordiffuse, according to its distribution in the histologic sections,without regard to the presence of nodules (Figure 2), a commoncopresenting feature.3

The large cell variant (Figure 3) of MB is defined in theliterature as a tumor composed of tumor cells with large, round,vesicular nuclei, prominent nucleoli, and variably abundanteosinophilic cytoplasm.14,25 Anaplasia (Figure 4) is identified by amitotically active tumor that both demonstrates elongatedhyperchromatic nuclei that are densely crowded and exhibitcharacteristic nuclear wrapping against adjacent tumor cells and

demonstrates abundant individual cell necrosis that also aggre-gates into geographic regions of necrosis. Tumors with features

of both large cells and severe anaplastic cells often occur together,lending the group the name ‘‘large cell/anaplastic.’’ 13,26–28 Noconsensus definition for moderate dysplasia exists, but ourexperience indicates that tumors that lack the full features of severe dysplasia most commonly demonstrate frequent individ-ual cell necrosis. Therefore our working definition of moderateanaplasia differs from severe anaplasia by the absence of geographic necrosis but features a ‘‘starry-sky’’ pattern of apoptosis dotting the profusion of small tumor cells. Similarly,no consensus definition exists concerning mild anaplasia.Therefore, our working definition was that mild anaplasia lacks

both geographic necrosis and single-cell apoptosis but doesexhibit nuclear molding and hyperchromasia. Tumors withround regular nuclei and a high overall cellular density wereconsidered to lack anaplasia. The degree of anaplasia was basedon the most severe component identified; its distribution wasgraded as absent, focal, or diffuse.

Atypical teratoid/rhabdoid tumor is a tumor characterized by

a polymorphous cytologic phenotype (Figure 5A), which en-compasses rhabdoid cells and expresses both neural and

Figure 1. A, Nodular medulloblastomas often demonstrate pale islands of tumor cells surrounded by smaller, more densely compacted regions of tumor cells. B, The pale islands frequently are outlined by strands of reticulin that surround the smaller cell population (hematoxylin-eosin, original magnification 3 10 [A]; Wilder reticulin, original magnification 3 20 [B]).

Figure 2. Some tumors that exhibit pericellular reticulin lack the pale islands. Despite the presence of diffuse pericellular reticulin, confusingly,these tumors are not considered desmoplastic by the World Health Organization; this designation is reserved for the nodular tumors (Wilder reticulin, original magnification 3 40).

Figure 3. Large cell medulloblastomas exhibit large, round nuclei with prominent nucleoli. They are distinguished from atypical teratoid/rhabdoid tumors by their minimal cytoplasm and frequent association with anaplastic change (hematoxylin-eosin, original magnification 3 60).




Figure 4. A, Severely anaplastic medulloblastomas (MBs) with dark, pleomorphic nuclei overlapping adjacent tumor cells. B, Severely anaplastic MBs with frequent apoptotic nuclei and geographic necrosis. C, Moderately anaplastic MBs with starry-sky apoptosis. D, Mildly anaplastic MBs with hyperchromatic nuclei and rare apoptotic cells (hematoxylin-eosin, original magnifications 3 40 [A and B] and 3 20 [C and D].

Figure 5. A, Epithelioid features and large, round, open nuclei with prominent nucleoli are features of the atypical teratoid/rhabdoid tumors. B,Scattered islands of immunoreactivity without obvious differentiation are also found (hematoxylin-eosin, original magnification 3 40 [A]; glial fibrillary acidic protein, original magnification 3 40 [B]).




nonneuroepithelial immunophenotypic markers, including notonly a lack of immunoreactivity for the INI1/BAF47 geneproduct, but possibly also scattered islands of immunoreactivityfor glial fibrillary acidic protein (Figure 5B), epithelial membraneantigen, and smooth muscle actin,3 among others.

Histochemical Methods

The Wilder method29 to detect the presence of reticulin wasused to define presence or absence of desmoplasia.

