Unusual malignant glioneuronal tumors of the cerebrum of adults: a clinicopathologic study of three cases

Acta Neuropathol (2006) 112:727–737

ORIGINAL PAPER

Unusual malignant glioneuronal tumors of the cerebrum of adults: a clinicopathologic study of three cases

Fausto J. Rodriguez · Bernd W. Scheithauer · John D. Port

Received: 7 June 2006 / Revised: 13 July 2006 / Accepted: 29 July 2006 / Published online: 7 September 2006© Springer-Verlag 2006

Abstract Malignant glioneuronal tumors of the brainare rare and poorly characterized. Herein, we reportthe clinicopathologic features of three examples withunusual morphologies including both glial and neuronaldiVerentiation. Hematoxylin and eosin-stained slideswere reviewed in all cases. Immunohistochemical stainswere performed on formalin-Wxed, paraYn-embeddedsections. Transmission electron microscopy (EM) wasperformed on both formalin-Wxed (n = 1) and paraYnembedded tissue (n = 2). The immunogold techniquefor localization of GFAP was also performed. Twopatients were male and one was female, age 66, 84, and34 years, respectively. Radiologic studies demonstratedhyperdensity on CT (n = 3), multicentricity (n = 2), anda cortical based solid component with a cystic extensioninto underlying white matter (n = 2). At surgery, allthree tumors were superWcial and relatively circum-scribed. Histologically, they were composed of largeepithelioid cells (n = 3), spindle cells (n = 1), and poorlydiVerentiated smaller cells with high nuclear/cytoplas-mic ratios (n = 1). Brisk mitotic activity and coagulativenon-palisading necrosis were present in all cases. Thetumors were immunopositive for GFAP (n = 3), S-100(n = 3), synaptophysin (n = 3), chromogranin (n = 3),Neu-N (n = 2), and neuroWlament protein (n = 2).Stains for EMA were negative. EM demonstrated con-vincing neurosecretory granules in one case, some in

Wlament-containing cells immunogold labeled forGFAP. Two patients expired 3–5 weeks after surgery.True malignant neoplasms with glial and neuronaldiVerentiation do occur in the central nervous systemof adults and may pursue a highly aggressive course.The use of minimal diagnostic criteria, e.g., immuno-reactivity for a single antigen like neuroWlament protein,may not be suYcient and should be discouraged.

Keywords Glioneuronal · Brain · Immunohistochemistry · Neuronal tumor · Glioma · Neuronal diVerentiation

Introduction

Primary central nervous system neoplasms that exhibitdiVerentiation along neuronal and glial lines are rela-tively rare, ganglioglioma being the most common andbest-characterized subtype [29, 32]. Recent years haveseen an increased interest in such tumors and the rec-ognition of new subtypes. This is largely due to theapplication of immunohistochemical and ultrastruc-tural criteria. The other members of the now expandedspectrum include extraventricular neurocytoma [1, 7]desmoplastic infantile ganglioglioma, pleomorphicxanthoastrocytoma [17, 21] as well as subependymalgiant cell astrocytoma [21, 33]. More recently, thetumor spectrum has come to include papillary glioneu-ronal tumor [30], and rosette-forming glioneuronaltumor of the fourth ventricle [31]. Of the above-men-tioned tumors, all are considered low grade/benign.The “glioneuronal tumor with neuropil-like islands”described by Teo et al. [50], represents a departure inbeing a malignant neoplasm.

F. J. Rodriguez · B. W. Scheithauer (&)Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street, SW, Rochester, MN 55905, USAe-mail: [email protected]

J. D. PortDivision of Neuroradiology, Mayo Clinic, Rochester, MN, USA

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Rather recently, Varlet et al. [51] expanded thesearch for glioneuronal tumors to include an evenbroader group of relatively benign and malignant glialneoplasms. These were morphologically classic gliomasubtypes but showed often-limited immunohistochemi-cal evidence of neuronal diVerentiation. The suggestionwas made that such tumors exhibited distinct clinical/radiographic features and perhaps a more favorableprognosis. In the present study, we report three cases ofmalignant mixed glioneuronal neoplasms with distinc-tive, albeit unusual histologic and immunohistochemicalfeatures. Unlike more ordinary glioneuronal tumors, alloccurred in adults. The literature regarding expansion ofthe glioneuronal tumor category is reviewed.

