Clarifying the Diffuse Gliomas An Update on the Morphologic ...

Am J Clin Pathol 2005;124:755-768 755755 DOI: 10.1309/6JNX4PA60TQ5U5VG 755

© American Society for Clinical Pathology

Anatomic Pathology / GLIOMA MARKERS

Clarifying the Diffuse Gliomas

An Update on the Morphologic Features and Markers That Discriminate Oligodendroglioma From Astrocytoma

Meenakshi Gupta, MD, Azita Djalilvand, MD, and Daniel J. Brat, MD, PhD

Key Words: Astrocytoma; Oligodendroglioma; Genetics; Immunohistochemistry; Brain tumor

DOI: 10.1309/6JNX4PA60TQ5U5VG

A b s t r a c t

Diffuse gliomas are the most common brain tumorsand include astrocytomas, oligodendrogliomas, andoligoastrocytomas. Their correct pathologic diagnosisrequires the ability to distinguish astrocytic fromoligodendroglial differentiation in histologic sections, achallenging feat even for the most experiencedneuropathologist. Interobserver variability in thediagnosis of diffuse gliomas has been high owing tosubjective diagnostic criteria, overlapping morphologicfeatures, and variations in training and practice amongpathologists. A select, albeit imperfect, group ofmolecular and immunohistochemical tests are availableto assist in diagnosis of these lesions. Combined loss ofchromosomes 1p and 19q is a genetic signature ofoligodendrogliomas, whereas gains of chromosome 7 inthe setting of intact 1p/19q are more typical ofastrocytomas. Detection of amplified epidermal growthfactor receptor favors the diagnosis of high-gradeastrocytomas over anaplastic oligodendroglioma, whichis especially relevant for small cell astrocytomas.Strong nuclear staining for p53 often reflects TP53mutation and is typical of low-grade astrocytomas. TheOlig family of transcription factors has notdemonstrated their diagnostic usefulness. Diffusegliomas remain a diagnostic challenge, and newmarkers are needed for proper classification anddirected therapies.

Infiltrative, or diffuse, gliomas are the most frequentprimary central nervous system (CNS) tumors and includeastrocytomas, oligodendrogliomas, and mixed oligoastro-cytomas.1-3 Their deserved reputation as devastating dis-eases is due in large part to their widespread invasivenessand their strong tendency toward biologic progression.Diffuse gliomas are nearly impossible to resect completelyand thus far have shown stubborn resistance to convention-al adjuvant therapies. Overall survivals for the diffusegliomas have not improved dramatically during the lastcentury, underscoring the needs for a better understandingof these neoplasms and a fresh approach to their treatment.Among the fundamental shortcomings in neuro-oncologythat need to be addressed to improve clinical care are thecurrent limitations and misconceptions involving thehistopathologic diagnosis of diffuse gliomas.4-6 We discussthe pathologic features of the 2 main subtypes—oligoden-droglioma and astrocytoma—with an emphasis on morpho-logic features and diagnostic markers that can assist thepathologist to distinguish them more reliably.

Histopathologic Features of DiffuseGliomas

The diagnosis of diffuse gliomas still hinges primarily onthe histopathologic analysis of H&E-stained slides.2,3 Ashared property of these tumors is the widespread infiltrationby individual cells through the CNS parenchyma ❚Image 1❚.Infiltrative properties are suggested by magnetic resonanceimaging, which generally shows expansion of the involvedbrain with associated signal abnormalities that are hypointense

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756 Am J Clin Pathol 2005;124:755-768756 DOI: 10.1309/6JNX4PA60TQ5U5VG


Gupta et al / GLIOMA MARKERS

on T1-weighted images and hyperintense on T2 and fluid-attenuated inversion recovery (FLAIR).7,8 Tumors occurthroughout the CNS neuroaxis but most commonly involvethe cerebral hemispheres. Only when these tumors becomehigh grade do they show significant contrast enhancement.9

Compared with astrocytomas, oligodendrogliomas are locatedmore frequently peripherally and generally are well-demarcat-ed and calcified. Nevertheless, the neuroimaging featuresoverlap significantly, and a tissue diagnosis is required toestablish tumor identity.

Histologic sections derived from neurosurgical resectionor stereotactic biopsy reveal a neoplastic population inter-spersed among the network of native neuronal and glialprocesses that comprise the delicate CNS neuropil, leading toarchitectural distortion and variable edema (Image 1). Once aninfiltrating pattern has been established, tumors are subclassi-fied based on their morphologic features as oligoden-droglioma, astrocytoma, or mixed oligoastrocytoma. A gradethen is applied to the infiltrating glioma depending on the cri-teria within each histologic category.10,11 Unfortunately,numerous studies have confirmed the suspicions of patholo-gists and clinicians that this method lacks a high degree ofreproducibility.6,12 Most concerning is the inability to reliablydistinguish oligodendroglial from astrocytic differentiation innonclassic or ambiguous gliomas, which account for a surpris-ingly high percentage of tumors. This is especially true among

low- and intermediate-grade gliomas. In one study, concor-dance among 4 experienced neuropathologists was only 50%in classifying and grading oligodendroglioma, astrocytoma,and oligoastrocytoma.13 Agreement improved modestly to70% after the pathologists reviewed the cases together anddiscussed diagnostic criteria to facilitate consistency. Otherstudies have demonstrated poor intraobserver reproducibilityand an unacceptably wide variation in the diagnostic frequen-cy of astrocytoma, oligodendroglioma, and mixed oligoastro-cytoma, even among the most experienced of neuropatholo-gists.14,15 Clearly, there is a problem.