For immunohistochemical assays, formalin-fixed, paraffin-embedded sections were cut at 5 mm, deparaffinized in xylenes,and brought to water through graded alcohols. Endogenousperoxides were blocked in 3% H2O2 in dH2O at roomtemperature for 10 minutes. For anti-OTX2 immunohistochem-istry, all sections were incubated in citrate buffer, pH 6.1, for

20 minutes at 100uC, blocked with 10% normal rabbit serum for1 hour, and incubated with OTX2-specific goat polyclonalantibody (dilution 1:80; AF1979, R&D, Minneapolis, Minnesota)for 45 minutes at room temperature, followed by 30 minutes of incubation with biotinylated rabbit anti-goat secondary antibodyat 1:200 dilution (BA5000, Vector Laboratories, Burlingame,California). Detection of antibody binding was performed usingVectastain Elite ABC reagent (PK-7100, Vector Laboratories)according to the protocol. For the slides to be tested for BAF47expression, the slides were placed in preheated Tris/EDTA,pH 9.0, and heated for 20 minutes in a 100uC water bath.Solutions and slides were cooled for 20 minutes, washed indeionized water, and placed in Tris-buffered saline, pH 7.5.Primary antibody mouse monoclonal BAF47 (1:200; B-D Biosci-ences, San Jose, California) or negative control reagent mouse

IgG was applied and incubated for 1 hour at room temperature.Detection of antibody binding was performed using horseradishperoxidase–conjugated anti-mouse Envision Plus (Dako, Carpin-teria, California) using diaminobenzidine. All slides werewashed with tap water; hematoxylin counterstain was applied,followed by dehydration with absolute alcohol, clearing withxylene, and coverslipping with a permanent mounting media.

Statistical Methods

Descriptive statistics were used to assess the interrateragreement on diagnostic and diagnostic feature calls. Agreementof diagnostic calls by the 3 pathologists was assessed indepen-dently and with respect to institutional diagnosis calls. Associ-ations of diagnostic value of consensus calls with OS and PFSdistributions were investigated. PFS was defined as the interval

from initiation of treatment to the earliest indication of diseaseprogression or death on study for patients who failed. OS wasdefined as the interval from initiation of treatment to death. Datafrom patients without failure for PFS or OS were censored at off-study or at the patient’s last date of follow-up. Distributions of PFS and OS were estimated using the Kaplan-Meier method andcompared using the log-rank test. An exact Cochran-Armitagetrend test was used to compare degree of anaplasia with thelikelihood of OTX2 presence. No multiplicity correction was usedfor the P values reported in this study, and because of thenumber of tests performed, statistical significance was calledonly if the P value was ,.002.

RESULTS

The patients, 41 girls and 55 boys, represented in thisstudy ranged in age from 2 to 36 months at the time

of enrollment. Median follow-up of the patients was17.2 months from diagnosis (range: 1.4–93 months).

Case Identification

Table 1 represents the diagnoses rendered by eachpathologist compared to the treating institution’s originaldiagnoses of MB or AT/RT (Table 1). As the centralreviewers were not provided with site of origin, MB,PNET, and pineoblastoma (PB) were combined into asingle diagnostic category. Of 96 patients treated onPBTC-001 protocol with pathology data, the institutionalpathologists reported 42 having MB. Of these 42 patients,the 3 central pathologists had perfect consensus on MB/PNET/PB for 38 patients (90.5%), and had at least 2-of-3consensus for 40 cases (95.2%). One case was diagnosed asAT/RT by 2 of the 3 central pathologists, and 1 case wasdiagnosed as pleomorphic xanthoastrocytoma by 2 of the3 central pathologists. Among the 54 cases for which theinstitutional diagnosis was not MB, 5 cases were diag-nosed as MB/PNET/PB by all 3 central pathologists, and3 cases were diagnosed as MB/PNET/PB by 2 of the 3central pathologists. The treatment protocols were basedon the diagnoses rendered by the treating institutions andno changes in diagnoses were made based on the results of these studies.

The 2-of-3 consensus data are slightly misleading in thatdiagnoses of MB were rendered on more than 42 cases by

the central reviewers shown in Table 1. When viewedfrom the perspective of the central pathologists who werereviewing the entire cohort of 96 cases, there were 55 casesof MB/PNET/PB. Out of these 55 patients, the 3 centralpathologists had perfect consensus on MB/PNET/PB for43 patients (78%), and at least 2-of-3 consensus for 50 cases(91%) indicating that there were an additional 13 cases forwhich we derived the diagnosis of MB/PNET/PB thatwere either PNETs or PBs by the institutional diagnosis.More informative for the purposes of this study are the 5non-MB/PNET/PB diagnoses on cases in which theinstitutional diagnosis was not MB, PNET, or PB. Forthese 5 cases, the differential diagnoses included AT/RT,malignant lymphoma, pleomorphic xanthoastrocytoma,

and glioblastoma.AT/RT proved to have a strong trend of recognizability

and resulted in reasonably accurate diagnoses (Table 1).The institutional pathologists reported 26 patients havingAT/RT. Out of these 26 patients, the 3 central pathologistshad perfect consensus on AT/RT for 15 patients (57.7%)and had at least 2-of-3 consensus for 23 cases (96.2%). Forthe remaining 3 cases, the differential diagnostic consid-erations from the central reviewers included malignantCNS tumor and MB/PNET/PB. Other diagnoses made bythe central reviewers included CNS germ cell tumor,choroid plexus carcinoma, anaplastic ependymoma, andmalignant meningioma, all of which reflect the epithelial

and occasionally papillary appearances manifested by thetumor.

