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

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1 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 Abstract Diffuse gliomas are the most common brain tumors and include astrocytomas, oligodendrogliomas, and oligoastrocytomas. Their correct pathologic diagnosis requires the ability to distinguish astrocytic from oligodendroglial differentiation in histologic sections, a challenging feat even for the most experienced neuropathologist. Interobserver variability in the diagnosis of diffuse gliomas has been high owing to subjective diagnostic criteria, overlapping morphologic features, and variations in training and practice among pathologists. A select, albeit imperfect, group of molecular and immunohistochemical tests are available to assist in diagnosis of these lesions. Combined loss of chromosomes 1p and 19q is a genetic signature of oligodendrogliomas, whereas gains of chromosome 7 in the setting of intact 1p/19q are more typical of astrocytomas. Detection of amplified epidermal growth factor receptor favors the diagnosis of high-grade astrocytomas over anaplastic oligodendroglioma, which is especially relevant for small cell astrocytomas. Strong nuclear staining for p53 often reflects TP53 mutation and is typical of low-grade astrocytomas. The Olig family of transcription factors has not demonstrated their diagnostic usefulness. Diffuse gliomas remain a diagnostic challenge, and new markers are needed for proper classification and directed therapies. Infiltrative, or diffuse, gliomas are the most frequent primary central nervous system (CNS) tumors and include astrocytomas, oligodendrogliomas, and mixed oligoastrocytomas. 1-3 Their deserved reputation as devastating diseases is due in large part to their widespread invasiveness and their strong tendency toward biologic progression. Diffuse gliomas are nearly impossible to resect completely and thus far have shown stubborn resistance to conventional adjuvant therapies. Overall survivals for the diffuse gliomas have not improved dramatically during the last century, underscoring the needs for a better understanding of these neoplasms and a fresh approach to their treatment. Among the fundamental shortcomings in neuro-oncology that need to be addressed to improve clinical care are the current limitations and misconceptions involving the histopathologic diagnosis of diffuse gliomas. 4-6 We discuss the pathologic features of the 2 main subtypes oligodendroglioma and astrocytoma with an emphasis on morphologic features and diagnostic markers that can assist the pathologist to distinguish them more reliably. Histopathologic Features of Diffuse Gliomas The diagnosis of diffuse gliomas still hinges primarily on the histopathologic analysis of H&E-stained slides. 2,3 A shared property of these tumors is the widespread infiltration by individual cells through the CNS parenchyma Image 1. Infiltrative properties are suggested by magnetic resonance imaging, which generally shows expansion of the involved brain with associated signal abnormalities that are hypointense Downloaded from Am J Clin Pathol 2005;124:

2 Gupta et al / GLIOMA MARKERS A B C Image 1 Neuroimaging and histopathologic features of diffuse gliomas. A and B, The normal brain is relatively dark on fluidattenuated inversion recovery (FLAIR) (B, right temporal lobe, left side of image [arrow]) and T 2 -weighted magnetic resonance imaging (MRI), and histologic sections (A) show homogeneous white matter with linear arrays of neuronal processes and regularly dispersed oligodendrocytes. B and C, Involvement of the brain (left temporal lobe) by an infiltrating glioma leads to its expansion and FLAIR and T 2 signal hyperintensity (B, arrow) on MRI, reflecting vasogenic edema that arises in response to invading tumor cells. C, Histologic sections show individual glioma cells (with astrocytic differentiation in this case) infiltrating among 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). on T 1 -weighted images and hyperintense on T 2 and fluidattenuated inversion recovery (FLAIR). 7,8 Tumors occur throughout the CNS neuroaxis but most commonly involve the cerebral hemispheres. Only when these tumors become high grade do they show significant contrast enhancement. 9 Compared with astrocytomas, oligodendrogliomas are located more frequently peripherally and generally are well-demarcated and calcified. Nevertheless, the neuroimaging features overlap significantly, and a tissue diagnosis is required to establish tumor identity. Histologic sections derived from neurosurgical resection or stereotactic biopsy reveal a neoplastic population interspersed among the network of native neuronal and glial processes that comprise the delicate CNS neuropil, leading to architectural distortion and variable edema (Image 1). Once an infiltrating pattern has been established, tumors are subclassified based on their morphologic features as oligodendroglioma, astrocytoma, or mixed oligoastrocytoma. A grade then is applied to the infiltrating glioma depending on the criteria within each histologic category. 10,11 Unfortunately, numerous studies have confirmed the suspicions of pathologists and clinicians that this method lacks a high degree of reproducibility. 6,12 Most concerning is the inability to reliably distinguish oligodendroglial from astrocytic differentiation in nonclassic or ambiguous gliomas, which account for a surprisingly high percentage of tumors. This is especially true among low- and intermediate-grade gliomas. In one study, concordance among 4 experienced neuropathologists was only 50% in classifying and grading oligodendroglioma, astrocytoma, and oligoastrocytoma. 13 Agreement improved modestly to 70% after the pathologists reviewed the cases together and discussed diagnostic criteria to facilitate consistency. Other studies have demonstrated poor intraobserver reproducibility and an unacceptably wide variation in the diagnostic frequency of astrocytoma, oligodendroglioma, and mixed oligoastrocytoma, even among the most experienced of neuropathologists. 14,15 Clearly, there is a problem. Diagnostic inadequacies have launched numerous investigations of potential genetic and immunohistochemical markers that might distinguish more reliably the 2 main subtypes of gliomas, thereby simplifying the task of pathologists and restoring confidence among clinicians. Proper classification is more than an academic exercise because prognosis and therapy are guided by diagnosis. Oligodendrogliomas generally have slower growth rates and are associated with a better prognosis than astrocytomas when compared grade for grade, and the presence of an oligodendroglioma component within an infiltrative glioma usually predicts longer survival. 16,17 Reproducible classification will become even more critical with the emergence of therapeutic regimens that are directed at specific neoplastic entities and molecular subtypes. For example, specific chemotherapies have shown effectiveness 756 Am J Clin Pathol 2005;124: Downloaded 756 from