Materials and methods

Three cases are presented. Clinical features wereabstracted by retrospective chart review. Neuroradio-logic studies and histopathology were evaluated in allinstances. Immunocytochemical stains were performedusing the streptavidin biotin peroxidase complex tech-nique and antibodies directed against GFAP (Dako,Carpinteria, CA; polyclonal; dilution 1:800), S100(Dako, polyclonal protein; 1:800), synaptophysin (ICN,Costa Mesa, CA; clone SY38; 1:40), Neu-N (Chemicon,Temecula, CA; 1:2,000), chromogranin (Roche, India-napolis, IN; clone LK2H10; 1:1,000), neuroWlamentprotein (NFP) (Dako, clone 2F11; 1:75), p53 protein(Dako, clone DO-7; 1:200), epithelial membrane anti-gen (EMA) (Dako, clone E29; 1:20), cytokeratin(CAM 5.2) (Becton Dickinson, San Jose, CA; 1:50),cytokeratin (CAM 5.2) (Becton Dickinson, San JoseCA; 1:50), CK7 (Dako, clone OB-TL 12/30; 1:200),CK20 (Dako, clone Ks20.8; 1:50), TTF-1 (Dako, clone8G7G3/1; 1:1000), desmin (Dako, clone DER11;1:100), smooth muscle actin (Dako, clone 1A4; 1:150),

and Ki67 antigen (Dako, 1:800, MIB-1). MIB-1 label-ing indices were determined using the CAS200 systemwith evaluation of 20 consecutive tumor Welds. Elec-tron microscopy was performed on formalin-Wxed tis-sue in case 1 and on formalin-Wxed/paraYn-embeddedtissue in cases 2 and 3 using standard processing meth-ods and a FEI company, Tecnai 12 model, transmissionelectron microscope. The immunogold labeling tech-nique [4, 35] was performed using a GFAP antibody(Dako, Carpinteria, CA; polyclonal, 1:100).

Case reports

Case 1

An 84-year-old male recently developed slurred speechand confusion. His past medical history was signiWcantfor a cerebral infarct involving the posterior circulationwith resultant homonymous hemianopsia. On physicalexam, the lungs were clear to auscultation and therewas no lymphadenopathy. Neurologic examinationshowed the patient to be confused about time, date,and place. Cranial nerves II–XII, as well as sensori-motor function, were intact. An MRI scan (Fig. 1a–c)demonstrated a 5.7 £ 5.6 cm left temporoparietal ring-enhancing lesion, surrounded by edema and exhibitingpronounced mass eVect. Two smaller satellite noduleswere also noted. A solid enhancing componentcentered in the cortex and a cystic, necrotic-appearingelement in sub-adjacent white matter characterized thelarge mass. The radiological appearance was mostconsistent with multicentric glioblastoma multiforme.None of the lesions were present on an MRI scanperformed 14 months prior. At left temporoparietalcraniotomy, the grossly red, necrotic and partially cystictumor was extensively debulked. The patient expired22 days after surgery. An autopsy was not performed.

Fig. 1 Case 1. Axial T2-weighted (a), FLAIR (b) and post-con-trast T1-weighted (c) MR images demonstrate a large, solid, andcystic, left temporoparietal lesion with a solid cortical component

and a subjacent cystic, necrotic-appearing element. Two smaller,solid, satellite nodules are also seen (arrows)

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Case 2

A 66-year-old male presented to the emergency roomwith a 7–10 day history of worsening dizziness, nausea,headache, gait diYculty, and drooping of the left face.His past medical history was noncontributory. Neuro-logic examination revealed slight left deviation of thetongue but was otherwise unremarkable. Brain imaging(Fig. 2a–c) demonstrated two enhancing masses, oneinvolving the right frontotemporal lobe and the otherthe left frontal lobe nearby the vertex. A solid enhanc-ing component centered in the right insular cortex and acystic component in subjacent white matter again char-acterized the dominant, right-sided mass. Both massesshowed moderate mass eVect and mild surroundingedema. Again, the radiologic appearance was mostconsistent with a multicentric glioblastoma. A CT scanof the chest, abdomen, and pelvis was unrevealing. Atright frontotemporal craniotomy, the dura appearednormal, as did the cerebral cortex. The tumor was quitevascular and largely demarcated except in the region ofthe sylvian Wssure. A postoperative MRI scan disclosedresidual tumor along inferior and deep margins of theresection, as well as the development of hemorrhagein association with the left frontal lesion. The patientwas frail after surgery and the family decided to providepalliative care only. He expired approximately 1 monthafter surgery. An autopsy was not performed.