Table 1. Comparison of Diagnostic Agreement Among Reference Pathologists Reviewing Hematoxylin-Eosin Slides of Representative Blocks of Tumor From the Entire Cohort of 96 Patients Versus the Referring Institutions’ Diagnoses as Either

Medulloblastoma (MB) or Atypical Teratoid/Rhabdoid Tumor (AT/RT)

Institutional Diagnosis Pathologist 1 Pathologist 2 Pathologist 3 3 of 3, No. (%) At Least 2 of 3, No. (%)

MB (n 5 42) 45 39 40 38 (90.5) 40 (95.2)AT/RT (n 5 26) 23 29 24 15 (57.7) 23 (88.5)




At the conclusion of the study, 27 patients with MB and9 with AT/RT were alive.

Medulloblastoma—Anaplasia and Nodularity

The qualitative assessment of the identification of nodular status, classical growth pattern, diffuse growthpattern, presence of anaplastic component, and presenceof large cell component all proved to produce close to

perfect consensus, with a minimum 2-of-3 consensus being at least 95% in any of these evaluations (Table 2).Anaplasia was identified by at least 2 of the reviewers in14 (25.5%) of 55 MB samples submitted for review.

Based on an agreement of 2 of 3 neuropathologists(Table 3), those patients whose tumors exhibited anyanaplastic features (mild to severe) included those withdiffuse large cell/anaplastic morphology (n 5 11);nodular with large cell/anaplastic morphology (n 5 12);and diffuse with anaplasia morphology (n 5 7). Thefindings of an analysis of the 3 central reviewers whocharacterized anaplasia as simply present or absentsuggested that anaplasia was not associated with OS orPFS in this age group (Table 3). However, analysis based

on degree of anaplasia suggests that patients with tumorsexhibiting moderate or severe anaplasia (Figure 5) mayhave a worse prognosis (Table 3). Similarly, MB patients

whose tumors exhibited OTX2 immunoreactivity mayhave a worse prognosis. OTX2 immunoreactivity wascompared against degree of anaplasia via an exactCochran-Armitage trend test that demonstrated a P valueof ,.001, which suggests that as anaplasia gets moresevere, there is a higher likelihood of OTX2 presence. Wealso undertook to further characterize anaplasia by extentof tumor involved. No further associations with survivaldistributions were suggested among these patients bysubgrouping according to absent anaplasia (n 5 20), focalanaplasia (n 5 17), or diffuse anaplasia (n 5 7).

Figure 6, A and B, suggests that nodularity may be apositive prognostic factor. Because MBEN is morpholog-ically distinctive, we further investigated the survivalassociations among patients whose tumors exhibitednodularity by comparing survival of MBEN (n 5 11)versus nodular MB (n 5 8) and found insignificantdifferences in PFS (P 5 .08) and OS (P 5 .74; Table 3).

We further analyzed effect on survival of both nodularpatterns versus diffuse MBs (n5 21), in which no nodulargrowth pattern was discernible (Figure 7, A and B).Among these growth patterns, tumors with any nodular-

ity exhibited a better OS (P5

.004; Table 3). In contrast, thepresence of fine reticulin-staining fibers coursing throughthe tumor, a finding that commonly accompanies nodularMBs in older children and adults, did not exhibit astatistically significant effect on either PFS (P 5 .13) or OS(P 5 .89). In this age group, desmoplasia, as identified by reticulin-positive fibers in the tumor, is biologicallydistinctive from nodularity (Figure 3). However, in adults,nodular tumors commonly display reticulin-positivefibers; we found that in this age group many diffusetumors also demonstrate pericellular reticulin. Thus, thepresence of nodularity is a prognostically beneficialfinding, but the presence of reticulin-staining fibers isnot. However, this may represent our lack of distinction as

to the source of pericellular reticulin from cases withleptomeningeal infiltration; it is difficult to know howmuch weight to place on this finding.

