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HAM56-Immunoreactive Macrophages in Untreated Infiltrating Gliomas.

In surgical neuropathology, a potential diagnostic pitfall is the failure to recognize macrophages. Freezing artifact, inadequate processing of tissue, and reactive astrocytosis may distort the characteristic morphologic features of macrophages and make interpretation based on hematoxylin-eosin features alone challenging. Studies using a variety of monoclonal antibodies against operationally specific markers for macrophages have suggested that macrophages are present in tumors of the central nervous system (CNS) including astrocytomas, oligodendrogliomas, ependymomas, meningiomas, medulloblastomas, acoustic neuromas, craniopharyngiomas, hemangioblastomas, CNS lymphomas, and tumors metastatic to the CNS.[1-12] Nonneoplastic lesions of the CNS that may mimic neoplasms clinically and histologically and may contain a significant macrophage population include infarcts and demyelinating disorders.[1]

The monoclonal antibody HAM56 labels bloodborne tissue macrophages such as hepatic Kupffer cells and pulmonary alveolar macrophages in addition to tingible body macrophages and interdigitating macrophages of lymph nodes. The reagent also labels a variable subpopulation of endothelial cells, most prominently those of capillaries and small blood vessels. Benign or reactive B or T lymphocytes are not labeled, although variable reactivity with neoplastic B cells has been noted. Neural tissue, smooth muscle cells, fibroblasts, and epithelia, except for occasional tubular epithelial cells of the kidney, are also nonreactive.[13]

Using the serial analysis of gene expression technique (SAGE) in a case of glioblastoma multiforme (GBM), the macrophage genes osteopontin (OPN) and macrophage-capping protein (MCP) were shown to be overexpressed 12-fold and eightfold, respectively, compared with normal brain tissue. A prominent macrophage population in this untreated GBM was admixed within areas of solid tumor and was confirmed with HAM56 immunohistochemistry. The present study is an extension of our prior studies of the utility of HAM56 in diagnostic neuropathology,[1] and evaluates the presence of HAM56-reactive macrophages within untreated gliomas.



Sixteen cases of untreated infiltrating gliomas from 1996-1999 were retrieved from the pathology files at Duke University Medical Center. No patients had any prior history of surgical intervention, radiation therapy, chemotherapy, or head trauma. All cases were diagnosed and graded according to criteria set forth by the World Health Organization[14] as well-differentiated astrocytoma (WDA, WHO grade II) (2 cases), well-differentiated oligodendroglioma (WDO, WHO grade II) (3 cases), anaplastic astrocytoma (AA, WHO grade III) (2 cases), anaplastic oligodendroglioma (AC), WHO grade III) (2 cases), and GBM (WHO grade IV) (7 cases).

SAGE Analysis

SAGE analysis of one case of GBM and of normal brain tissue was performed as described[15] using the SAGE protocol as originally described.[16] To summarize, SAGE involves the formation of quantitative libraries of genes and their levels of expression. The SAGE method begins with the preparation of total RNA from tissue followed by the conversion of messenger RNA (mRNA) to double-stranded complementary DNA (cDNA) using a biotinylated oligo (dT) primer. The cDNA is digested with a restriction enzyme (NlaIII) (anchoring enzyme) that cleaves the cDNA into fragments of approximately 256 base pairs. The 3' portion of the cDNA fragments are isolated and bound to streptavidin beads, and this provides the tags from a defined position within each cDNA that is necessary for the ultimate identification of the corresponding genes. The cDNA is divided in half, and the 5' ends are ligated to 2 different linkers, each of which contains a restriction site for a tagging enzyme (BsmFI), an anchoring enzyme overhang, and a priming site for polymerase chain amplification. The linker-cDNA sequences are digested with the tagging enzyme, causing the release of fragments consisting of linker and a short piece of cDNA. The released tags are ligated to each other and serve as polymerase chain reaction templates. The amplification products contain 2 tags (one ditag) that can be concatenated by ligation, cloned, and sequenced. In order to identify the tags, the sequences obtained are compared with sequences in different genome databases.[15-19] SAGEmap is a public database of SAGE data, including data presented in this manuscript, and can be accessed at the SAGEmap Web site ([15,19]


