Combined Granular Cell And Small Cell Malignant Astrocytoma Teresa Ribalta, M.D., Ph.D Hospital Clinic Barcelona, Spain

Diagnostic Features: Infiltrating, heterogeneous tumor exhibiting two separate components that blend into each other minimally at the interface: a predominant population (70%) made up of benign-looking, large, eosinophilic cells, with distinctive granular cytoplasm, and a less extensive component of highly cellular, uniformly small cells with abundant mitoses. A narrow transition zone of well-differentiated neoplastic astrocytes interspersed with each cell population is present. Granular cell nuclei are slightly irregular, sometimes eccentric, and contain small nucleoli. Cytoplasmic granules are small, non-refringent, PAS-positive and diastase-resistant, and in some cells, are confined to the peripheral area of cytoplasm. In the small cell areas, a well-developed focal capillary network, occasional perinuclear haloes, and psammoma-type calcifications may suggest oligodendroglial tumor. EM: Abundant lysosomes (dense bodies, autophagic vacuoles) and bundles of intermediate filaments in granular cells. IHC: GFAP is strongly positive in the atypical astrocytes of the border zone, focally positive in the small cells, and weakly to moderately positive in granular cells; CD68, a-1-antitrypsin, a-1-antiquimotrypsin, and ubiquitin only positive in granular cells; MIB-1 LI ranging from 50% in small cells to 13% in granular cells. EGFR overexpression and p53-negative staining in both tumor areas. FISH: intact 1p/19q and EGFR amplification in both tumor areas. Figure 1 Click to view with ImageScope Click to view with a Web-Based Viewer Differential Diagnosis Taken separately, the granular cells simulate a reactive condition or a benign granular cell tumor. However, the surgical specimen in our case clearly revealed a neoplastic process, so the main differential diagnosis is that of an anaplastic oligodendroglioma (WHO grade III) with a granular cell component or macrophage infiltration. An intact 1p/19q and EGFR overexpression do not exclude the possibility of an oligodendroglial tumor, since the codeletion is present in only 50-70% of anaplastic oligodendrogliomas and EGFR overexpression is observed in about 50% of oligodendroglial tumors; however, genetic amplification of EGFR is not a feature in these tumors (Reifenberger 1996). Instead, EGFR amplification is present in about half of glioblastomas (GBM), particularly in the small cell variant. In our case, demonstration of EGFR amplification without deletion of chromosomes 1p and 19q is highly consistent with GBM. EGFR amplification was also clearly present in the granular cells, thus confirming their neoplastic nature and suggesting a common genetic origin with the small cell GBM component. Histologic Variants of Glioblastoma As is well known, the histologic and cytologic spectrum that GBM may display is among the widest in neuro-oncology. The tumor cells can vary from poorly differentiated small cells to spindle, round or pleomorphic cells to multinucleated giant cells. A significant intra- and intertumoral morphologic heterogeneity is also characteristic. However, there are a number of GBM variants with peculiar morphologic features which are less frequently encountered and can cause confusion with other tumor types or non-neoplastic lesions (Fuller 2004). Some of these variants are recognized by the current WHO 2000 classification of CNS tumors (Kleihues 2000), while others have been characterized more recently. The distinctive pathologic features, differential diagnoses, and diagnostic markers of GBM variants are summarized in the Table. In the present case, a recently recognized small cell variant was seen in combination with a granular cell astrocytoma, a rare and still not fully characterized entity. Both variants share a deceptively bland histology and a poor prognosis, and should not be misdiagnosed as less aggressive conditions. Small Cell Astrocytoma/Glioblastoma Variant GBMs usually contain variable proportions of small, primitive cells in appearance, with markedly hyperchromatic nuclei; however, the small cell GBM variant, as outlined recently (Burger 2001), has been defined as a highly cellular, cytologically monotonous neoplasm with bland nuclei and a brisk