Atypical Teratoid/Rhabdoid Tumor

As a result of the previous studies of Haberler et al,21

BAF47 immunohistochemistry was performed on 32tumors for which at least 1 pathologist made a diagnosisof MB; BAF47 reactivity was diffuse in 17 cases, multifocalin 14 cases, and absent in 1 case. The diagnosis of theBAF47-negative MB (60775; shown in Figure 8, A and B)was agreed upon by 2 of the 3 central reviewers; it had noepithelial elements and was histologically identical to adiffuse MB without anaplasia. To compare the immuno-

reactivity of BAF47 in these tumors versus the AT/RTs, asample of 6 AT/RTs as diagnosed by the originatinginstitution were analyzed for BAF47 expression; BAF47was absent in 5 of 6 (Table 4). The remaining casediagnosed by the outside institution as AT/RT (107487;Figure 9, A and B) was diagnosed as PNET by 2 of 3central reviewers. Survival analysis based on the diagno-ses of the originating institutions revealed significantdifferences in PFS and OS (P , .001 and P , .001,respectively) with regard to making a diagnosis of MBversus AT/RT (Figure 10).

COMMENT

Among infants and children less than 5 years, CNStumors affect more than 1300 each year in the United

Table 2. Comparison of Diagnostic AgreementAmong Reference Pathologists for Various Descriptors

in Medulloblastomas

3-of-3Consensus, %

At Least 2-of-3Consensus, %

Classic component 21.4 97.6Diffuse component 45.2 100Apoptoses present 69 100Large cell component 57.1 100Anaplastic component 47.6 100Necrosis present 64.3 100Nodular status 64.3 97.6

Table 3. Log-Rank Test P Values to Compare VariousSubgroups and Pathologic Features

Variable of Interest PFS OS

Anaplasia

Anaplasia versus no anaplasia .29 .79Absent versus focal versus diffuse .57 .90None/mild versus moderate/severe .02 .25

Nodularity

None versus not extensive versus extensive .01 .01(None or not extensive) versus extensive .005 .03None versus (not extensive or extensive) .02 .004

Reticulin

Negative versus multifocal versus diffuse .13 .89

Large cell/anaplastic versus nodular

LCA2 /Nod.2 versus LCA+ /Nod.2 versusLCA2 /Nod.+ versus LCA+ /Nod.+ .02 .01

LCA2 /Nod.+ versus others ,.001 .02LCA versus others .01 .001

Diagnostic category

MB versus AT/RT ,.001 ,.001

Abbreviations: AT/RT, atypical teratoid/rhabdoid tumor; LCA, large

cell/anaplastic; MB, medulloblastoma; Nod, nodular; OS, overallsurvival; PFS, progression-free survival.




States; 320 of these tumors will be embryonal tumors,1 MBand AT/RT being the most common types. These 2 tumortypes have diagnostic overlap.22,30 Because diagnostic

variants of MB seem to have widely variable prognosticimplications,5,12,23 proper classification is important. ThePBTC drug trial 001, investigating the utility of intrathecalmafosfamide to supplement intensive chemotherapy andradiation therapy in infants and children younger than3 years with embryonal tumors and metastatic ependy-momas, offered a resource whereby these tumors and thefeatures used to evaluate them could be further studied.

The patients entered into this protocol exhibited tumorsthat represented the spectrum of embryonal tumors of childhood, including MB, AT/RT, PB, CNS PNET,ependymoblastoma, and choroid plexus carcinoma. Inthe present study, we first undertook to determine the

reproducibility of making histologic distinctions betweenMB and AT/RT. Next, we proceeded to analyze the

reliability of characterization of histologic features used tocharacterize the embryonal tumors in general and MBsspecifically.

First, it was important to determine if the morphologicfeatures used by the central reviewers could be replicatedamong the pathologists conducting the central review.Among the factors examined, both nodular morphologyand anaplastic features proved to be reliable andreproducible. In a number of cases, the central neuropa-thology review committee disagreed with the diagnosissubmitted by the original institution. Because the centralreviewers were blinded as to anatomic site, thesediscrepancies appeared to be related to site in themisclassification of MB as PNET. In a reevaluation of theresults in which all cases classified as PNET wererecombined with tumors classified as MB and PB, the

number of disagreements diminished, both between thecentral reviewers and the submitting institutions and

Figure 6. A, Progression-free survival estimates by degrees of anaplasia (medulloblastoma patients, P 5 .02). B, Overall survival by degrees of anaplasia (medulloblastoma patients, P 5 .25).