Diagnostic inadequacies have launched numerous inves-tigations of potential genetic and immunohistochemical mark-ers that might distinguish more reliably the 2 main subtypes ofgliomas, thereby simplifying the task of pathologists andrestoring confidence among clinicians. Proper classification ismore than an academic exercise because prognosis and thera-py are guided by diagnosis. Oligodendrogliomas generallyhave slower growth rates and are associated with a better prog-nosis than astrocytomas when compared grade for grade, andthe presence of an oligodendroglioma component within aninfiltrative glioma usually predicts longer survival.16,17

Reproducible classification will become even more criticalwith the emergence of therapeutic regimens that are directedat specific neoplastic entities and molecular subtypes. Forexample, specific chemotherapies have shown effectiveness

A B C

❚Image 1❚ Neuroimaging and histopathologic features of diffuse gliomas. A and B, The normal brain is relatively dark on fluid-attenuated inversion recovery (FLAIR) (B, right temporal lobe, left side of image [arrow]) and T2-weighted magnetic resonanceimaging (MRI), and histologic sections (A) show homogeneous white matter with linear arrays of neuronal processes andregularly dispersed oligodendrocytes. B and C, Involvement of the brain (left temporal lobe) by an infiltrating glioma leads to itsexpansion and FLAIR and T2 signal hyperintensity (B, arrow) on MRI, reflecting vasogenic edema that arises in response toinvading tumor cells. C, Histologic sections show individual glioma cells (with astrocytic differentiation in this case) infiltratingamong the native neuronal processes and glial cell bodies of the white matter, leading to disruption of neuropil architecture,slight edema, and hypercellularity (H&E, ×400).

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against a subset of oligodendrogliomas with chromosomallosses of 1p and 19q.18,19 Other therapies directed at the over-expressed epidermal growth factor receptor (EGFR) in high-grade astrocytomas are finding clinical application.20,21 Properclassification will be necessary to direct tumors for moleculartesting and to provide patients with optimal care.

Oligodendroglioma

Oligodendrogliomas account for 4% of primary braintumors and 12% to 20% of infiltrating gliomas.1,3 Their inci-dence seems to have increased during the past 2 decades, butit remains unclear whether this increase reflects a diagnostictrend among pathologists or a meaningful epidemiologic shift.

In one of the original classifications of glial neoplasmsby Bailey and Cushing22 and later in the article“Oligodendrogliomas of the Brain” by Bailey and Bucy,23 aunique cerebral hemispheric tumor of adults with histologicfeatures unlike the other gliomas was described.22,23 In thesedescriptions, oligodendrogliomas contained cells with nucleithat “are almost all perfectly round and of a fairly constantsize”23(p746) and are “surrounded by a ring of cytoplasmwhich stains very feebly”22(p88); they have a “network of finecapillaries”23(p746) and “are prone to become calcified.”22(p88)

Our current concept of oligodendroglioma retains most of

these essential features, yet there is an impression amongsome authorities that the diagnostic criteria of present daypathologists have expanded to gradually encompass noncon-ventional morphologic features.5,11,13,24

Restricting the diagnosis of oligodendroglioma to mor-phologically classic lesions that fulfill the original descrip-tions22,23 likely would eliminate at least some of the currentconfusion in neuro-oncology. Nevertheless, these early arti-cles also foreshadowed some of the real diagnostic dilemmasthat we continue to encounter: “There are also many cellswhich appear to be transitions between gigantic oligoden-droglia and astrocytes. It is impossible definitely to classifythem as belonging in either group.”23(p748) “Practically everystage of gradual transition from typical oligodendroglia to typ-ical astrocytes can be found.”23(p748)

Current diagnosis of oligodendroglioma requires the iden-tification of infiltrating glioma cells that have round, regular,and monotonous nuclei, with little cell-to-cell variability ❚Image

2❚.11,24 As most pathologists are aware, tumor cell cytoplasmtends to swell during routine formalin fixation and paraffinembedding, resulting in cells with well-defined cell mem-branes, cytoplasm clearing, and a central spherical nucleus—acombination that gives rise to the classic “fried egg” cell (alsodescribed as “honeycomb” and “woody plant” histologic fea-tures by Bailey and Bucy23). It should be emphasized that peri-nuclear cytoplasmic clearing is helpful for establishing the