mitotic activity, that can closely mimic anaplastic oligodendroglioma. The small cell phenotype is commonly associated with EGFR amplification and it is considered an important component of primary GBMs. Given the improved prognosis and therapeutic responsiveness of patients with oligodendroglial tumors, particularly for those with the combined deletion of chromosomes 1p and 19q, the distinction between the two entities is crucial. Recently, Perry et al. (2004) have further characterized this variant in a clinicopathologic and genetic study of 71 small cell malignant astrocytomas, which included 33% of tumors that, as in our case, were classified as anaplastic astrocytoma (Grade III) using WHO criteria. Interestingly, these anaplastic tumors progressed rapidly, with mortality rates that were indistinguishable from the grade IV astrocytomas. Therefore, small cell astrocytoma is an aggressive histologic variant that behaves like primary GBM, even in the absence of microvascular proliferation and necrosis. Grading following the current WHO criteria is problematic but as Perry suggests, such neoplasms are GBM molecularly, although not yet histologically. None of the 71 small cell astrocytomas in his series had 1p/19q codeletions, whereas EGFR amplification and 10q deletions were present in 69% and 97% of tumors, respectively. Small cell GBMs expressed EGFR (83%) and EGFR-vIII (50%) more commonly than non-small cell GBMs (35% and 21%, respectively). Granular Cell Astrocytoma/Glioblastoma Granular cell astrocytoma (GCA) is an uncommon variant of infiltrative glioma, characterized histopathologically by a prominent population of bland, granular astrocytoma cells and clinically by aggressive behavior. Granular cells are large, round, with eccentric nuclei, and are packed with eosinophilic, PAS-positive and diastase-resistant granules. Since upon initial inspection, the granular cells resemble large macrophages, GCA may be mistaken for non-neoplastic lesions, such as evolving infarct and demyelinating disease (Zagzag 1993), especially in small biopsy specimens. GCA shares the cytologic features with granular cell tumors of pituitary stalk and elsewhere in the body. However, despite the morphologic resemblance, granular cell tumors seem to be biologically heterogeneous (Rickert 1997). The histopathologic spectrum and biological behavior of GCAs has been recently studied by Brat et al (2002) in 22 cases, the largest series to date. Of the 22 cases (age range 29-75 y.o.; 17 men), 6 were pure GCA and the rest showed transition to a more conventional astrocytoma. None of the 22 GCAs showed a combined small cell glioma component as in our case. Focal GFAP staining of the granular cells was seen in all but one tumor. The majority of cases were also immunoreactive for S-100 protein, KP-1, ubiquitin, and EMA. In all cases, MIB-1 labeling index was significantly lower in the granular cell component than in the conventional astrocytoma cells. Following the WHO 2000 criteria, four tumors were classified grade II, 7 were grade III, and 11 were grade IV. However, the vast majority of tumors progressed to death in less than one year, independently of the tumor grade. The cause of this apparent aggressiveness of GCA is currently unknown. It has been suggested, by analogy to gemistocytic astrocytomas, that non-proliferative granular cells, which reflect severe abnormalities in cell cycle regulation, could be a morphologic marker of poor prognosis in diffuse astrocytoma (Chorny 2000). No genetic alterations specific for GCA were found in a subgroup of 11 tumors in Brat's series (Castellano-Sanchez 2003). In contrast with our case, EGFR amplification was not observed in any tumor. Either, no specific chromosomal imbalances were observed in another series of 7 cases studied by CGH (Rickert 2002). Instead, a high frequency of allelic loss, especially on 9p and 10q, occurred in most of the cases, including low grade lesions, which could confer aggressive growth potential to these tumors. Although the distinction from reactive macrophages can be difficult in small biopsy specimens, a number of traits are useful in recognizing the neoplastic character of the granular cells in the GCA variant: granular tumor cells are significantly larger than macrophages, show nuclear