Figure 7. A, Progression-free survival estimates by degrees of nodularity (medulloblastoma patients, P 5 .02). B, Overall survival by degrees of nodularity (medulloblastoma patients, P 5 .01).




among the central reviewers themselves. The data supportthe historical contention that knowledge of tumor locationis essential in making a diagnosis using light microscopy.

Desmoplasia

Medulloblastomas with nodules are currently classified by the World Health Organization using the compoundterm ‘‘desmoplastic/nodular.’’ These tumors are ‘‘charac-

terized by nodular reticulin-free zones (‘pale islands’)surrounded by densely packed, highly proliferative cellswith hyperchromatic and moderately pleomorphic nucleiwhich produce a dense intercellular reticulin fibernetwork.’’ 3 The definition indicates that the presence of intercellular reticulin without nodules should not beconsidered a desmoplastic/nodular variant. The presentstudy undertook an analysis of the effect of the presence of nodules alone and desmoplasia alone and found thatnodules, regardless of the presence of reticulin oranaplastic content, were associated with a good OS.

Nodular MBs (Figure 1) comprised 19 of 42 MBs (45%) inthe present study, a rate similar to the 57% rate previously

reported in infants.

31

The nodular MB variant, MBEN, is atumor of infancy that is known to be associated with a good

outcome.12 Some have suggested that the nodular tumorsare more likely to be lateral, thus more accessible to acomplete resection and therefore having a better associatedsurvival.32 However, other reports suggest the biologicresponse of MBENs to therapy may be different from thatof other MBs; McManamy and colleagues31 hypothesizedthat the limited growth potential of nodular MBs wasrelated to their differentiation capacity.

An attempt was made to determine if the findings of thepresent study were somehow a drug treatment–relatedeffect versus an age-related effect, that is, whether therewere more MBEN types in this cohort compared to otherMB treatment trials because MBENs are predominatelyinfantile tumors.31,33 The rarity of this tumor in patientsolder than 3 years makes such a comparison difficult andnot readily found in the literature. Furthermore, the lack of radiation therapy in this trial (and other recent trialsinvolving infants) also poses a problem in the comparison.The initial hopeful report of Giangaspero and colleagues12

all involved children younger than 4 years, and the reportsof Eberhardt et al,34 Grill and colleagues,35 Rutkowski and

colleagues,

36

and McManamy and colleagues

31

all empha-size the good prognostic effect of MBEN histology in

Figure 8. A, Tumor from patient 60775 reveals no epithelioid or rhabdoid features by hematoxylin-eosin. B, Negative BAF47 reactivity is noted in the tumor with intact endothelial cell immunoreactivity (hematoxylin-eosin, original magnification 3 40 [A]; anti-BAF47, original magnification 3 40 [B]).

Figure 9. A, Tumor from patient 107487 diagnosed by institution as atypical teratoid/rhabdoid tumor. B, Diffuse BAF47 immunoreactivity is present (hematoxylin-eosin, original magnification 3 40 [A]; BAF47, original magnification 3 40 [B]).




infants. Less sanguine reports involved children of olderage, although McManamy and colleagues31 noted only oneexample of MBEN arising in a child older than 3 years. Inthe absence of an evaluation of MBEN histology andprognosis occurring in older children, it seems sufficiently

conservative to say that MBEN histology occurring ininfants is predictive of good PFS and OS when confrontedwith the intensive therapeutic regimen of this protocol. Incontrast, the presence of reticulin, a finding frequentlyassociated with nodular phenotype, was not associatedwith a good prognosis, a finding incorporated into theWorld Health Organization definition as well as support-ed by Lamont et al5 and by McManamy and colleagues.31

Anaplasia

To our knowledge, no previous studies have analyzedthe interobserver reproducibility of anaplasia as a histo-logic feature. The highly aggressive large cell variant of