Anatomic Pathology / REVIEW ARTICLE

A B C D

❚Image 2❚ Histopathologic features of oligodendroglioma. A, Tumor cells of oligodendroglioma cells are monotonous in histologicsections owing to the similarity of their round, regular nuclei and the presence of perinuclear cytoplasmic clearing (halos). Tumorcells have a tendency to infiltrate the cerebral cortex, where they often aggregate around neuronal cell bodies (arrow;perineuronal satellitosis) (H&E, ×400). B, Oligodendroglioma can show a purely infiltrating pattern or can have a back-to-backpattern of growth (“honeycomb” histologic features) (H&E, ×400). C, Microcalcifications are typical of but not specific foroligodendroglioma and reflect their relatively slow growth (arrow) (H&E, ×400). D, The delicate vascular pattern associated witholigodendroglioma is seen most often within involved cerebral cortex and has been referred to as a “chicken-wire” pattern(arrow) (H&E, ×400).

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diagnosis of oligodendroglioma but is not a feature that isrequired, sufficient, or constant. Not all tumor cells in an oligo-dendroglioma will show cytoplasmic clearing; in contrast, occa-sional tumors with classic astrocytic differentiation might con-tain perinuclear halos owing to fixation artifact. More importantfor proper classification are the nuclei: oligodendrogliomashave round, regular, generally bland nuclei with open or delicatechromatin, whereas astrocytoma nuclei are enlarged, elongated,hyperchromatic, and irregular in shape and hue.

Quite literally, “oligodendroglia” means “glia with fewprocesses.” In tissue sections and especially on smear prepara-tions, there is a paucity of glial processes emerging fromoligodendrogliomas compared with the long, finely fibrillarprocesses that are in abundance in cytologic and histologicpreparations of most astrocytic neoplasms ❚Image 3❚.25 Forreasons that are not entirely clear, a delicate, branching capil-lary network with a “chicken-wire” appearance is noted morefrequently in oligodendrogliomas (Image 2). Other commonbut nonspecific features include cortical involvement, perinu-clear satellitosis by tumor cells, microcalcifications, andmicrocysts filled with mucin.

Grading systems typically have divided oligoden-drogliomas into 2, 3, or 4 grades depending on cellularity,cytologic atypia, mitotic activity, vascular proliferation, andnecrosis.11,24,26-28 Criteria for grading are not as clearlydefined for oligodendrogliomas as they are for astrocytomas.The current World Health Organization (WHO) Classificationrecognizes 2 grades: oligodendroglioma (grade II) andanaplastic oligodendrogliomas (grade III).24,27

Grade II tumors vary from low to moderate cellularity.These tumors have a tendency to involve the cerebral cortex,and, as they progress, they often grow in a distinctly nodularpattern ❚Image 4❚. Nodular growth is compatible with a gradeII lesion but might represent a transition to a higher grade,especially when the nodules coalesce into regions of confluenthypercellularity. Grade II oligodendrogliomas can show occa-sional mitotic figures and cytologic atypia, but marked mitot-ic activity, microvascular proliferation, or necrosis is consis-tent with a WHO grade III, anaplastic oligodendroglioma(Image 4). A recent study of prognostic features in oligoden-droglioma identified endothelial hypertrophy, necrosis, and 6or more mitotic figures per high-power field as significant uni-variate markers of poor outcome, providing a solid frameworkfor establishing the diagnosis of anaplastic oligodendroglioma(grade III).29 Previous multivariate analyses have suggestedthat necrosis is the single most important feature of aggressiveclinical behavior.30,31 Pseudopalisading of tumor cells aroundnecrosis is an acceptable finding in anaplastic oligoden-droglioma and does not suggest a diagnosis of glioblastoma(Image 4).

Infiltrating “Diffuse” Astrocytomas

Much like oligodendrogliomas, infiltrative or diffuseastrocytomas represent a spectrum ranging from low grade tohighly malignant. Although numerous grading systems havebeen used, the current WHO system uses a 3-tiered scheme

A B

❚Image 3❚ Cytologic features of oligodendroglioma and astrocytoma. A, On smear preparations, oligodendrogliomas display apaucity of fibrillar glial processes and instead show tumor cells embedded within a delicate neuropil matrix. The nuclei are roundand have delicate or open chromatin (H&E, ×1,000). B, Astrocytomas, in contrast, have an abundance of long, fine to coarsefibrillar processes that emerge from individual tumor cell bodies. A variable amount of pink cytoplasm can be noted in the cellbodies of most astrocytoma cells, and their nuclei are more irregular, elongated, and hyperchromatic (H&E, ×1,000).

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that spans from grade II to grade IV (glioblastoma).10,32-37

Infiltrative astrocytomas are more common than oligoden-drogliomas and, when all grades are considered, account forone third of all primary brain tumors and 70% to 75% of dif-fuse gliomas.1,10 Astrocytomas have a tendency to involve sub-cortical regions and typically are centered in the white matter.