Case 3

The patient, a 34-year-old female with a 6 year historyof grand mal seizures, presented to a local emergencydepartment with progressive severe headaches, diplopia,nausea, and vomiting. A CT scan without contrast dem-onstrated a right frontal lobe abnormality interpretedas a hematoma associated with an 8 mm shift of the

midline. A right frontal craniotomy achieved gross totalresection of a superWcially situated vascular tumor withdural attachment. Aside from mild residual diplopia, therecovery was uneventful. Recommendations were madefor cranial irradiation followed by chemotherapy.

Pathology

Case 1

The tumor was received in multiple fragmentsdescribed as blood tinged, tan-gray, gritty, andnecrotic. On H&E stain, it consisted of cells disposedin large, irregularly shaped nodules composed centrallyof a primitive component of small cells with a highmitotic rate and frequent apoptosis. At the peripheryof the nodules, the most remarkable morphologic fea-ture was a peripheral zone of larger cells with gener-ous, eosinophilic, and occasionally eccentric cytoplasm,nuclei with open chromatin and prominent nucleoli(Fig. 3). Outside of the nodules, the larger cells wereoccasionally disposed in aggregates, but exhibited amore “ganglioid” appearance, with open chromatin,larger nucleoli, and scant Nissl substance (Fig. 3). Thedegree of clustering and cytologic abnormalities wasstrong morphologic evidence that these cells were partof the neoplastic process. A dense, intercellular, reticu-lin-rich meshwork was present in internodular regions.No rosettes were identiWed. Convincing endothelialproliferation was not identiWed. Coagulative non-pali-sading necrosis was a frequent Wnding.

Case 2

Grossly, the tumor aggregated 5 £ 3 £ 1.5 cm and con-sisted of multiple tan or gray-white fragments, some

Fig. 2 Case 2. A non-contrast CT scan (a) shows a hyperdense,right lateral frontal lobe tumor with the solid cortical mass. Theunderlying cystic white matter component is best seen on the

coronal post-contrast T1-weighted MR images (b). A second masswas also noted in the posterosuperior left frontal lobe and showedearly cystic change (c, post-contrast T1-weighted MR image)

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with hemorrhage. Microscopically, it was comprisedmainly of eosinophilic cells with eccentric cytoplasmresembling gemistocytic astrocytes. A variety ofarchitectural patterns were identiWed including nests,ribbons, and pseudopapillae (Fig. 4). Single cell inWl-tration, including “Indian Wling” in portions of thetumor imparted a resemblance to inWltrating carci-noma (Figs. 4, 5). A reticulin-rich, Wbrosarcoma-likecomponent showed markedly increased mitotic activity(21 mitoses/10 hpf). Endothelial proliferation was absent.Coagulative non-palisading necrosis was abundant.

Case 3

The tumor consisted of two tumor fragments measur-ing 3.7 and 2.5 cm in greatest dimension. Attachedto a fragment of dura, the largest specimen was wellcircumscribed, pink-red, and mottled red-tan on cutsurface. Microscopically, it was composed of epitheli-oid cells with ample eosinophilic cytoplasm, clumpedchromatin, and inconspicuous nucleoli. Focally, thecells were spindled or markedly enlarged and pleomor-phic with eccentric cytoplasm and nuclear pseudo-

inclusions (Fig. 6). Perivascular pseudorosettes, briskmitotic activity, and coagulative non-palisading necro-sis were present. Endothelial vascular proliferation wasabsent. No granular bodies were identiWed.

Immunohistochemistry

Essential immunohistochemical features of the threetumors are summarized in Table 1.