Sections from formalin-fixed, paraffin-embedded blocks were cut at 4 to 5 [micro]m, placed on positively charged glass slides, deparaffinized in organic solvents, treated with methanolic [H.sub.2][O.sub.2] to quench endogenous peroxidase activity, and rehydrated. Sections were pretreated with nonimmune serum (normal goat serum, 1: 20, for 15 minutes). Sections were reacted with HAM56 (1:30, mouse immunoglobulin M [IgM] monoclonal, Dako Corporation, Carpinteria, Calif). Mouse IgM isotype (Dako) was used as a negative control, and appropriate positive tissue controls were also tested. The unlabeled bound primary antibody was linked with biotinylated goat anti-mouse IgM (1:100; Vector Laboratories, Burlingame, Calif), and detected with horseradish peroxidase--labeled streptavidin (1:800; Jackson Immuno Research Inc, West Grove, Pa). Immunoreactivity was visualized using diaminobenzidine as the chromogen, with Harris modified hematoxylin as the counterstain. Macrophages were defined as round cells with foamy cytoplasm, distinct cell borders, and an eccentric bland nucleus.

The frequency of HAM56-positive cells was quantified with an ocular grid by counting 1000 cells from selected high-power fields (400x) in areas of solid infiltrating tumor remote from areas of necrosis. The HAM56-positive cells within foci of microvascular proliferation or the rim of pseudopalisading cells abutting necrosis were not counted.


Demographic and pathologic data of the untreated gliomas are summarized in the Table. HAM56 reactivity was present within the delicate endothelial cells of WDA tumors (Figure 1) and WDO tumors, but no unequivocal macrophages were identified. In AA tumors (Figure 2) and AO tumors, rare HAM56-positive macrophages were identified within infiltrating tumor (mean labeling index, 0.4% range, 0.1%-1.2%), in addition to rare positive cells among foci of microvascular proliferation. The GBMs showed a range of HAM56 reactivity from 0% to 24.5% (mean labeling index, 8.6%) in areas of solid tumor remote from necrotic foci. In the GBM analyzed by SAGE (Table, case 11), numerous HAM56-positive macrophages (labeling index, 24.5%) were present in areas of solid tumor (Figure 3). In all cases of GBM, nonquantitated HAM56-positive macrophages were readily identified in foci of microvascular proliferation and within the pseudopalisading rim of cells abutting necrosis.

Features of Cases of Untreated Supratentorial
Infiltrating Gliomas(*)

Case Age, y Sex Site Diagnosis Grade HAM56
 Index, %

 1 42 M L frontal WDA II 0
 2 34 M L temporal WDA II 0
 3 58 F R insular WDO II 0
 4 28 M R temporal WDO II 0
 5 27 F R frontal WDO II 0
 6 42 M R frontal AA III 0.2
 7 29 M L parietal AA III 0.1
 8 37 M R temporal AO III 0.1
 9 54 M L parietal AO III 1.2
 10 53 M L temporal GBM IV 0
 11 61 M R frontal GBM IV 24.5
 12 53 M L frontal GBM IV 15.3
 13 62 F L parietal GBM IV 4.8
 14 56 F R parietal GBM IV 7.7
 15 85 F R frontal GBM IV 2.5
 16 65 F L temporal GBM IV 5.2

(*) WHO indicates World Health Organization; M, male; F, female;
L, left; R, right; WDA, well-differentiated astrocytoma; WDO,
well-differentiated oligodendroglioma; AA, anaplastic astrocytoma;
AO, anaplastic oligodendroglioma; and GBM, glioblastoma multiforme.

SAGE analysis of one untreated GBM showed that gene transcripts of the macrophage-related genes OPN and MCP were overexpressed 12-fold and eightfold, respectively, compared with normal brain tissue. Tag sequences and gene symbols of OPN and MCP that were identified are as follows: AATAGAAATT, SPP1 (OPN) and CTCCCCTGCC, CAPG (MCP).


Previous studies comparing the relative sensitivity and specificity among a variety of anti-macrophage/microglial cell antibodies found HAM56 to exhibit the highest sensitivity and specificity in identifying CNS macrophages.[1] HAM56 was found to be particularly useful in labeling the macrophage populations of demyelinating diseases[20] and infarcts, with no cross-reactivity against reactive astrocytes.[1]

In agreement with others,[3,6-9,11,21,22] the present study demonstrates that the percentage of macrophages in infiltrating gliomas increases with higher tumor grades. No HAM56-labeled macrophages were present in WDA and WDO tumors. Rare HAM56-labeled macrophages were found in AA and AO tumors, but they were neither as plentiful nor as microscopically obvious as those found in inflammatory or vascular diseases. On the other hand, the HAM56 macrophage population in GBM tumors was significant. As previously described,[5,11] our cases of GBM showed a striking population of macrophages in areas occupied by solid formations of tumor remote from areas of necrosis. Despite the presence of a significant number of macrophages, the admixture of anaplastic glial cells should obviate misinterpretation of tumor for an inflammatory process, even when one is confronted with a small stereotactic biopsy. Our diagnostic experience, and that of others, has found that the pseudopalisading foci of GBM are often more easily identified at lower powers of magnification, with higher powers of magnification often disclosing a population of viable cells abutting necrosis. This study confirms the high-magnification impression of living cells and identifies a significant population of these cells as macrophages.

Macrophages participate in the clearance of necrotic tissue as seen in both the treated and untreated GBM. The distribution of macrophages within necrotic tissue as a result of surgical or therapeutic intervention appears as dense sheets of foamy macrophages (Figure 4). Alternatively, infiltrating microglia near a focus of radiation necrosis may be mistaken for a malignancy in a treated glioma. In needle biopsies, the infiltrating rim may be sampled without the characteristic findings seen in large excisional biopsy samples, leading to a risk of misdiagnosis. We have found HAM56 a useful supplement to glial fibrillary acidic protein to characterize the cells. Previous studies from our institution have identified persistent macrophage infiltrates as responsible for locally increased uptake of fluorine-18-fluorodeoxyglucose on positron emission tomography scanning, resulting in false-positive signals for recurrent glioma.[23] Studies are now under way on a large series of tumors to characterize not only the distribution of these cells in these lesions but also the time frame of their appearance and disappearance.


SAGE is a technique that allows the analysis and quantitative profile of cellular gene expression in normal, developmental, and disease states. Potential applications of SAGE data include the characterization of disease at the molecular level and the identification of therapeutic targets and

diagnostic markers.[15-19] SAGE quantifies a tag (a nucleotide sequence of a defined length) that represents the transcription product of a gene. The data product of SAGE is a list of tags with their count values and thus is a digital representation of cellular gene expression. Osteopontin is a secreted glycoprotein that has been suggested to function in adhesive interactions at the tumor-host border and to potentially influence tumor invasion and metastasis.[24] Osteopontin has also been hypothesized to play an important role in the development of type 1 (cell-mediated) immunity.[25] Osteopontin mRNA has been identified in macrophages at the tumor-stroma border and in areas of tumor necrosis in a wide variety of human carcinomas, including carcinomas of the colon, stomach, duodenum, pancreas, breast, lung, bladder, prostate, ovary, and thyroid.[24] In human gliomas, OPN mRNA and proteins were detected in transformed cell lines of various malignancy grades with high expression in malignant astrocytomas, but low expression in benign astrocytomas and nonneoplastic tissue.[26,27] SAGE also showed overexpression of MCP, which was originally isolated from macrophage extracts.[28-30] Increased levels of MCP mRNA during the development of promyelocytes and monocytes into macrophages have suggested that MCP may play an important role in macrophage function.[31] In addition to OPN and MCP, other SAGE tag sequences that were compared for GBM and normal brain tissue include cell cycle regulators, transcription factors, angiogenesis factors, neurotransmission factors, and others that have been previously reported.[15]

In summary, HAM56-positive macrophages were identified in high-grade but not low-grade untreated infiltrating gliomas. In an untreated GBM, SAGE data showed overexpression of 2 proteins that have been reported as secretory products of macrophages, and the presence of abundant macrophages was confirmed with HAM56. The present study lends support for the ability of HAM56 to identify macrophages in untreated gliomas.

The authors thank James L. Burchette, HT(ASCP) for his immunohistochemical expertise.


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Accepted for publication December 11, 2000.

From the Department of Pathology, Duke University Medical Center, Durham, NC.

Presented in part at the Annual Meeting of the American Association of Neuropathologists, Atlanta, Ga, June 9, 2000.

Corresponding author: Thomas J. Cummings, MD, Department of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710 (e-mail:
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Title Annotation:monoclonal antibody analysis in glioma diagnosis
Author:Cummings, Thomas J.; Hulette, Christine M.; Bigner, Sandra H.; Riggins, Gregory J.; McLendon, Roger
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:1USA
Date:May 1, 2001
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