The diagnosis of infiltrative astrocytoma (WHO grade II)is applied when individual tumor cells showing astrocytic dif-ferentiation are seen invading CNS parenchyma with a lowdegree of cellularity and proliferative activity (Image 1),❚Image 5❚, and ❚Image 6❚.35 Astrocytic differentiation is bestdetermined morphologically by the presence of nuclei that areelongated, hyperchromatic, and irregular, having angulatedand distorted contours (Images 3 and 5). If oligoden-drogliomas have nuclei shaped like Florida oranges, thenthose of astrocytomas are akin to Idaho potatoes. In addition,these nuclei generally lack prominent nucleoli and perinuclearhalos and have an ominous dark purple or black coloration fol-lowing H&E staining. Cytologic preparations are extremelyhelpful for detecting the nuclear features and the fibrillarity ofastrocytic neoplasms (Image 3). Some astrocytomas, such asgranular cell, giant cell, gliosarcoma, and gemistocytic sub-types, contain an abundance of large cells with prominent pinkcytoplasm and are rarely confused with oligodendrogliomas(Image 5).38-41 More problematic are fibrillary, protoplasmic,and small cell variants, which are composed of smaller cellsthat often have only minimal perinuclear fibrillarity and,therefore, can demonstrate considerable diagnostic overlapwith oligodendroglioma.42,43

Many of the previous grading systems for the diffuseastrocytomas did not allow any mitotic figures to be present ina grade II tumor, and the finding of a single mitotic figure wassufficient to establish the diagnosis of anaplastic astrocytoma(AA; grade III). Clinicopathologic studies have demonstratedthat astrocytomas with 0 or 1 mitosis have similar clinicalbehaviors, and, therefore, current criteria for grade II astrocy-tomas allow 0 or 1 mitotic figures, but generally not more.44

AA (WHO grade III) has higher cellularity, a greaterdegree of nuclear pleomorphism and atypia, and increasedproliferation. Mitotic activity (>1 mitosis) should be identifiedto apply the diagnosis of AA.36 The number of mitoses identi-fied necessarily depends on sample size, number of tissue lev-els examined, and intensity of the search. One study suggest-ed that fifty 400× fields should be evaluated to ensure morethan 90% sensitivity in the identification of mitoses.45

Glioblastoma (WHO grade IV) is the highest grade form ofinfiltrating astrocytoma.37 In addition to the histopathologicfindings of AA, either microvascular hyperplasia or necrosis,often with pseudopalisading, is required for the diagnosis ofglioblastoma (Image 5). Glioblastomas with microvascularhyperplasia but no necrosis have been shown to have behaviorsimilar to that of glioblastomas with necrosis.34,46

Oligoastrocytomas

Oligoastrocytomas contain distinct regions of oligoden-droglial and astrocytic differentiation and account for 5% to


A B

❚Image 4❚ Tumor progression in oligodendroglioma. A, Oligodendrogliomas often progress with a nodular pattern of growthwithin the cerebral cortex (arrow). Nodules contain a higher cell density, and individual cells are more proliferative. Nodulargrowth is compatible with grade II oligodendroglioma but might indicate that the tumor is in the process of progressing to ahigher grade (H&E, ×100). B, Anaplastic oligodendroglioma (grade III) is characterized by hypercellularity, foci of necrosis (arrow),microvascular hyperplasia, and increased mitotic activity (H&E, ×200).

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10% of infiltrative gliomas.47,48 They may be biphasic, withthe 2 components separate, or intermingled, with the 2 neo-plastic cell types in proximity.

The minimal percentage of each component required forthe diagnosis of a mixed glioma has been debated, resulting inhighly variable diagnostic criteria and poor interobserver repro-ducibility for this group of neoplasms. Perhaps more problem-atic is the tendency for pathologists to “dump” diagnosticallychallenging infiltrating gliomas into the wastebasket of the

mixed oligoastrocytoma category. It should be emphasizedthat a mixed oligoastrocytoma is not equivalent to a morpho-logically ambiguous glioma, but rather refers to a tumor con-taining cells with 2 distinct histologic patterns. One studysuggested that a single 100× field filled with an oligoden-droglioma component could be used as a threshold for thediagnosis of mixed oligoastrocytomas because this criteri-on identifies a subset with a better prognosis than astrocy-toma and results in improved interobserver concordance.13

A B

C D

❚Image 5❚ Histopathologic features of infiltrative astrocytomas. A, Many variants of infiltrative astrocytoma exist, but theprototype is the fibrillary astrocytoma, which contains scant amounts of perinuclear cytoplasm, a high degree of glial fibrillarity,and irregular “Idaho potato” nuclei (H&E, ×600). B, Gemistocytic astrocytomas are composed of individual tumor cells withlarge amounts of eosinophilic cytoplasm and eccentrically placed nuclei (H&E, ×600). These and other astrocytic variants withabundant cytoplasm are rarely confused with oligodendroglioma. C, Small cell astrocytomas are a biologically aggressive variantthat often is confused with anaplastic oligodendroglioma owing to its hypercellularity, low degree of fibrillarity, and deceptivelybland nuclei (H&E, ×600). D, Regardless of the subtype of astrocytoma morphologic features, progression to glioblastoma ischaracterized by necrosis (with pseudopalisading in this case, arrow) or microvascular proliferation (arrowhead) (H&E, ×400).