atypia or at least nuclear enlargement, and, often, prominent nucleoli. The cytoplasmic granules are smaller and more uniform than those of macrophages. In some cells, a circular disposition of granules at the cell periphery produces a "targetoid" appearance. The granular change is due to accumulation of lysosomes in the cytoplasm and it is currently considered a degenerative phenomenon that can occur in a wide variety of tumor types (Geddes 1996). In addition to astrocytic neoplasms, granular cells have been described in other glial and nonglial CNS tumors (Giangaspero 1999). Since CD68 (KP-1) is an indiscriminant lysosomal marker (Hulette 1992), it is not useful for distinguishing GCA from macrophage infiltration. GFAP and S-100 proteinreactivities coupled with the above mentioned morphologic features are strong evidence of glial neoplasia. In the majority of specimens, a close examination will disclose the presence, at least focally, of conventional infiltrating atypical astrocytes, obscured by the granular cell component. Another mimic of GCA cells is eosinophilic granular bodies (EGBs), which are considered a useful diagnostic finding to distinguish pilocytic astrocytoma and other low-grade astrocytic lesions from malignant gliomas, although an occasional EGB may be observed in GBM. EGB granules are refringent, irregular in size and usually are seen associated with Rosenthal fibers. Follow-up This case was seen in consultation and, according the information provided by the referring pathologist, the patient was treated with postoperative irradiation (60 Gy) and chemotherapy with carmustine (BCNU), and she is alive and without clinical or radiological evidence of recurrence 16 months after diagnosis. Conclusion Both small cell and granular cell GBM variants, which were encountered together in this unique tumor, are highly aggressive neoplasms with morphologic features that overlap with anaplastic oligodendroglioma and benign conditions, respectively. Given the greatly different treatment and survival of these groups of patients, accurate diagnosis is critical. Awareness of these variants and careful assessment of conventional light microscopy, along with ancillary techniques, including immunohistochemistry, cytogenetics, and in some cases electron microscopy, are keys to avoid misdiagnosis in uncommon GBM variants. Table 1 - Morphologic variants of glioblastoma (GBM). Pathologic features, differential diagnoses, and helpful diagnostic markers GBM Variant Distinguishing Pathologic Features Differential Diagnoses Diagnostic Markers Granular cell Large granular cells, peripheral rims of eosinophilic granularity, nuclear atypia Infarct, multiple sclerosis, progressive multifocal leukoencephalopathy, benign granular cell tumors Nuclear atypia, GFAP, S-100 protein, electron microscopy Small cell Small glial cells, sometimes with bland nuclei Anaplastic oligodendroglioma, low-grade glioma (rarely PNET, lymphoma, carcinoma, melanoma) Intact 1p/19q, EGFR amplification (70%), 10q deletion (>95%) Giant cell Relatively circunscribed tumor, predominance of bizarre, multinucleated giant cells Pleomorphic xanthoastrocytoma Mitoses, microvascular proliferation, necrosis, p53 mutations (>75%) Spindle cell Bipolar, fusiform cells Sarcoma, gliosarcoma, malignant meningioma, carcinoma GFAP, S-100 protein Gliosarcoma Biphasic tissue pattern (alternating areas of GBM and sarcoma), metaplastic epithelium, bone, muscle, or cartilage GBM invading meninges, spindle cell GBM, fibrosarcoma GFAP, reticulin, actin Adenoid Cohesive small epithelial cells, glandular structures Carcinoma GFAP Epithelioid Superficial, relatively circunscribed tumor, cohesive rounded cells without processes Amelanotic melanoma, carcinoma, meningioma GFAP Lipid-Rich Relatively circunscribed tumor, cohesive epithelioid cells with foamy cytoplasm Balloon cell melanoma, pleomorphic xanthoastrocytoma GFAP, reticulin Myxoid Striking myxoid background Sarcoma, chordoid glioma, myxoid meningioma, other myxoid tumors Mitoses, microvascular proliferation, GFAP Inflammatory Prominent neutrophil or macrophage infiltration Abscess and other inflammatory disorders, infarct, demyelinating disease Atypical astrocytes, GFAP References Brat DJ et al. 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