3 Anatomic Pathology / REVIEW ARTICLE against a subset of oligodendrogliomas with chromosomal losses of 1p and 19q. 18,19 Other therapies directed at the overexpressed epidermal growth factor receptor (EGFR) in highgrade astrocytomas are finding clinical application. 20,21 Proper classification will be necessary to direct tumors for molecular testing and to provide patients with optimal care. Oligodendroglioma Oligodendrogliomas account for 4% of primary brain tumors and 12% to 20% of infiltrating gliomas. 1,3 Their incidence seems to have increased during the past 2 decades, but it remains unclear whether this increase reflects a diagnostic trend among pathologists or a meaningful epidemiologic shift. In one of the original classifications of glial neoplasms by Bailey and Cushing 22 and later in the article Oligodendrogliomas of the Brain by Bailey and Bucy, 23 a unique cerebral hemispheric tumor of adults with histologic features unlike the other gliomas was described. 22,23 In these descriptions, oligodendrogliomas contained cells with nuclei that are almost all perfectly round and of a fairly constant size 23(p746) and are surrounded by a ring of cytoplasm which stains very feebly 22(p88) ; they have a network of fine capillaries 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 among some authorities that the diagnostic criteria of present day pathologists have expanded to gradually encompass nonconventional morphologic features. 5,11,13,24 Restricting the diagnosis of oligodendroglioma to morphologically classic lesions that fulfill the original descriptions 22,23 likely would eliminate at least some of the current confusion in neuro-oncology. Nevertheless, these early articles also foreshadowed some of the real diagnostic dilemmas that we continue to encounter: There are also many cells which appear to be transitions between gigantic oligodendroglia and astrocytes. It is impossible definitely to classify them as belonging in either group. 23(p748) Practically every stage of gradual transition from typical oligodendroglia to typical astrocytes can be found. 23(p748) Current diagnosis of oligodendroglioma requires the identification 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 cytoplasm tends to swell during routine formalin fixation and paraffin embedding, resulting in cells with well-defined cell membranes, cytoplasm clearing, and a central spherical nucleus a combination that gives rise to the classic fried egg cell (also described as honeycomb and woody plant histologic features by Bailey and Bucy 23 ). It should be emphasized that perinuclear cytoplasmic clearing is helpful for establishing the A B C D Image 2 Histopathologic features of oligodendroglioma. A, Tumor cells of oligodendroglioma cells are monotonous in histologic sections owing to the similarity of their round, regular nuclei and the presence of perinuclear cytoplasmic clearing (halos). Tumor cells 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-back pattern of growth ( honeycomb histologic features) (H&E, 400). C, Microcalcifications are typical of but not specific for oligodendroglioma and reflect their relatively slow growth (arrow) (H&E, 400). D, The delicate vascular pattern associated with oligodendroglioma is seen most often within involved cerebral cortex and has been referred to as a chicken-wire pattern (arrow) (H&E, 400). Downloaded from Am J Clin Pathol 2005;124:

4 Gupta et al / GLIOMA MARKERS diagnosis of oligodendroglioma but is not a feature that is required, sufficient, or constant. Not all tumor cells in an oligodendroglioma will show cytoplasmic clearing; in contrast, occasional tumors with classic astrocytic differentiation might contain perinuclear halos owing to fixation artifact. More important for proper classification are the nuclei: oligodendrogliomas have round, regular, generally bland nuclei with open or delicate chromatin, whereas astrocytoma nuclei are enlarged, elongated, hyperchromatic, and irregular in shape and hue. Quite literally, oligodendroglia means glia with few processes. In tissue sections and especially on smear preparations, there is a paucity of glial processes emerging from oligodendrogliomas compared with the long, finely fibrillar processes that are in abundance in cytologic and histologic preparations of most astrocytic neoplasms Image For reasons that are not entirely clear, a delicate, branching capillary network with a chicken-wire appearance is noted more frequently in oligodendrogliomas (Image 2). Other common but nonspecific features include cortical involvement, perinuclear satellitosis by tumor cells, microcalcifications, and microcysts filled with mucin. Grading systems typically have divided oligodendrogliomas into 2, 3, or 4 grades depending on cellularity, cytologic atypia, mitotic activity, vascular proliferation, and necrosis. 11,24,26-28 Criteria for grading are not as clearly defined for oligodendrogliomas as they are for astrocytomas. The current World Health Organization (WHO) Classification recognizes 2 grades: oligodendroglioma (grade II) and anaplastic 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 nodular pattern Image 4. Nodular growth is compatible with a grade II lesion but might represent a transition to a higher grade, especially when the nodules coalesce into regions of confluent hypercellularity. Grade II oligodendrogliomas can show occasional mitotic figures and cytologic atypia, but marked mitotic activity, microvascular proliferation, or necrosis is consistent with a WHO grade III, anaplastic oligodendroglioma (Image 4). A recent study of prognostic features in oligodendroglioma identified endothelial hypertrophy, necrosis, and 6 or more mitotic figures per high-power field as significant univariate markers of poor outcome, providing a solid framework for establishing the diagnosis of anaplastic oligodendroglioma (grade III). 