Case 1

The large peripherally situated eosinophilic cells stronglyco-expressed GFAP and S-100 protein, as well as synapt-ophysin, and chromogranin in rare foci. The morecentrally situated, primitive small cells showed variablereactivity for these same antigens (Fig. 3). The neuro-Wlament protein immunostain demonstrated few axons,indicating that the neoplasm was largely solid. Cytokera-tins CAM 5.2, CK7 and CK20 as well as TTF-1 werenegative. Moderate numbers of CD68-immunopositive

Fig. 3 Case 1. The tumor was largely composed of irregular nodules, poorly diVerentiated cells with scant cytoplasm and hyperchromatic nuclei. A key feature of the tumor was rim-ming of these nodules by large cells with eccentric nuclei, prominent nucleoli and eosin-ophilic cytoplasm (upper left). Variably enlarged cells, some with a “ganglioid” appearance were occasionally seen both at the center of nodules and forming aggregates (upper right). In addition to synapto-physin, rimming cells strongly expressed GFAP. Synapto-physin was also expressed, but to a lesser extent, in the prim-itive central small cells, and in “ganglioid” cell aggregates. The latter cells also showed convincing, albeit focal chro-mogranin expression

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macrophages were noted at the center of the nodules.The MIB-1 labeling index was 47%.

Case 2

As in case 1, the large, gemistocytic-appearing cellsstrongly co-expressed GFAP and S-100 protein and,to a lesser extent, synaptophysin and Neu-N as well(Fig. 5). Only rare cells were chromogranin reactive.The sarcomatous component lacked expression for allbut focal S-100 protein staining. NeuroWlament proteinstains labeled rare tumor cells at the center of thelesion and scant overrun axons, mainly at the interfaceof the tumor with brain parenchyma. The MIB-1 label-ing index ranged from 19 (glioneuronal component) to25% (sarcomatous component).

Case 3

Strong co-expression of S-100 protein and synaptophy-sin, as well as marked neuroWlament protein expres-sion of cell aggregates, were the main Wndings in thistumor (Fig. 6). Partial, but convincing GFAP staining

was also noted, especially nearby vessels. The MIB-1labeling index was 20%.

Electron microscopy

Case 1

Scant dense core granules were identiWed within bothcytoplasm and cellular processes, some being seen incells with abundant Wlaments (Fig. 7). Grouped,aligned microtubules were identiWed within the largecells with abundant cytoplasm and cell processes.These same cells often also contained numerous inter-mediate Wlaments labeled with GFAP by the immuno-gold technique. The small cells were undiVerentiated inappearance with high nuclear/cytoplasmic ratios andonly scant organelles.

Cases 2 and 3

The cytoplasm of the epithelioid cells containedstructures consistent with neurosecretory granules, but

Fig. 4 Case 2. Eosinophilic cells arranged in “Zellballen-like” nests (a) and ribbons (b) were distinctive features of the neoplasm, as was Indian Wling (c). A high-power view demonstrates nests of cells with eccentric, eosinophilic cytoplasm (d). Nearby zones of necrosis, cohesive cells formed perivascular pseudo-papillae (e). A sarcomatous element is seen abutting the predominant nested compo-nent of the tumor (f)

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artifacts related to routine processing and paraYnembedding obscured their morphology. The large cellsin case 2 demonstrated abundant intracytoplasmic Wla-ments immunogold labeled for GFAP. The sarcoma-tous component was not ultrastructurally evaluated.

Discussion

Malignant primary central nervous system tumors ofadulthood exhibiting neuronal and glial diVerentiationare rare and poorly characterized. Historically, malig-nant tumors featuring primarily a neuronal phenotypewere termed “cerebral neuroblastomas” [3, 22]. Mono-morphous, rather small cell examples were lumpedinto the then nebulous category of primitive neuro-ectodermal tumor (PNET) [42]. With the advent ofimmunohistochemistry, it became clear that, on occa-sion, these neoplasms, whether occurring in the cere-bellum [36] or cerebrum, clearly showed bidirectionalor glioneuronal diVerentiation [2, 11, 15, 49].