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Low-grade oligoastrocytomas (WHO grade II) can containoccasional mitotic figures, low to moderate cellularity, and mildto moderate cytologic atypia.47 The WHO criteria for anaplasticoligoastrocytoma (WHO grade III) are not well defined but sug-gest that “histological features of anaplasia” should be pres-ent.48 This list includes nuclear atypia, cellular pleomorphism,high cellularity, high mitotic activity, microvascular prolifera-tion, and necrosis. Anaplasia can be present in the astrocyticcomponent, the oligodendroglial component, or both.

Frozen Section Diagnosis of DiffuseGliomas

Distinguishing oligodendroglioma from astrocytoma ischallenging enough on permanent sections. The difficulty iscompounded at the time of frozen section because this prepa-ration does not optimally reveal the histologic features thattypically are used to separate these entities.49-53 The perinu-clear halos, delicate chromatin patterns, and nuclear regulari-ty of oligodendrogliomas are not as evident in frozen sections❚Image 7❚. Ice crystal artifact resulting from CNS edema inthese lesions can further degrade the morphologic features. Itshould be remembered that making the distinction betweenastrocytic and oligodendroglial morphologic features at thetime of frozen section is not usually essential for intraopera-tive patient management. The diagnosis of infiltrating glialneoplasm together with a general degree of histologic differ-entiation (well, moderately, or poorly differentiated) or grade(low, intermediate, or high) is the best that can be communi-cated in many, perhaps most, circumstances.50-53

The process of freezing brain tissue during the frozen sec-tion procedure introduces artifacts that remain in permanentsections and limits their interpretation. Most notably, nucleiappear more hyperchromatic and atypical in previously frozentissue; perinuclear halos of oligodendroglioma are not as evi-dent; and the cytologic resolution is lower (Image 7).49 Takentogether, these changes impart an “astrocytic” appearance tootherwise classic oligodendrogliomas. Therefore, it is alwaysrecommended to submit tissue for permanent sections that hasnot been frozen. If it is not clear that additional tissue will beavailable for permanent sections, it is prudent to freeze onlyhalf of the tissue submitted at frozen section.49,54

Oligodendroglial and Astrocytic Markers

Immunohistochemical Analysis

Because distinguishing among diffuse gliomas is notalways straightforward based on morphologic features alone,reliable immunohistochemical or genetic markers of differen-tiation have been highly sought. Many studies have been guid-ed by the principle that specific proteins must be expresseddifferentially by glial cells with distinct developmental pro-grams and cellular function. The intermediate filament glialfibrillary acidic protein (GFAP) is a marker of glial differenti-ation that is expressed consistently by resting astrocytes andgreatly overexpressed in reactive astrocytosis. GFAP has beeninvaluable as a marker of glial differentiation in neoplasmsinvolving the CNS. Led by the mistaken assumption thatoligodendrocytes do not express GFAP, some have suggested


A B

❚Image 6❚ Immunohistochemical stains are of limited usefulness in the classification of diffuse gliomas. A and B, Strong nuclearp53 staining (B, arrow) may favor the diagnosis of an astrocytoma in morphologically ambiguous infiltrating gliomas becauseTP53 mutations are much more frequent in astrocytomas than oligodendrogliomas (A, H&E, ×1,000; B, p53, ×1,000).

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that GFAP might be a marker that could distinguish astrocy-tomas from oligodendrogliomas. However, immunohisto-chemical and ultrastructural studies have concluded firmlythat oligodendrogliomas also express GFAP and that somemorphologic subtypes, such as gliofibrillary oligodendro-cytes and minigemistocytes, express high levels.55-57 WhetherGFAP-positive oligodendroglioma cells represent a transi-tional form between an oligodendrocytic and astrocytic dif-ferentiation is debated. Nevertheless, GFAP is not currentlyconsidered a useful marker for distinguishing among infiltrat-ing gliomas.58

Alterations of p53 are more common in astrocytomasthan in oligodendrogliomas, suggesting that it might be a dis-criminating marker.10,59,60 The most frequent disruptions areinactivating point mutations of the TP53 gene, which occur in50% to 60% of grade II astrocytomas but in only 5% to 10%

of grade II oligodendrogliomas. TP53 mutations on 1 alleleusually are accompanied by loss of heterozygosity (LOH) onchromosome 17p involving the other allele, leading to thecomplete loss of normal TP53 and production of only mutantp53 protein. This mutated form is altered physically in a man-ner that reduces its degradation, which makes it immunohisto-chemically detectable in the nucleus of tumor cells. Stainingfor p53 can be diagnostically useful in some cases. For exam-ple, the presence of strong, nuclear p53 staining could indicatean underlying TP53 mutation and favor the diagnosis of astro-cytoma (Image 6). However, mechanisms other than TP53mutation can cause p53 protein overexpression, and p53immunoreactivity does not correlate perfectly with the pres-ence of TP53 mutations (concordance of 65%-75%).61,62

Therefore, a positive p53 immunostain requires interpretationin the context of other clinical and pathologic data.