29 Previous multivariate analyses have suggested that necrosis is the single most important feature of aggressive clinical behavior. 30,31 Pseudopalisading of tumor cells around necrosis is an acceptable finding in anaplastic oligodendroglioma and does not suggest a diagnosis of glioblastoma (Image 4). Infiltrating Diffuse Astrocytomas Much like oligodendrogliomas, infiltrative or diffuse astrocytomas represent a spectrum ranging from low grade to highly malignant. Although numerous grading systems have been 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 a paucity of fibrillar glial processes and instead show tumor cells embedded within a delicate neuropil matrix. The nuclei are round and have delicate or open chromatin (H&E, 1,000). B, Astrocytomas, in contrast, have an abundance of long, fine to coarse fibrillar processes that emerge from individual tumor cell bodies. A variable amount of pink cytoplasm can be noted in the cell bodies of most astrocytoma cells, and their nuclei are more irregular, elongated, and hyperchromatic (H&E, 1,000). 758 Am J Clin Pathol 2005;124: Downloaded 758 from

5 Anatomic Pathology / REVIEW ARTICLE that spans from grade II to grade IV (glioblastoma). 10,32-37 Infiltrative astrocytomas are more common than oligodendrogliomas and, when all grades are considered, account for one third of all primary brain tumors and 70% to 75% of diffuse gliomas. 1,10 Astrocytomas have a tendency to involve subcortical 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 differentiation are seen invading CNS parenchyma with a low degree of cellularity and proliferative activity (Image 1), Image 5, and Image Astrocytic differentiation is best determined morphologically by the presence of nuclei that are elongated, hyperchromatic, and irregular, having angulated and distorted contours (Images 3 and 5). If oligodendrogliomas have nuclei shaped like Florida oranges, then those of astrocytomas are akin to Idaho potatoes. In addition, these nuclei generally lack prominent nucleoli and perinuclear halos and have an ominous dark purple or black coloration following H&E staining. Cytologic preparations are extremely helpful for detecting the nuclear features and the fibrillarity of astrocytic neoplasms (Image 3). Some astrocytomas, such as granular cell, giant cell, gliosarcoma, and gemistocytic subtypes, contain an abundance of large cells with prominent pink cytoplasm and are rarely confused with oligodendrogliomas (Image 5) More problematic are fibrillary, protoplasmic, and small cell variants, which are composed of smaller cells that often have only minimal perinuclear fibrillarity and, therefore, can demonstrate considerable diagnostic overlap with oligodendroglioma. 42,43 Many of the previous grading systems for the diffuse astrocytomas did not allow any mitotic figures to be present in a grade II tumor, and the finding of a single mitotic figure was sufficient to establish the diagnosis of anaplastic astrocytoma (AA; grade III). Clinicopathologic studies have demonstrated that astrocytomas with 0 or 1 mitosis have similar clinical behaviors, and, therefore, current criteria for grade II astrocytomas allow 0 or 1 mitotic figures, but generally not more. 44 AA (WHO grade III) has higher cellularity, a greater degree of nuclear pleomorphism and atypia, and increased proliferation. Mitotic activity (>1 mitosis) should be identified to apply the diagnosis of AA. 36 The number of mitoses identified necessarily depends on sample size, number of tissue levels examined, and intensity of the search. One study suggested that fifty 400 fields should be evaluated to ensure more than 90% sensitivity in the identification of mitoses. 45 Glioblastoma (WHO grade IV) is the highest grade form of infiltrating astrocytoma. 37 In addition to the histopathologic findings of AA, either microvascular hyperplasia or necrosis, often with pseudopalisading, is required for the diagnosis of glioblastoma (Image 5). Glioblastomas with microvascular hyperplasia but no necrosis have been shown to have behavior similar to that of glioblastomas with necrosis. 34,46 Oligoastrocytomas Oligoastrocytomas contain distinct regions of oligodendroglial 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 growth within the cerebral cortex (arrow). Nodules contain a higher cell density, and individual cells are more proliferative. Nodular growth is compatible with grade II oligodendroglioma but might indicate that the tumor is in the process of progressing to a higher 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). Downloaded from Am J Clin Pathol 2005;124:

6 Gupta et al / GLIOMA MARKERS 10% of infiltrative gliomas. 47,48 They may be biphasic, with the 2 components separate, or intermingled, with the 2 neoplastic cell types in proximity. The minimal percentage of each component required for the diagnosis of a mixed glioma has been debated, resulting in highly variable diagnostic criteria and poor interobserver reproducibility for this group of neoplasms. Perhaps more problematic is the tendency for pathologists to dump diagnostically challenging infiltrating gliomas into the wastebasket of the mixed oligoastrocytoma category. It should be emphasized that a mixed oligoastrocytoma is not equivalent to a morphologically ambiguous glioma, but rather refers to a tumor containing cells with 2 distinct histologic patterns. One study suggested that a single 100 field filled with an oligodendroglioma component could be used as a threshold for the diagnosis of mixed oligoastrocytomas because this criterion identifies a subset with a better prognosis than astrocytoma 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 the prototype 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 with large amounts of eosinophilic cytoplasm and eccentrically placed nuclei (H&E, 600). These and other astrocytic variants with abundant cytoplasm are rarely confused with oligodendroglioma. C, Small cell astrocytomas are a biologically aggressive variant that often is confused with anaplastic oligodendroglioma owing to its hypercellularity, low degree of fibrillarity, and deceptively bland nuclei (H&E, 600). D, Regardless of the subtype of astrocytoma morphologic features, progression to glioblastoma is characterized by necrosis (with pseudopalisading in this case, arrow) or microvascular proliferation (arrowhead) (H&E, 400). 760 Am J Clin Pathol 2005;124: Downloaded 760 from