Aside from these malignant, primarily pediatriccases, several other neoplasms occurring in adults and

exhibiting glioneuronal features have been describedin recent years [34]. These include benign or low-gradelesions e.g., “papillary glioneuronal tumor” [30],“rosette-forming glioneuronal tumor of the fourth ven-tricle” [31], and ependymomas with neuropil islands[16]. Malignant examples e.g., “glioneuronal tumorwith neuropil-like islands” [5, 19, 27, 50] are less oftenencountered. The Wrst three neoplasms are morpho-logically unique, whereas the fourth is regarded as anaggressive neoplasm resembling an inWltrating astrocy-toma with the addition of the obvious neuropil islandsthat lend the tumor its name. An abundance of litera-ture has accumulated describing mixed glioneuronaltumors in which, with few exceptions [12, 24], themalignant component is phenotypically glial. In theWHO classiWcation a small provision has been madefor gangliogliomas with grade IV (glioblastoma)changes in the glial component [38]. There have beenseveral reports in which these tumors have been vari-ously classiWed as anaplastic ganglioglioma [13, 37, 46],glioblastomatous transformation of ganglioglioma [20,28], and ganglion cell-giant cell glioblastoma [10].There have been rare reports of tumors interpreted as

Fig. 5 Case 2. Immunohisto-chemical Wndings. The cells showed strong, diVuse expres-sion of GFAP and to a lesser extent synaptophysin and Neu-N. NeuroWlament pro-tein and chromogranin stain-ing was limited to rare isolated cells. Convincing expression of cytokeratin (CAM 5.2) was a focal Wnding

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“anaplastic transformation of dysembryoplastic neuro-epithelial tumor” [18, 43], a phenomenon of which weare skeptical.

Russell and Rubinstein [44] reported malignantchange in gangliogliomas to occur in 10% of theircases, with the caveat that the frequency of this changemay be greater than expected in their series due toreferral bias. Although some of these reports clearlydocument glial malignancy in an otherwise typical gan-glion cell tumor, others, especially those reported priorto the widespread use of immunohistochemistry, arethought to represent either inWltrating gliomas withentrapped normal neurons or tumoral giant cells super-Wcially resembling ganglion forms [8, 26].

Of interest, there was one report of four patientswith tumors that resembled low grade inWltratinggliomas, but expressed MAP-2 and synaptophysin inaddition to GFAP by immunohistochemistry [52]. Infollow-up correspondence [53], it was documented thatall four tumors underwent radiologic/histopathologicprogression.

More recently, the sizeable series of Varlet et al. [51]and a few case reports [47] brought attention to focal

immunohistochemical evidence of neuronal diVerenti-ation in otherwise typical glial tumors, usually in theform of neuroWlament protein staining. The tumorswere reported as having distinct clinicopathologic fea-tures, including: (a) gross, although not microscopiccircumscription that might facilitate resection and, (b)a paradoxic increased rate of leptomeningeal spread[51]. Two of our three cases showed some similarities.However, the tumor in case 1, despite showing con-vincing immunophenotypic and ultrastructural evi-dence of neuronal diVerentiation, lacked convincingexpression of NFP; and, despite the surgical impressionof discrete margins, the extensively resected tumor ofcase 2 followed an aggressive course.

Although the Wndings of Varlet et al. [51] are inter-esting and thought provoking, we are reluctant to placea neoplasm into the glioneuronal category based solelyon the results of a single immunostain labeling rarecells. It is our practice to require multiple immunomar-kers and perhaps electron microscopy studies to sup-port the diagnosis. The issue of classifying glioneuronaltumors is complicated by the existence of tumors thatexhibit positivity for neuronal markers but have not

Fig. 6 Case 3. InWltrating eosinophilic cells with ample cytoplasm (upper left). Pseudo-papillae formation was a prominent feature (upper right). Individual neoplastic cells expressed neuroWlament protein (NF), synaptophysin, and GFAP. p53 was strongly expressed in many cells, including giant forms

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734 Acta Neuropathol (2006) 112:727–737

been generally regarded or classiWed as glioneuronal innature [6]. Examples include: (a) oligodendrogliomaswith synaptophysin-positivity and rare examples show-ing rosette formation [41], (b) pleomorphic xanthoas-trocytomas showing synaptophysin and neuroWlamentprotein staining or ultrastructural features of neuronalT

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Fig. 7 Case 1. Electron microscopy. Large cells with prominentprocesses and cytoplasmic vacuoles correspond to those compris-ing the rim surrounding nodules of small cells with scant cyto-plasm and few organelles (a). Occasional dense-core granuleswere seen within process of the large cells (b) as were cytoplasmicbundles of Wlaments (c). Dense core granules were also identiWedin large cells containing intermediate Wlaments (d). The latterdemonstrated GFAP staining by the immunogold technique (e)

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Acta Neuropathol (2006) 112:727–737 735

diVerentiation [17, 21], as well as (c) subependymalgiant cell astrocytomas with neuronal cytoskeletal pro-teins and neurotransmitter substance immunostainingand/or ultrastructural neuronal features [21, 33]. Con-versely, there are distinct neuronal tumors that oftenshow partial expression of glial markers but are stillconsidered neuronal in nature, for example, neuro-cytoma [7, 25]. Interestingly, the capacity of neuro-cytomas to undergo glial diVerentiation has also beendemonstrated in vitro [23]. Thus, we are inclined to rec-ommend combinations of more stringent criteria indica-tive of a neuronal phenotype. These include not onlyconventional morphology and batteries of immunohisto-chemical markers but ultrastructural studies as well.

The occurrence of supratentorial neoplasms with aglioneuronal phenotype should not be surprising, andmay be explained by the existence of putative precursorsor multipotential stem cells in several regions of thebrain, including the subventricular zone and subcorticalwhite matter. Such cells are capable of diVerentiationalong both glial and neuronal cell lines [14, 45, 48].

Given the multicentric imaging Wndings, theadvanced age of patients 1 and 2, and the epithelialmorphology most accentuated in case 2, a Wnal consid-eration must be given to the possibility of metastaticneuroendocrine carcinoma. In case 1 one of the mostinteresting morphologic Wndings was a considerablerim of cells with generous eosinophilic cytoplasm. It isknown that reactive astrocytes may border the inter-face of metastases and even demonstrate remarkablereactive changes simulating neoplasia. However, we dointerpret these cells in case 1 to be neoplastic becauseof the degree of clustering, cytologic atypia, lack ofconspicuous processes, and proliferation as demon-strated by MIB-1. Furthermore, at the ultrastructurallevel, the demonstration of intermediate Wlamentslabeling for GFAP and dense core granules was limitedto these larger cells.

Focal cytokeratin CAM 5.2 expression was noted incases 2 and 3. This should not be surprising given thatmRNA for cytokeratins has been detected in astrocytictumor cells [9], and in fact even reactive astrocytes arefrequently immunopositive for broad-spectrum kera-tins. Even though cytokeratin CAM 5.2 is relativelymore speciWc for metastatic carcinoma, some primaryglial tumors might react with it in rare instances [9, 39],especially adenoid glioblastoma [40]. Conversely, cases2 and 3 did demonstrate convincing GFAP expression,which, although reported [39], tends to be unusual incarcinomas other than of myoepithelial origin and gen-erally limited to rare cells.

The lack of vascular proliferation and pseudopallisad-ing necrosis as well as the unusual epithelioid cytology

and pseudopapillary architecture exempliWed in cases2 and 3, although making necessary to exclude meta-static carcinoma as a possibility, may actually representmorphologic clues suggesting neuronal diVerentiationon a malignant neoplasm that has convincing glial fea-tures, and should probably trigger the incorporation ofneuronal immunostains in the diagnostic histopatho-logic evaluation.

In conclusion, we describe three unusual, malignantglioneuronal neoplasms, two occurring in older adultsand following an aggressive course. Given their lightmicroscopic, immunohistochemical and/or ultrastruc-tural features indicating a glial and neuronal pheno-type, we consider them somewhat unique and notreadily classiWed. They certainly do not Wt well into thecategory of supratentorial primitive neuroectodermaltumor which are generally round blue cell tumors withneuroblastic or advanced neuronal diVerentiation,features lacking in the morphologic distinct tumorsdescribed herein. However, clear, accepted minimal“gold standards” of neuronal diVerentiation have yetto be agreed upon. Further studies expanding the spec-trum of immunomarkers over those applied by Varletet al. [51] and applying ultrastructure will probablyprove to be useful. These three cases fulWll the criteriaof glioneuronal tumor in every aspect and may repre-sent standards against which other cases can be com-pared.

Acknowledgments The authors would like to thank MargaretSpringett for assistance with electron microscopy and immunola-beling, as well as Drs. John Averyt, Jorge Pellegrini, and JeVreyRoberts.

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