MB was first reported by Giangaspero and colleagues,14

who also described the poor OS associated with thepresence of anaplasia and large cell morphology and itsassociation with MYC oncogenic amplification. Addition-al reports have indicated no prognostic difference among

cases with large cell morphology versus anaplasticmorphology, and, indeed, many tumors exhibited largecell morphology intermixed with areas of anaplasticmorphology, thus leading to the conjoined term anaplas-tic/large cell MB.23,27,37 Although anaplasia was associated

with a significantly worse survival in this study, our initialanalysis suggested that its impact was not independent of nonnodular morphology. This discrepancy was furtherinvestigated. Using a working definition of moderateanaplasia as an MB with starry-sky apoptosis but lackinggeographic necrosis and mild anaplasia as a tumor withnuclear molding but lacking both starry-sky apoptosis andgeographic necrosis, a reanalysis of our cases subsequent-ly supported the historic findings that moderate andsevere anaplasia inferred a worse PFS and OS.23,27,37 Wealso examined the antigenic expression of OTX2, adevelopmentally regulated transcription factor that hasrecently been described to be overexpressed in anaplastic

MBs.15,38

OTX2 immunoreactivity correlated with anapla-sia in this cohort of patients and was associated with astrong trend toward a poor PFS, suggesting a need for alarger study of the prognostic application of this immu-nohistochemical marker in diffuse MB.

Atypical Teratoid/Rhabdoid Tumor

Previous studies have identified the difficulty indiagnosing AT/RT and in distinguishing the tumor fromMB. The propensities of the tumor to exhibit both neuronaland glial markers (as well as epithelial markers) plus itslesser-recognized ability to mimic the features of diffuseMB compound this difficulty. We sought to determine theinterobserver variation among a group of expert neuropa-

thologists in making these diagnoses and to study theimmunoreactivity profile among a group of AT/RTs andMBs. As noted in Table 1, the 3-of-3 concordance amongMBs was clearly better than the concordance seen whendiagnosing AT/RTs. However, when 2-of-3 consensus wasexamined, the diagnostic variance between MBs and AT/RTs diminished considerably. MB and other ‘‘small bluecell’’ tumors frequently entered into the differentialdiagnosis of AT/RT. Thus, the subjective distinction of an AT/RT from other small blue cell tumors is prone toerror. The BAF47 antibody has proven its utility inobjectively identifying the nonexpressing AT/RTs fromother expressing tumors in the differential diagnosis with

rare exception. Therefore, among the AT/RTs and MBsstudied, we performed BAF47 staining on a sample of 6

Table 4. Medulloblastomas (MBs) and Atypical Teratoid/Rhabdoid Tumors (AT/RTs) With Diagnoses by Referees andBAF47 for the Conflicting Cases (at Least 1 Atypical Teratoid/Rhabdoid Call)

Accession No. Institutional Call BAF47 Call by Pathologist 1 Call by Pathologist 2 Call by Pathologist 3

5371 AT/RT Absent AT/RT AT/RT MM7804 AT/RT Absent AT/RT AT/RT CNSNeo

28608 AT/RT Absent AT/RT AT/RT CNSNeo52807 AT/RT Absent AT/RT CPC AT/RT82673 AT/RT Absent MB AT/RT AT/RT

107487 AT/RT Diffuse PNET AT/RT PNET15091 MB Multifocal MB AT/RT MB31442 MB Diffuse MB AT/RT AT/RT31442 MB Diffuse MB AT/RT AT/RT60775 MB Absent MB MB AT/RT49615 PNET Diffuse PNET AT/RT PNET

Abbreviations: CNSNeo, central nervous system neoplasm, not otherwise specified; CPC, choroid plexus carcinoma; MM, malignant melanoma;PNET, primitive neuroectodermal tumor.

Figure 10. Overall survival (OS; P , .001) and progression-free

survival (PFS; P,

.001) estimates by diagnostic category (medulloblastomas [MB] versus atypical teratoid/rhabdoid tumors [ATRT]).




AT/RTs as well as on 32 MBs. In support of the findings byKraus and colleagues30 and by Edgar and Rosenblum,22 5 of 6 AT/RTs (as diagnosed by the institutional pathologists)demonstrated negative immunoreactivity, as did 1 diffuseMB, possibly a cryptic AT/RT (Figure 8, A and B). This MBwas lacking in both anaplastic features and cytologicfeatures of epithelioid or rhabdoid differentiation. Theliterature has indicated that the otherwise typical-appear-ing MBs with diffuse histology, but negative expression of BAF47, all proved to have poor clinical outcomes, andwere best considered AT/RTs. However, to date, nostudies have examined the polyphenotypic profiles of thesmall cell AT/RTs seen in the more typical examples.

This study was supported by the Pediatric Brain TumorConsortium. Dr McLendon is supported by the Pediatric BrainTumor Foundation and by NIH/NINDS grants 5P50-NS-20023-26 and 5 R01 CA118822-05.

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