7 Anatomic Pathology / REVIEW ARTICLE Low-grade oligoastrocytomas (WHO grade II) can contain occasional mitotic figures, low to moderate cellularity, and mild to moderate cytologic atypia. 47 The WHO criteria for anaplastic oligoastrocytoma (WHO grade III) are not well defined but suggest that histological features of anaplasia should be present. 48 This list includes nuclear atypia, cellular pleomorphism, high cellularity, high mitotic activity, microvascular proliferation, and necrosis. Anaplasia can be present in the astrocytic component, the oligodendroglial component, or both. Frozen Section Diagnosis of Diffuse Gliomas Distinguishing oligodendroglioma from astrocytoma is challenging enough on permanent sections. The difficulty is compounded at the time of frozen section because this preparation does not optimally reveal the histologic features that typically are used to separate these entities The perinuclear halos, delicate chromatin patterns, and nuclear regularity of oligodendrogliomas are not as evident in frozen sections Image 7. Ice crystal artifact resulting from CNS edema in these lesions can further degrade the morphologic features. It should be remembered that making the distinction between astrocytic and oligodendroglial morphologic features at the time of frozen section is not usually essential for intraoperative patient management. The diagnosis of infiltrating glial neoplasm together with a general degree of histologic differentiation (well, moderately, or poorly differentiated) or grade (low, intermediate, or high) is the best that can be communicated in many, perhaps most, circumstances The process of freezing brain tissue during the frozen section procedure introduces artifacts that remain in permanent sections and limits their interpretation. Most notably, nuclei appear more hyperchromatic and atypical in previously frozen tissue; perinuclear halos of oligodendroglioma are not as evident; and the cytologic resolution is lower (Image 7). 49 Taken together, these changes impart an astrocytic appearance to otherwise classic oligodendrogliomas. Therefore, it is always recommended to submit tissue for permanent sections that has not been frozen. If it is not clear that additional tissue will be available for permanent sections, it is prudent to freeze only half of the tissue submitted at frozen section. 49,54 Oligodendroglial and Astrocytic Markers Immunohistochemical Analysis Because distinguishing among diffuse gliomas is not always straightforward based on morphologic features alone, reliable immunohistochemical or genetic markers of differentiation have been highly sought. Many studies have been guided by the principle that specific proteins must be expressed differentially by glial cells with distinct developmental programs and cellular function. The intermediate filament glial fibrillary acidic protein (GFAP) is a marker of glial differentiation that is expressed consistently by resting astrocytes and greatly overexpressed in reactive astrocytosis. GFAP has been invaluable as a marker of glial differentiation in neoplasms involving the CNS. Led by the mistaken assumption that oligodendrocytes 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 nuclear p53 staining (B, arrow) may favor the diagnosis of an astrocytoma in morphologically ambiguous infiltrating gliomas because TP53 mutations are much more frequent in astrocytomas than oligodendrogliomas (A, H&E, 1,000; B, p53, 1,000). Downloaded from Am J Clin Pathol 2005;124:

8 Gupta et al / GLIOMA MARKERS that GFAP might be a marker that could distinguish astrocytomas from oligodendrogliomas. However, immunohistochemical and ultrastructural studies have concluded firmly that oligodendrogliomas also express GFAP and that some morphologic subtypes, such as gliofibrillary oligodendrocytes and minigemistocytes, express high levels Whether GFAP-positive oligodendroglioma cells represent a transitional form between an oligodendrocytic and astrocytic differentiation is debated. Nevertheless, GFAP is not currently considered a useful marker for distinguishing among infiltrating gliomas. 58 Alterations of p53 are more common in astrocytomas than in oligodendrogliomas, suggesting that it might be a discriminating marker. 10,59,60 The most frequent disruptions are inactivating point mutations of the TP53 gene, which occur in 50% to 60% of grade II astrocytomas but in only 5% to 10% of grade II oligodendrogliomas. TP53 mutations on 1 allele usually are accompanied by loss of heterozygosity (LOH) on chromosome 17p involving the other allele, leading to the complete loss of normal TP53 and production of only mutant p53 protein. This mutated form is altered physically in a manner that reduces its degradation, which makes it immunohistochemically detectable in the nucleus of tumor cells. Staining for p53 can be diagnostically useful in some cases. For example, the presence of strong, nuclear p53 staining could indicate an underlying TP53 mutation and favor the diagnosis of astrocytoma (Image 6). However, mechanisms other than TP53 mutation can cause p53 protein overexpression, and p53 immunoreactivity does not correlate perfectly with the presence of TP53 mutations (concordance of 65%-75%). 61,62 Therefore, a positive p53 immunostain requires interpretation in the context of other clinical and pathologic data. A B C Image 7 Frozen section artifacts in diffuse gliomas. A, Frozen sections of brain tissue involved by an infiltrating glioma often show ice crystal artifact, which distorts the architecture and cytologic features of the neoplasm and makes the definitive diagnosis of astrocytoma or oligodendroglioma difficult or impossible. Tumor cell nuclei often appear dark and irregular, and the morphologic features that typically are relied on in the diagnosis of oligodendroglioma, such as perinuclear halos and nuclear regularity, are not evident on frozen sections (H&E, 600). Permanent sections of an oligodendroglioma that previously was frozen for intraoperative consultation (B) and adjacent tissue that was not previously frozen (C) demonstrates that the histologic and cytologic features of the tumor are considerably different (B, H&E, 600; C, H&E, 600). Previously frozen tissue has nuclei that are irregular and hyperchromatic (features that are more typical of astrocytoma), whereas unfrozen tissue shows the round nuclei, delicate chromatin, and perinuclear halos typical of oligodendroglioma. 762 Am J Clin Pathol 2005;124: Downloaded 762 from