A B

C ❚Image 7❚ Frozen section artifacts in diffuse gliomas. A, Frozensections of brain tissue involved by an infiltrating glioma oftenshow ice crystal artifact, which distorts the architecture andcytologic features of the neoplasm and makes the definitivediagnosis of astrocytoma or oligodendroglioma difficult orimpossible. Tumor cell nuclei often appear dark and irregular,and the morphologic features that typically are relied on in thediagnosis of oligodendroglioma, such as perinuclear halos andnuclear regularity, are not evident on frozen sections (H&E,×600). Permanent sections of an oligodendroglioma thatpreviously was frozen for intraoperative consultation (B) andadjacent tissue that was not previously frozen (C) demonstratesthat the histologic and cytologic features of the tumor areconsiderably different (B, H&E, ×600; C, H&E, ×600).Previously frozen tissue has nuclei that are irregular andhyperchromatic (features that are more typical of astrocytoma),whereas unfrozen tissue shows the round nuclei, delicatechromatin, and perinuclear halos typical of oligodendroglioma.

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A wide array of proteins expressed by nonneoplasticoligodendrocytes have been studied for their usefulness asmarkers of oligodendrogliomas, including myelin basic pro-tein, proteolipid protein, NG2, myelin-associated glycopro-tein, galactocerebroside, Leu7, and cyclic nucleotide-3'-phos-phatase.63,64 Although the rationale for their study has beensolid, none have proven useful for routine diagnostic work.More recent studies have focused on oligodendrocyte-lineage(Olig) genes, Olig1, Olig2, and Olig3, which encode for basichelix-loop-helix transcription factors that regulate neural-derived cell proliferation and differentiation.65 Of these, Olig1and Olig2 show the highest brain specificity. Both areexpressed in the periventricular zone during embryogenesisand are essential for proper oligodendrocyte maturation, butalso have more general roles in nervous system development.

Initial in situ hybridization studies demonstrated highlyspecific Olig1 and Olig2 expression in nonneoplastic oligoden-drocytes and oligodendroglioma cells, with only weak or absentOlig expression in astrocytomas.66,67 Mixed oligoastrocytomaswere reported as having expression limited to the oligoden-droglial component.66 Follow-up study using semiquantitativereverse transcriptase–polymerase chain reaction for Oligexpression have been less promising. Increased Olig expressionindeed was identified in glial neoplasms, but levels did not dif-fer substantially between astrocytomas and oligoden-drogliomas.68-70 Subsequent immunohistochemical studieshave similarly demonstrated that Olig1 and Olig2 are expressed

by glial neoplasms, with nuclear patterns that would be expect-ed for transcription factors.69,71 Immunoreactivity generally ismore intense in oligodendrogliomas than in astrocytomas, butthe degree of staining overlaps to an extent that precludes theiruse as discriminating markers by themselves. Correlation ofOlig and GFAP staining in diffuse gliomas has suggested thatthe Olig+/GFAP– immunophenotype identifies a morphologi-cally “pure” form of oligodendroglioma.72,73 Nevertheless, theabundance of evidence indicates that Olig expression has limit-ed usefulness in the subclassification of infiltrating gliomas.

Genetic Markers

The last 2 decades of brain tumor research have demon-strated that each histologic category of infiltrating glioma con-tains multiple distinct molecular genetic subsets.10,59,74,75

Some genetic alterations have been exploited in the diagnos-tic arena and are used to assist with pathologic classificationand provide independent prognostic data. Each technique forgenetic testing has its own set of advantages and disadvan-tages. Most often used are LOH (traditional gel-based assaysor capillary electrophoresis), fluorescence in situ hybridiza-tion (FISH), and comparative genomic hybridization(CGH)18,19,76,77 ❚Image 8❚ and ❚Image 9❚.

These tests demonstrate excellent concordance (73%-99%), and the choice depends largely on the preferences of thepathologist, department, and institution.77 LOH analysis andFISH have the highest concordance (>93%) and are used most


A B C

19p1319q13

1p321q42

CEP7EGFR

❚Image 8❚ Genetic analysis of infiltrating gliomas can aid in diagnosis. A, Oligodendrogliomas typically show loss ofchromosomes 1p and 19q by fluorescence in situ hybridization (FISH; 2 green 19p13 signals and 1 red 19q13 signal in mostnuclei indicate a loss of 19q in this oligodendroglioma). B, Astrocytic neoplasms normally display intact 1p and 19q (2 green1p32 and 2 red 1q42 signals in most nuclei). C, Amplification analysis of epidermal growth factor receptor (EGFR) by FISHsometimes is useful for differentiating high-grade astrocytomas (especially small cell astrocytoma) from anaplasticoligodendrogliomas. The centromere for chromosome 7 is green (chromosome enumeration probe 7) and EGFR is red;numerous red signals are demonstrated within most astrocytoma nuclei. (FISH images courtesy of Arie Perry, MD.)