9 Anatomic Pathology / REVIEW ARTICLE A wide array of proteins expressed by nonneoplastic oligodendrocytes have been studied for their usefulness as markers of oligodendrogliomas, including myelin basic protein, proteolipid protein, NG2, myelin-associated glycoprotein, galactocerebroside, Leu7, and cyclic nucleotide-3'-phosphatase. 63,64 Although the rationale for their study has been solid, 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 basic helix-loop-helix transcription factors that regulate neuralderived cell proliferation and differentiation. 65 Of these, Olig1 and Olig2 show the highest brain specificity. Both are expressed in the periventricular zone during embryogenesis and are essential for proper oligodendrocyte maturation, but also have more general roles in nervous system development. Initial in situ hybridization studies demonstrated highly specific Olig1 and Olig2 expression in nonneoplastic oligodendrocytes and oligodendroglioma cells, with only weak or absent Olig expression in astrocytomas. 66,67 Mixed oligoastrocytomas were reported as having expression limited to the oligodendroglial component. 66 Follow-up study using semiquantitative reverse transcriptase polymerase chain reaction for Olig expression have been less promising. Increased Olig expression indeed was identified in glial neoplasms, but levels did not differ substantially between astrocytomas and oligodendrogliomas Subsequent immunohistochemical studies have similarly demonstrated that Olig1 and Olig2 are expressed by glial neoplasms, with nuclear patterns that would be expected for transcription factors. 69,71 Immunoreactivity generally is more intense in oligodendrogliomas than in astrocytomas, but the degree of staining overlaps to an extent that precludes their use as discriminating markers by themselves. Correlation of Olig and GFAP staining in diffuse gliomas has suggested that the Olig+/GFAP immunophenotype identifies a morphologically pure form of oligodendroglioma. 72,73 Nevertheless, the abundance of evidence indicates that Olig expression has limited usefulness in the subclassification of infiltrating gliomas. Genetic Markers The last 2 decades of brain tumor research have demonstrated that each histologic category of infiltrating glioma contains multiple distinct molecular genetic subsets. 10,59,74,75 Some genetic alterations have been exploited in the diagnostic arena and are used to assist with pathologic classification and provide independent prognostic data. Each technique for genetic testing has its own set of advantages and disadvantages. Most often used are LOH (traditional gel-based assays or capillary electrophoresis), fluorescence in situ hybridization (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 the pathologist, department, and institution. 77 LOH analysis and FISH have the highest concordance (>93%) and are used most A B C 19p13 19q13 1p32 1q42 CEP7 EGFR Image 8 Genetic analysis of infiltrating gliomas can aid in diagnosis. A, Oligodendrogliomas typically show loss of chromosomes 1p and 19q by fluorescence in situ hybridization (FISH; 2 green 19p13 signals and 1 red 19q13 signal in most nuclei indicate a loss of 19q in this oligodendroglioma). B, Astrocytic neoplasms normally display intact 1p and 19q (2 green 1p32 and 2 red 1q42 signals in most nuclei). C, Amplification analysis of epidermal growth factor receptor (EGFR) by FISH sometimes is useful for differentiating high-grade astrocytomas (especially small cell astrocytoma) from anaplastic oligodendrogliomas. 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.) Downloaded from Am J Clin Pathol 2005;124:

10 Gupta et al / GLIOMA MARKERS N T 1,600 1, ,600 1, frequently for diagnostic purposes on tissue derived from histologic sections FISH has some advantages from a pathologist s perspective (Image 8): (1) Analysis is based on the morphologic identification of genetic alterations within tumor cell nuclei. (2) Nonneoplastic cells (positive controls) are almost always present within the tissue sections examined (eg, normal endothelial cells, neurons). (3) FISH does not require microdissection of normal and tumor tissue before analysis. (4) Genetic gains and losses in infiltrative tumors with a low ratio of neoplastic/normal cells can be analyzed by FISH, whereas these alterations might not be detected by polymerase chain reaction based analysis (LOH studies) owing to overwhelming amounts of normal DNA. One major disadvantage 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 oligodendroglial tumors, generally with increasing frequency at higher grades, and, therefore, are not useful markers for discriminating histologic subtypes. 74,75,80 These alterations, including loss of 9p21 (p16/cdkn2a) and losses involving chromosome 10 (PTEN/DMBT1), might provide prognostic information for a given tumor but are not specific to 1 histologic pattern and will not be considered further. 1p and 19q One of the best-recognized molecular signatures among diffuse gliomas, which also has prognostic significance, is the T N + 3,200 3,600 4,000 4,400 LOH 1p Image 9 Loss of heterozygosity (LOH) analysis demonstrates loss 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 unstained histologic slides after comparison with an H&E-stained section. The polymerase chain reaction products for a microsatellite marker at 1p36.21 analyzed by capillary electrophoresis show 2 allele peaks for tissue derived from for the normal brain (allelic balance, upper panel). Only 1 allele peak is noted for tissue derived from the infiltrating glioma, indicating allelic loss of 1p36.21 (arrow, lower panel). + combined loss of genetic material from chromosomes 1p and 19q in oligodendrogliomas q losses occur in 60% to 80% in oligodendrogliomas, but also are detected in approximately 30% to 40% of astrocytomas, indicating that this may be a shared genetic alteration in gliomagenesis. 19,77,85 Losses on the short arm of 1p also are frequent in oligodendrogliomas (50%-80%) but are much more specific because they are detected in only 10% to 18% of astrocytomas. 18,19,77 Almost always, losses on 1p and 19q coexist in oligodendroglioma, and the combination can be detected in 60% to 80%. In most cases, the entire arms of the affected chromosome are deleted, allowing reliable detection by LOH, FISH, or CGH (Images 8 and 9). Combined loss of 1p and 19q is a strong predictor of response to chemotherapy and overall survival in low- and high-grade oligodendrogliomas. 18,19 In contrast, oligodendrogliomas with deletions of p16/cdkn2a (9p21), LOH 10q, or EGFR amplifications, but without loss of 1p and 19q, are associated with poor overall survival. 18,86 Initial studies of oligodendrogliomas with 1p and 19q deletion showed an increased responsiveness of these tumors to PCV chemotherapy (procarbazine, CCNU [lomustine], vincristine), but it now is evident that this molecular subset is more responsive to other therapies, including radiation and temozolamide. 18,19,87,88 The combined loss of 1p and 19q occurs much less frequently in astrocytic tumors (only in 1%-10%). Nevertheless, it should be remembered that the incidence of astrocytomas is nearly 8 times greater than that of oligodendrogliomas. 1 One issue that continues to be debated is whether genetic loss of 1p and 19q has prognostic significance in nonoligodendroglial tumors including astrocytomas. The majority of studies have concluded the following 19,78,89 : (1) Combined 1p and 19q loss does not predict enhanced survival for astrocytomas or mixed oligoastrocytomas. (2) The frequency of 1p and 19q loss is so low that a survival effect cannot be demonstrated. In contrast, Ino et al 90 suggested that loss of chromosome 1p might be associated with longer survival rates in a subset of high-grade astrocytomas and oligoastrocytomas. Similarly, combined loss of 1p and 19q predicted prolonged survival in 1 study of 97 glioblastomas. 91,92 Currently, combined 1p and 19q loss is best viewed as a marker of oligodendroglial differentiation and a marker of therapeutic responsiveness in these tumors. Additional studies will be needed to demonstrate the clinical usefulness, if any, for 1p and 19q testing in other primary brain tumors. Epidermal Growth Factor Receptor Amplifications of the EGFR gene occur in approximately 40% of glioblastomas and can be detected by FISH, CGH, and polymerase chain reaction based tests 93,94 (Image 8). Amplifications are much less frequent in lower grade astrocytomas and are considered a late genetic event in the progression 764 Am J Clin Pathol 2005;124: Downloaded 764 from

11 Anatomic Pathology / REVIEW ARTICLE of tumors to glioblastoma. Wild-type and mutated forms of EGFR can be amplified, and, in either case, messenger RNA and cell surface protein levels are increased markedly. The most common EGFR amplification is a mutated form lacking exons 2 through 7, which results in a truncated cell surface protein with constitutive tyrosine kinase activity (EGFRvIII). 95,96 Therapies directed at the overexpressed EGFR in glioblastomas are finding their way into neurooncology practice. 20,97 EGFR amplifications are rare in oligodendroglial tumors, and analysis of EGFR status has proven useful for distinguishing high-grade astrocytomas from anaplastic oligodendrogliomas in some cases. 15,98 For the majority of glioblastomas, a correct diagnosis can be made based on morphologic examination alone, and EGFR testing is not necessary. However, the recently described small cell astrocytoma is a high-grade astrocytoma that has a great deal of morphologic similarity to anaplastic oligodendroglioma and might require ancillary tests for correct diagnosis. 42,43 Both of these tumors contain a high density of neoplastic cells with scant cytoplasm, uniform and deceptively bland nuclei, and a high proliferation index. It is important to recognize small cell astrocytomas because they are biologically aggressive, behaving clinically as glioblastomas even in the absence of necrosis and microvascular hyperplasia. Small cell astrocytomas are characterized by a high frequency of EGFR amplification and chromosome 10 losses, but they have intact chromosomes 1p and 19q. In contrast, anaplastic oligodendrogliomas show the opposite genetic alterations, having a high frequency of 1p and 19q deletions but only rare EGFR amplifications and chromosome 10 losses. Immunohistochemical analysis for EGFR and EGFRvIII also is possible in high-grade gliomas, but this application has not been studied as a tool for distinguishing histologic subtypes. 99,100 Chromosome 7 Alterations on chromosome 7 are common in astrocytomas, and amplification of EGFR at 7p12 is the best known example. Although they are frequent in glioblastoma, EGFR amplifications are less common in grades II and III astrocytomas, limiting the usefulness of EGFR as a marker for distinguishing lower-grade gliomas. In contrast, classic cytogenetic and CGH studies of grades II and III astrocytomas have demonstrated that chromosome 7 gains are among the most common genetic alterations (40%-66%), either as entire chromosome gains (trisomy/polysomy 7) or as gains of 7p or 7q alone These gains already are present in 40% to 50% of grade II astrocytomas, suggesting that it is an early tumorigenic event, and its presence is associated with shorter survivals. Chromosome 7 gains are less common in oligodendrogliomas, occurring as the whole chromosome in roughly 10% and as gains of 7p or 7q in 20%. 80 When present, they occur in a subset of oligodendrogliomas that also have chromosome 10 losses but not 1p and 19q losses, indicating that these gains of 7 might occur in a biologically distinct class of tumors, perhaps more related to astrocytomas. 107,108 Chromosome 7 gains also are more frequent in anaplastic oligodendrogliomas (grade III), which might indicate that this genetic alteration is a shared feature of malignant progression in astrocytomas and oligodendrogliomas. 80,107,108 Thus, the detection of chromosome 7 gains occasionally might be helpful for distinguishing low-grade astrocytomas from oligodendrogliomas, especially in the absence of 1p and 19q losses, but it cannot be used reliably as a discriminating marker among high-grade gliomas. Summary Even with ancillary tests, the diagnosis of diffuse gliomas remains a challenge, and the search for more specific and diagnostically useful markers continues. Emerging technologies, like DNA, complementary DNA, and protein microarrays, are demonstrating potential for brain tumor classification and for marker discovery. For example, complementary DNA microarray analysis coupled with computational algorithms has been used to successfully classify high-grade gliomas into histologic groups. 109 Moreover, this technique was found to be superior to histologic classification in predicting the prognosis of morphologically ambiguous tumors. Diagnostic microarrays containing a limited number of relevant genes could be designed to classify tumors, predict their clinical behavior, and rank therapeutic options. Such approaches have already revealed new markers with the potential for diagnostic use that await validation. 110 The development of therapies targeted at the molecular underpinnings of these diseases ultimately will require a molecular component in the diagnosis of the diffuse gliomas. From the Departments of Pathology and Laboratory Medicine and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA. Address reprint requests to Dr Brat: Dept of Pathology and Laboratory Medicine, Emory University Hospital, H-176, 1364 Clifton Rd NE, Atlanta, GA References 1. Central Brain Tumor Registry of the United States. Statistical Report: Primary Brain Tumors in the United States, Chicago, IL: Central Brain Tumor Registry of the United States; Burger PC, Scheithauer BW, Vogel FS. Surgical Pathology of the Nervous System and Its Coverings. 4th ed. New York, NY: Churchill Livingstone; Downloaded from Am J Clin Pathol 2005;124:

12 Gupta et al / GLIOMA MARKERS 3. Kleihues P, Cavenee WK, eds. Pathology and Genetics of Tumours of the Nervous System. 2nd ed. Lyon, France: IARC Press; World Health Organization Classification of Tumours. 4. Perry A. Oligodendroglial neoplasms: current concepts, misconceptions, and folklore. Adv Anat Pathol. 2001;8: Burger PC. What is an oligodendroglioma? Brain Pathol. 2002;12: Bruner JM, Inouye L, Fuller GN, et al. Diagnostic discrepancies and their clinical impact in a neuropathology referral practice. Cancer. 1997;79: Burger PC, Nelson JS, Boyko OB. Diagnostic synergy in radiology and surgical neuropathology: neuroimaging techniques and general interpretive guidelines. Arch Pathol Lab Med. 1998;122: Burger PC, Nelson JS, Boyko OB. Diagnostic synergy in radiology and surgical neuropathology: radiographic findings of specific pathologic entities. Arch Pathol Lab Med. 1998;122: Hoffman JM. New advances in brain tumor imaging. Curr Opin Oncol. 2001;13: Cavenee WK, Furnari FB, Nagane M, et al. Diffusely infiltrating astrocytomas. In: Kleihues P, Cavenee WK, eds. Pathology and Genetics of Tumours of the Nervous System. 2nd ed. Lyon, France: IARC Press; 2000: World Health Organization Classification of Tumours. 11. Burger PC, Scheithauer BW. Tumors of the Central Nervous System. Washington, DC: Armed Forces Institute of Pathology; Atlas of Tumor Pathology; Third Series, Fascicle Prayson RA, Agamanolis DP, Cohen ML, et al. Interobserver reproducibility among neuropathologists and surgical pathologists in fibrillary astrocytoma grading. J Neurol Sci. 2000;175: Coons SW, Johnson PC, Scheithauer BW, et al. Improving diagnostic accuracy and interobserver concordance in the classification and grading of primary gliomas. Cancer. 1997;79: Mittler MA, Walters BC, Stopa EG. Observer reliability in histological grading of astrocytoma stereotactic biopsies. J Neurosurg. 1996;85: Fuller CE, Schmidt RE, Roth KA, et al. Clinical utility of fluorescence in situ hybridization (FISH) in morphologically ambiguous gliomas with hybrid oligodendroglial/astrocytic features. J Neuropathol Exp Neurol. 2003;62: Shaw EG, Scheithauer BW, O Fallon JR, et al. Mixed oligoastrocytomas: a survival and prognostic factor analysis. Neurosurgery. 1994;34: Shaw EG, Scheithauer BW, O Fallon JR. Supratentorial gliomas: a comparative study by grade and histologic type. J Neurooncol. 1997;31: Cairncross JG, Ueki K, Zlatescu MC, et al. Specific genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendrogliomas. J Natl Cancer Inst. 1998;90: Smith JS, Perry A, Borell TJ, et al. Alterations of chromosome arms 1p and 19q as predictors of survival in oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas. J Clin Oncol. 2000;18: Rich JN, Reardon DA, Peery T, et al. Phase II trial of gefitinib in recurrent glioblastoma. J Clin Oncol. 2004;22: Phuphanich S, Brat DJ, Olson JJ. Delivery systems and molecular targets of mechanism-based therapies for GBM. Expert Rev Neurother. 2004;4: Bailey P, Cushing H. Tumors of the Glioma Group. Philadelphia, PA: Lippincott; Bailey P, Bucy PC. Oligodendrogliomas of the brain. J Pathol Bacteriol. 1929;32: Reifenberger G, Kros JM, Burger PC, et al. Oligodendroglioma. In: Kleihues P, Cavenee WK, eds. Pathology and Genetics of Tumours of the Nervous System. 2nd ed. Lyon, France: IARC Press; 2000: World Health Organization Classification of Tumours. 25. Burger PC. Use of cytological preparations in the frozen section diagnosis of central nervous system neoplasia. Am J Surg Pathol. 1985;9: Bigner DD, McClendon RE, Bruner JM, eds. Russell and Rubinstein s Pathology of Tumors of the Nervous System. 6th ed. New York, NY: Oxford University Press; Reifenberger G, Kros JM, Burger PC, et al. Anaplastic oligodendroglioma. In: Kleihues P, Cavenee WK, eds. Pathology and Genetics of Tumours of the Nervous System. 2nd ed. Lyon, France: IARC Press; 2000: World Health Organization Classification of Tumours. 28. Smith MT, Ludwig CL, Godfrey AD, et al. Grading of oligodendrogliomas. Cancer. 1983;52: Giannini C, Scheithauer BW, Weaver AL, et al. Oligodendrogliomas: reproducibility and prognostic value of histologic diagnosis and grading. J Neuropathol Exp Neurol. 2001;60: Mork SJ, Halvorsen TB, Lindegaard KF, et al. Oligodendroglioma: histologic evaluation and prognosis. J Neuropathol Exp Neurol. 1986;45: Burger PC, Rawlings CE, Cox EB, et al. Clinicopathologic correlations in the oligodendroglioma. Cancer. 1987;59: Kernohan JW, Sayre GP. Tumors of the Central Nervous System. Washington, DC: Armed Forces Institute of Pathology, Atlas of Tumor Pathology; Series 1, Fascicle Ringertz N. Grading of gliomas. Acta Pathol Microbiol Scand. 1950;27: Daumas-Duport C, Scheithauer B, O Fallon J, et al. Grading of astrocytomas: a simple and reproducible method. Cancer. 1988;62: Kleihues P, Davis RL, Ohgaki H, et al. Diffuse astrocytoma. In: Kleihues P, Cavenee WK, eds. Pathology and Genetics of Tumours of the Nervous System. 2nd ed. Lyon, France: IARC Press; 2000: World Health Organization Classification of Tumours. 36. Kleihues P, Davis RL, Coons SW, et al. Anaplastic astrocytoma. In: Kleihuis P, Cavenee WK, eds. Pathology and Genetics of Tumours of the Nervous System. 2nd ed. Lyon, France: IARC Press; 2000: World Health Organization Classification of Tumours. 37. Kleihues P, Burger PC, Collins VP, et al. Glioblastoma. In: Kleihues P, Cavenee WK, eds. Pathology and Genetics of Tumours of the Nervous System. 2nd ed. Lyon, France: IARC Press; 2000: World Health Organization Classification of Tumours. 38. Brat DJ, Scheithauer BW, Medina-Flores R, et al. Infiltrative astrocytomas with granular cell features (granular cell astrocytomas): a study of histopathologic features, grading, and outcome. Am J Surg Pathol. 2002;26: Kosel S, Scheithauer BW, Graeber MB. Genotype-phenotype correlation in gemistocytic astrocytomas. Neurosurgery. 2001;48: Reis RM, Konu-Lebleblicioglu D, Lopes JM, et al. Genetic profile of gliosarcomas. Am J Pathol. 2000;156: Am J Clin Pathol 2005;124: Downloaded 766 from