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frequently for diagnostic purposes on tissue derived from his-tologic sections.77-79 FISH has some advantages from apathologist’s perspective (Image 8): (1) Analysis is based onthe morphologic identification of genetic alterations withintumor cell nuclei. (2) Nonneoplastic cells (positive controls)are almost always present within the tissue sections examined(eg, normal endothelial cells, neurons). (3) FISH does notrequire microdissection of normal and tumor tissue beforeanalysis. (4) Genetic gains and losses in infiltrative tumorswith a low ratio of neoplastic/normal cells can be analyzed byFISH, whereas these alterations might not be detected bypolymerase chain reaction–based analysis (LOH studies)owing to overwhelming amounts of normal DNA. One majordisadvantage of FISH is that it can be highly labor-intensive,and automation has not yet reached all of its applications.

Some genetic alterations occur in astrocytic and oligo-dendroglial tumors, generally with increasing frequency athigher grades, and, therefore, are not useful markers for dis-criminating histologic subtypes.74,75,80 These alterations,including loss of 9p21 (p16/CDKN2A) and losses involvingchromosome 10 (PTEN/DMBT1), might provide prognosticinformation for a given tumor but are not specific to 1 histo-logic pattern and will not be considered further.

1p and 19qOne of the best-recognized molecular signatures among

diffuse gliomas, which also has prognostic significance, is the

combined loss of genetic material from chromosomes 1p and19q in oligodendrogliomas.81-84 19q losses occur in 60% to80% in oligodendrogliomas, but also are detected in approxi-mately 30% to 40% of astrocytomas, indicating that this maybe a shared genetic alteration in gliomagenesis.19,77,85 Losseson the short arm of 1p also are frequent in oligodendrogliomas(50%-80%) but are much more specific because they aredetected in only 10% to 18% of astrocytomas.18,19,77 Almostalways, losses on 1p and 19q coexist in oligodendroglioma,and the combination can be detected in 60% to 80%. In mostcases, the entire arms of the affected chromosome are deleted,allowing reliable detection by LOH, FISH, or CGH (Images 8and 9). Combined loss of 1p and 19q is a strong predictor ofresponse to chemotherapy and overall survival in low- andhigh-grade oligodendrogliomas.18,19 In contrast, oligoden-drogliomas with deletions of p16/CDKN2A (9p21), LOH 10q,or EGFR amplifications, but without loss of 1p and 19q, areassociated with poor overall survival.18,86 Initial studies ofoligodendrogliomas with 1p and 19q deletion showed anincreased responsiveness of these tumors to “PCV”chemotherapy (procarbazine, CCNU [lomustine], vincristine),but it now is evident that this molecular subset is more respon-sive to other therapies, including radiation and temozo-lamide.18,19,87,88

The combined loss of 1p and 19q occurs much less fre-quently in astrocytic tumors (only in 1%-10%). Nevertheless,it should be remembered that the incidence of astrocytomas isnearly 8 times greater than that of oligodendrogliomas.1 Oneissue that continues to be debated is whether genetic loss of 1pand 19q has prognostic significance in nonoligodendroglialtumors including astrocytomas. The majority of studies haveconcluded the following19,78,89: (1) Combined 1p and 19q lossdoes not predict enhanced survival for astrocytomas or mixedoligoastrocytomas. (2) The frequency of 1p and 19q loss is solow that a survival effect cannot be demonstrated. In contrast,Ino et al90 suggested that loss of chromosome 1p might beassociated with longer survival rates in a subset of high-gradeastrocytomas and oligoastrocytomas. Similarly, combined lossof 1p and 19q predicted prolonged survival in 1 study of 97glioblastomas.91,92 Currently, combined 1p and 19q loss isbest viewed as a marker of oligodendroglial differentiationand a marker of therapeutic responsiveness in these tumors.Additional studies will be needed to demonstrate the clinicalusefulness, if any, for 1p and 19q testing in other primarybrain tumors.

Epidermal Growth Factor ReceptorAmplifications of the EGFR gene occur in approximately

40% of glioblastomas and can be detected by FISH, CGH, andpolymerase chain reaction–based tests93,94 (Image 8).Amplifications are much less frequent in lower grade astrocy-tomas and are considered a late genetic event in the progression

N

T

1,600

1,200

800

400

0

1,6001,200

960640320

0

T

N +

+

3,200 3,600 4,000 4,400

LOH 1p

❚Image 9❚ Loss of heterozygosity (LOH) analysis demonstratesloss of 1p by capillary electrophoresis on an infiltrating glioma.LOH analysis can be performed using normal (N) and tumoral(T) tissue that has been microdissected from unstainedhistologic slides after comparison with an H&E-stained section.The polymerase chain reaction products for a microsatellitemarker at 1p36.21 analyzed by capillary electrophoresis show 2allele peaks for tissue derived from for the normal brain (allelicbalance, upper panel). Only 1 allele peak is noted for tissuederived from the infiltrating glioma, indicating allelic loss of1p36.21 (arrow, lower panel).

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of tumors to glioblastoma. Wild-type and mutated forms ofEGFR can be amplified, and, in either case, messenger RNAand cell surface protein levels are increased markedly.

The most common EGFR amplification is a mutated formlacking exons 2 through 7, which results in a truncated cellsurface protein with constitutive tyrosine kinase activity(EGFRvIII).95,96 Therapies directed at the overexpressedEGFR in glioblastomas are finding their way into neuro-oncology practice.20,97 EGFR amplifications are rare in oligo-dendroglial tumors, and analysis of EGFR status has provenuseful for distinguishing high-grade astrocytomas fromanaplastic oligodendrogliomas in some cases.15,98

For the majority of glioblastomas, a correct diagnosis canbe made based on morphologic examination alone, and EGFRtesting is not necessary. However, the recently described “smallcell astrocytoma” is a high-grade astrocytoma that has a greatdeal of morphologic similarity to anaplastic oligodendrogliomaand might require ancillary tests for correct diagnosis.42,43 Bothof these tumors contain a high density of neoplastic cells withscant cytoplasm, uniform and deceptively bland nuclei, and ahigh proliferation index. It is important to recognize small cellastrocytomas because they are biologically aggressive, behav-ing clinically as glioblastomas even in the absence of necrosisand microvascular hyperplasia. Small cell astrocytomas arecharacterized by a high frequency of EGFR amplification andchromosome 10 losses, but they have intact chromosomes 1pand 19q. In contrast, anaplastic oligodendrogliomas show theopposite genetic alterations, having a high frequency of 1p and19q deletions but only rare EGFR amplifications and chromo-some 10 losses. Immunohistochemical analysis for EGFR andEGFRvIII also is possible in high-grade gliomas, but this appli-cation has not been studied as a tool for distinguishing histolog-ic subtypes.99,100

Chromosome 7Alterations on chromosome 7 are common in astrocy-

tomas, and amplification of EGFR at 7p12 is the best knownexample. Although they are frequent in glioblastoma, EGFRamplifications are less common in grades II and III astrocy-tomas, limiting the usefulness of EGFR as a marker for dis-tinguishing lower-grade gliomas. In contrast, classic cytoge-netic and CGH studies of grades II and III astrocytomas havedemonstrated that chromosome 7 gains are among the mostcommon genetic alterations (40%-66%), either as entirechromosome gains (trisomy/polysomy 7) or as gains of 7p or7q alone.101-106 These gains already are present in 40% to50% of grade II astrocytomas, suggesting that it is an earlytumorigenic event, and its presence is associated with short-er survivals.

Chromosome 7 gains are less common in oligoden-drogliomas, occurring as the whole chromosome in roughly10% and as gains of 7p or 7q in 20%.80 When present, they

occur in a subset of oligodendrogliomas that also have chro-mosome 10 losses but not 1p and 19q losses, indicating thatthese gains of 7 might occur in a biologically distinct class oftumors, perhaps more related to astrocytomas.107,108

Chromosome 7 gains also are more frequent in anaplasticoligodendrogliomas (grade III), which might indicate that thisgenetic alteration is a shared feature of malignant progressionin astrocytomas and oligodendrogliomas.80,107,108

Thus, the detection of chromosome 7 gains occasionallymight be helpful for distinguishing low-grade astrocytomasfrom oligodendrogliomas, especially in the absence of 1p and19q losses, but it cannot be used reliably as a discriminatingmarker among high-grade gliomas.

Summary

Even with ancillary tests, the diagnosis of diffuse gliomasremains a challenge, and the search for more specific anddiagnostically useful markers continues. Emerging technolo-gies, like DNA, complementary DNA, and protein microar-rays, are demonstrating potential for brain tumor classificationand for marker discovery. For example, complementary DNAmicroarray analysis coupled with computational algorithmshas been used to successfully classify high-grade gliomas intohistologic groups.109 Moreover, this technique was found tobe superior to histologic classification in predicting the prog-nosis of morphologically ambiguous tumors. Diagnosticmicroarrays containing a limited number of relevant genescould be designed to classify tumors, predict their clinicalbehavior, and rank therapeutic options. Such approaches havealready revealed new markers with the potential for diagnos-tic use that await validation.110 The development of therapiestargeted at the molecular underpinnings of these diseases ulti-mately will require a molecular component in the diagnosisof the diffuse gliomas.

From the Departments of Pathology and Laboratory Medicine andWinship Cancer Institute, Emory University School of Medicine,Atlanta, GA.

Address reprint requests to Dr Brat: Dept of Pathology andLaboratory Medicine, Emory University Hospital, H-176, 1364Clifton Rd NE, Atlanta, GA 30322.

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