13 Anatomic Pathology / REVIEW ARTICLE 41. Peraud A, Watanabe K, Schwechheimer K, et al. Genetic profile of the giant cell glioblastoma. Lab Invest. 1999;79: Perry A, Aldape KD, George DH, et al. Small cell astrocytoma: an aggressive variant that is clinicopathologically and genetically distinct from anaplastic oligodendroglioma. Cancer. 2004;101: Burger PC, Pearl DK, Aldape K, et al. Small cell architecture; a histological equivalent of EGFR amplification in glioblastoma multiforme? J Neuropathol Exp Neurol. 2001;60: Giannini C, Scheithauer BW, Burger PC, et al. Cellular proliferation in pilocytic and diffuse astrocytomas. J Neuropathol Exp Neurol. 1999;58: Coons SW, Pearl DK. Mitosis identification in diffuse gliomas: implications for tumor grading. Cancer. 1998;82: Barker FG II, Davis RL, Chang SM, et al. Necrosis as a prognostic factor in glioblastoma multiforme. Cancer. 1996;77: Reifenberger G, Kros JM, Burger PC, et al. Oligoastrocytoma. In: Kleihues P, Cavenee WK, eds. Pathology and Genetics of Tumours of the Nervous System. 2nd ed. Lyon, France: IARC Press; 2000: World Health Organization Classification of Tumours. 48. Reifenberger G, Kros JM, Burger PC, et al. Oligoastrocytoma. In: Kleihues P, Cavenee WK, eds. Pathology and Genetics of Tumours of the Nervous System. 2nd ed. Lyon, France: IARC Press; 2000: World Health Organization Classification of Tumours. 49. Burger PC, Vogel FS. Frozen section interpretation in surgical neuropathology, I: intracranial lesions. Am J Surg Pathol. 1978;2: Reyes MG, Homsi MF, McDonald LW, et al. Imprints, smears, and frozen sections of brain tumors. Neurosurgery. 1991;29: Martinez AJ, Pollack I, Hall WA, et al. Touch preparations in the rapid intraoperative diagnosis of central nervous system lesions: a comparison with frozen sections and paraffinembedded sections. Mod Pathol. 1988;1: Shah AB, Muzumdar GA, Chitale AR, et al. Squash preparation and frozen section in intraoperative diagnosis of central nervous system tumors. Acta Cytol. 1998;42: Colbassani HJ, Nishio S, Sweeney KM, et al. CT-assisted stereotactic brain biopsy: value of intraoperative frozen section diagnosis. J Neurol Neurosurg Psychiatry. 1988;51: Burger PC, Nelson JS. Stereotactic brain biopsies: specimen preparation and evaluation. Arch Pathol Lab Med. 1997;121: Herpers MJ, Budka H. Glial fibrillary acidic protein (GFAP) in oligodendroglial tumors: gliofibrillary oligodendroglioma and transitional oligoastrocytoma as subtypes of oligodendroglioma. Acta Neuropathol (Berl). 1984;64: Kros JM, Van Eden CG, Stefanko SZ, et al. Prognostic implications of glial fibrillary acidic protein containing cell types in oligodendrogliomas. Cancer. 1990;66: Nakopoulou L, Kerezoudi E, Thomaides T, et al. An immunocytochemical comparison of glial fibrillary acidic protein, S-100p and vimentin in human glial tumors. J Neurooncol. 1990;8: Kros JM, de Jong AA, van der Kwast TH. Ultrastructural characterization of transitional cells in oligodendrogliomas. J Neuropathol Exp Neurol. 1992;51: Hunter SB, Brat DJ, Olson JJ, et al. Alterations in molecular pathways of diffusely infiltrating glial neoplasms: application to tumor classification and anti-tumor therapy. Int J Oncol. 2003;23: von Deimling A, Fimmers R, Schmidt MC, et al. Comprehensive allelotype and genetic analysis of 466 human nervous system tumors. J Neuropathol Exp Neurol. 2000;59: van Meyel DJ, Ramsay DA, Casson AG, et al. p53 mutation, expression, and DNA ploidy in evolving gliomas: evidence for two pathways of progression. J Natl Cancer Inst. 1994;86: Watanabe K, Sato K, Biernat W, et al. Incidence and timing of p53 mutations during astrocytoma progression in patients with multiple biopsies. Clin Cancer Res. 1997;3: Nakagawa Y, Perentes E, Rubinstein LJ. Immunohistochemical characterization of oligodendrogliomas: an analysis of multiple markers. Acta Neuropathol (Berl). 1986;72: Schwechheimer K, Gass P, Berlet HH. Expression of oligodendroglia and Schwann cell markers in human nervous system tumors: an immunomorphological study and Western blot analysis. Acta Neuropathol (Berl). 1992;83: Rowitch DH, Lu QR, Kessaris N, et al. An oligarchy rules neural development. Trends Neurosci. 2002;25: Marie Y, Sanson M, Mokhtari K, et al. OLIG2 as a specific marker of oligodendroglial tumour cells. Lancet. 2001;358: Lu QR, Park JK, Noll E, et al. Oligodendrocyte lineage genes (OLIG) as molecular markers for human glial brain tumors. Proc Natl Acad Sci U S A. 2001;98: Bouvier C, Bartoli C, Aguirre-Cruz L, et al. Shared oligodendrocyte lineage gene expression in gliomas and oligodendrocyte progenitor cells. J Neurosurg. 2003;99: Ohnishi A, Sawa H, Tsuda M, et al. Expression of the oligodendroglial lineage associated markers Olig1 and Olig2 in different types of human gliomas. J Neuropathol Exp Neurol. 2003;62: Riemenschneider MJ, Koy TH, Reifenberger G. Expression of oligodendrocyte lineage genes in oligodendroglial and astrocytic gliomas. Acta Neuropathol (Berl). 2004;107: Ligon KL, Alberta JA, Kho AT, et al. The oligodendroglial lineage marker OLIG2 is universally expressed in diffuse gliomas. J Neuropathol Exp Neurol. 2004;63: Azzarelli B, Miravalle L, Vidal R. Immunolocalization of the oligodendrocyte transcription factor 1 (Olig1) in brain tumors. J Neuropathol Exp Neurol. 2004;63: Mokhtari K, Paris S, Aguirre-Cruz L, et al. Olig2 expression, GFAP, p53 and 1p loss analysis contribute to glioma subclassification. Neuropathol Appl Neurobiol. 2005;31: Hartmann C, Mueller W, von Deimling A. Pathology and molecular genetics of oligodendroglial tumors. J Mol Med. 2004;82: Reifenberger G, Collins VP. Pathology and molecular genetics of astrocytic gliomas. J Mol Med. 2004;82: Kros JM, van Run PR, Alers JC, et al. Genetic aberrations in oligodendroglial tumours: an analysis using comparative genomic hybridization (CGH). J Pathol. 1999;188: Smith JS, Alderete B, Minn Y, et al. Localization of common deletion regions on 1p and 19q in human gliomas and their association with histological subtype. Oncogene. 1999;18: Downloaded from Am J Clin Pathol 2005;124: