Journal of Histochemistry and Cytochemistry, Vol. 48, 1103-1110, August 2000, Copyright © 2000, The Histochemical Society, Inc.


ARTICLE

Glioma-associated Antigen Expression in Oligodendroglial Neoplasms: Tenascin and Epidermal Growth Factor Receptor

Roger E. McLendona, Carol J. Wikstranda, Mark R. Matthewsa, Raid Al-Baradeia, Sandra H. Bignera, and Darell D. Bignera
a Department of Pathology, Duke University Medical Center, Durham, North Carolina

Correspondence to: Roger E. McLendon, DUMC 3712, Durham, NC 27710. E-mail: roger.mclendon@duke.edu


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Epidermal growth factor receptor (EGFR), its variant, EGFRvIII, and tenascin are glioma-associated antigens that are hyperexpressed by neoplastic glial cells relative to normal brain, making them attractive antigenic targets for immunotherapy. Preliminary surveys indicate that oligodendroglial tumors also produce these proteins, although the exact patterns and degrees of reactivity are not known. In this study we examined the immunoreactivity of tenascin among 50 oligodendroglial tumors, including 25 well-differentiated oligodendrogliomas (WDOs) and 12 glioblastomas (GBMs) exhibiting high proportions of oligodendroglia-like cells. We used well-characterized immunoreagents with defined specificities against the target antigens on formalin-fixed, paraffin-embedded archival tissue. The tumors were graded according to WHO guidelines. Immunoreactivity was reported on a 1–3 scale according to staining intensity multiplied by a 1–3 distribution scale distribution within tumor as focal (1), multifocal (2), and diffuse (3) for both the parenchymal and the perivascular components. Although there is considerable overlap in antigen production among the grades of tumor, this study establishes the production of tenascin and wild-type EGFR (but not EGFR vIII) in oligodendroglial neoplasms and supports the concept that antigen production increases with tumor grade.

(J Histochem Cytochem 48:1103–1110, 2000)

Key Words: epidermal growth factor, receptor, glioblastoma, immunotherapy, oligodendroglioma, tumor grade


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Oligodendrogliomas are tumors composed of cells that histologically resemble the mature oligodendrocyte, occurring predominately in the cerebral hemispheres. Constituting approximately 5% of all intracranial CNS tumors, oligodendrogliomas are most commonly diagnosed during the fourth and fifth decades of life, with many patients having a multi-year history of epileptic seizure activity that suggests a prolonged prediagnostic period with tumor (Chin et al. 1980 ). Even in patients with such a prolonged history, postoperative survivals are disproportionately short. However, progress in the therapy of these tumors is being made. Recent work on oligodendrogliomas has revealed that they typically respond initially to the chemotherapeutic protocols that depend on alkylating chemotherapeutic agents such as PCV (Levin et al. 1980 ; Cairncross et al. 1994 ). At present, the mean postdiagnostic survival for low-grade oligodendroglioma is 10 years, although for high-grade tumors, the survival is only 5 years (Paleologos and Cairncross 1999 ). Other methods of therapy are needed if postoperative survivals in high-grade tumors are to be extended.

One avenue of therapy exploits tumor-specific antigens that can be targeted by monoclonal antibodies (MAbs) conjugated with either radiohalides or cytotoxins. Required antigenic characteristics that identify a target antigen for such a therapeutic approach include hyperexpression of the antigen by tumor cells, little or no expression in normal tissues, and cytologic location preferably on the cell surface or in stable extracellular matrix. Few antigens qualify with these strict criteria. An operationally specific approach identifies antigens with these characteristics within a compartmental administration setting. For CNS tumors, this approach requires the identification of antigens present on neoplastic but not benign CNS tissues, whereby restricted routes of administration such as intrathecal or intracavitary routes (within tumor cyst cavities) limit the distribution of the toxic therapy. Tenascin (TN), wild-type epidermal growth factor receptor (EGFRwt), and its variant, epidermal growth factor variant III (EGFRvIII), all represent antigens with operationally specific, tumor-restricted presentations in adult brains.

The glioma-associated isoform of tenascin, tenascin C, was originally identified with MAb 81C6 (Bourdon et al. 1983 ). It has since been demonstrated to be an effective therapeutic target for the majority of GBMs, the most common and most deadly of primary intracranial CNS tumors (Blasberg et al. 1987 ; Wikstrand et al. 1987 ; Lee et al. 1988 ; Lastoria et al. 1990 ; Riva et al. 1992 , Riva et al. 1993 ; Ventimiglia et al. 1992 ; Brown et al. 1996 ; Bigner et al. 1995 , Bigner et al. 1998 ). The elevated synthesis of TN in greater than 90% of glioblastomas and the extremely low-level synthesis in normal brain make TN an attractive target for immunological therapy.

Both EGFRwt and EGFRvIII also represent targets in astrocytic neoplasia because a high percentage of astrocytic tumors express either antigen or both. Although EGFRwt is present on the benign surface leptomeninges, no studies to date have identified the antigen within the CNS parenchyma. Up to 90% of high-grade astrocytic gliomas express the EGFRwt antigen, a hyperexpression associated with EGFR gene amplification in greater than 50% of glioblastomas. Previous studies indicate that oligodendrogliomas also express the antigen, although genomic amplification is generally absent (Reifenberger et al. 1996 ; Broholm et al. 1999 ). Approximately 50% of high-grade astrocytomas contain the EGFRvIII rearrangement, a rearrangement that results in a 145-kD molecule with a unique primary sequence characterized by an inserted glycine at position 6 between in-frame-deleted amino acids 5 and 274. Being a unique antigen in the CNS, EGFRvIII would be a very attractive target if identified in oligodendroglial tumors. To date, one study has identified one oligodendroglial tumor with genomic rearrangement (Reifenberger et al. 1996 ). Despite generally negative molecular studies, this antigen apparently has not been studied immunohistochemically in these tumors.

On the basis of our previous work in astrocytic gliomas, we hypothesize that TN, EGFR, and EGFRvIII, if expressed in oligodendroglial tumors, may offer attractive targets for immunotherapeutic approaches to these tumors. In this study we sought to characterize TN, EGFR, and EGFRvIII protein distribution and intensity among 50 low- and high-grade oligodendroglial tumors and 10 glioblastomas exhibiting a significant population of cells with oligodendroglial features. Furthermore, this study undertook to characterize TN, EGFRwt, and EGFRvIII distribution and heterogeneity in the vascular and parenchymal components among the grades of these tumors.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Archival formalin-fixed, paraffin-embedded brain tumor tissues from 50 adults (Table 1 Table 2 Table 3) diagnosed as having oligodendrogliomas (well-differentiated oligodendroglioma or WDO, n = 25; anaplastic oligodendroglioma or AO, n = 25) and treated at Duke University Medical Center with gross total or subtotal open resection were used (needle biopsies were not used in this study). The patients included 33 males and 17 females ranging in age from 6 to 75 years (mean ± SD 39.82 ± 13.6). The study also included 12 patients diagnosed with glioblastoma (WHO Grade IV) (eight men, four women, age range 31–67, mean 48 ± 12.5). The cases previously constituted a molecular study of oligodendrogliomas, with additional cases added subsequently (Bigner et al. 1999 ).


 
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Table 1. Well-differentiated oligodendrogliomas a


 
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Table 2. Anaplastic oligodendrogliomas a


 
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Table 3. Glioblastomas a

H&E-stained sections were used to characterize the tumors histologically with regard to overall cellularity, nuclear hyperchromasia, mitotic activity, vascular proliferation, and necrosis with or without pseudopalisading of tumor cells. Diagnoses for this study were made according to WHO criteria (Kleihues et al. 1993 ). In addition, 12 glioblastomas were included because of a high predominance of oligodendroglia-like cells composing the tumors.

Anti-tenascin Polyclonal Antibody Production and Purification
Tenascin Purification. Tenascin was purified according to techniques described previously (Ventimiglia et al. 1992 ). Briefly, TN was purified from U-251 MG-C3 supernatant by immunoaffinity chromatography using a murine anti-TN MAb 81C6 (Bourdon et al. 1983 ). Cultured supernatant was passed over an 81C6-Sepharose 4B affinity column at room temperature; the column was washed with 10 mM Tris plus 500 mM sodium chloride (pH 8.0), and the TN was eluted with 100 mM CAPS in 500 mM NaCl (pH 11.0) into tubes containing 30 mg glycine/ml eluate to neutralize the pH to approximately 8.3. TN used for polyclonal antibody preparation was subjected to an additional glycerol gradient sedimentation purification step (Erickson and Taylor 1987 ).

Production of Polyclonal Antiserum. Polyclonal antiserum to TN was prepared against affinity-purified TN. Five µg of TN in Freund's complete adjuvant was injected SC into rabbits. Nine subsequent monthly IV boosts of 5 µg were administered, with high titers (1:50,000 against purified human TN) noted after the second boost. Antiserum from blood drawn 11 days after the second boost was used for all studies. No reactivity of this antiserum to ZO + 10% FBS or to purified human fibronectin was noted on immunoblots (Erickson and Taylor 1987 ).

Purification of Anti-tenascin Polyclonal Antiserum. The resulting polyclonal antiserum was purified by immunoaffinity chromatography over a TN–Sepharose 4B affinity column at room temperature; the column was then washed with 0.115 phosphate buffer. Bound antibody was eluted and equilibrated as described for TN purification. Antibody fractions were pooled and dialyzed against 115 mM of phosphate buffer and stored at 4C. The specificity of the affinity-purified polyclonal antiserum was characterized utilizing Western blotting techniques on both benign brain and malignant glioma cell lines as previously described (Ventimiglia et al. 1992 ). The affinity-purified polyclonal antiserum identifies multiple antigenic sites on the TN molecule and recognizes multiple isoforms of the TN molecule (Ventimiglia et al. 1992 ).

Immunohistochemistry (Tenascin, EGFRwt, and EGFRvIII)
The present study used the polyclonal antibody against TN described above as well as MAb E30 (Ab No-207M; BioGenex, San Ramon, CA) against EGFRwt and MAb L8A4 against EGFRvIII prepared and purified at Duke University Medical Center as previously described (Wikstrand et al. 1995 ). Whereas MAb E30 exhibits operational specificity for EGFRwt in Western blotting assay under reducing conditions, recent flow cytometric studies against the cell line NR6M set 3 (Batra et al. 1995 ), which has been genetically altered to express EGFRvIII only and no EGFRwt, revealed labeling of the cells with MAb E30 (data not shown).

Immunohistochemistry was performed using 5-µm-thick slides cut onto Plus glass (Fisher Scientific; Atlanta, GA). Briefly, the slides were brought to water through prolonged deparaffinization in serial xylenes and graded alcohols. For the anti-TN polyclonal antibody and for the anti-EGFRvIII MAb L8A4, antigen retrieval was performed using 1HC citrate buffer (Fisher Scientific) with microwave (Kenmore model 999111; Sears Roebuck, Chicago, IL) heating at medium power for 10 min, then cooled at room temperature for 30 min. For the anti-EGFRwt MAb E30, antigen retrieval was performed with pepsin digestion using 0.25% pepsin (Pepsin A; Sigma Chemical, St Louis, MO) diluted in Automation Buffer (Biomeda; Foster City, CA), pH 2.0, at 40C for 7 min. After antigen retrieval, the slides were washed in running d-H2O and one change of PBS. Nonspecific protein binding was blocked with 10% normal goat serum at RT for 30 min. Primary antibodies were used at the designated concentrations: polyclonal anti-TN Ig and normal rabbit Ig 10 µg/ml; E30 (IgG1) and isotype-matched control 1.2 µg/ml; and L8A4 (IgG1) and isotype-matched control 5 µg/ml. Primary antibodies were incubated for 2 hr with these reagents. Antibody detection was performed using the peroxidase-conjugated streptavidin system according to the manufacturer's recommendation, followed by incubation with diaminobenzidine (3,3'-diaminobenzidine #34065; Pierce Chemical, Rockford, IL) for 5 min. Nuclear counterstaining was accomplished with Harris' modified hematoxylin (Fisher Scientific; Pittsburgh, PA). Controls consisted of a human glioma xenograft (D245) with known TN immunoreactivity and human glioblastoma tissues previously shown to express EGFRwt and EGFRvIII run in each batch.


  Results
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Tenascin
Histologically, 25 WDOs, 25 AOs, and 12 glioblastomas with extensive oligodendroglia-like elements comprised the study material. Only one case, a WDO, was entirely negative for TN localization; the remaining 49 oligodendrogliomas and 12 glioblastomas exhibited TN reactivity (Table 1 and Table 2), at least focally, in some part of the tissue (98% oligodendroglioma reactivity). Tenascin is expressed in the cell cytoplasm and on cell membranes and is extensively elaborated into the extracellular matrix. Oligodendroglial tumor tissues often exhibited reactivity in the cytoplasm as well as the membranes (Fig 1A and Fig 1B), an observation also seen in other gliomas (Zagzag 1995 ). In the AOs (Fig 1A and Fig 1B) and glioblastomas, the ECM reactivity was often pronounced, resulting in a fibrillar netlike pattern of reactivity entrapping the tumor cells (Fig 1). Perivascular reactivity was also noted, but typically not to the degree of intensity or multiplicity that was noted in the tumor cell parenchymal component. The perivascular reactivity never extended beyond the regions of dense staining, arguing against the notion that the reactivity began as a primarily perivascular event with secondary extension to the parenchyma. Cytologically, the reactivity in oligodendrogliomas reflected patterns reported for other gliomas, with the reactivity localized both in the extracellular matrix and within the cytoplasmic outlines. However, because oligodendrogliomas often exhibit cytoplasmic clearing ("fried egg" appearance), cytoplasmic reactivity was rarely seen. The results of the semi-quantitative scoring for tenascin are shown in Table 4. Among oligodendrogliomas, 24/25 well-differentiated tumors and 25/25 anaplastic tumors exhibited at least some positivity. Immunoreactivity in both the mesenchymal and perivascular components increased with tumor grade.



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Figure 1. (A) Tenascin in WDO. This tumor exhibits a moderate (2), diffuse (3) net-like membranous pattern also found in other gliomas, resulting in a mesenchymal score of 6. Evidence of cytoplasmic reactivity is seen, although not as prominent as that found in astrocytic gliomas given the fixation artifact to which oligodendroglial cells are prone. Original magnification x132. (B) Tenascin in AO. This tumor exhibits strong (3) multifocal (2) mesenchymal immunoreactivity (mesenchymal score of 6) and strong (3) multifocal (2) perivascular immunoreactivity (perivascular score of 6). Original magnification x50.

Figure 2. (A) EGFRwt in WDO. This tumor exhibits moderate (2), multifocal (2) immunoreactivity for EGFRwt in a net-like mesenchymal pattern. (B) EGFRwt in AO. This tumor exhibits strong (3), multifocal (2) immunoreactivity in a net-like pattern. Original magnifications x100.


 
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Table 4. Immunohistochemical score for tenascin by component

EGFRwt and EGFRvIII
Greater than 90% of oligodendrogliomas in this study exhibited immunoreactivity for EGFRwt, at least focally (Fig 2A and Fig 2B; Table 1 and Table 2), whereas none of the tumors (including 0/5 GBM) exhibited any expression for EGFRvIII as detected by MAb L8A4. Immunoreactivity of low-grade tumors revealed either occasional cells exhibiting low intensity of EGFRwt staining or small groups of reactive tumor cells exhibiting low or moderate intensity (Fig 2A and Fig 2B). AOs tended to show higher percentages of reactive cells with higher staining intensities (Fig 2B), occasionally involving the entire tumor section. In contrast to the perivascular patterns seen in slides reacted against polyclonal anti-81C6 (anti-tenascin), the parenchyma around vessels occasionally exhibited perivascular clearing of antigen reactivity in slides developed with anti-EGFRwt. Although the immunohistochemical scoring system increased from WDO to AO, there was no distinction between AO and glioblastoma (Table 5). In this study, the only benign tissues to show reactivity were the leptomeninges overlying the cortex; perivascular arachnoid was nonreactive.


 
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Table 5. Immunohistochemical score for EGFRwt by component


  Discussion
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Materials and Methods
Results
Discussion
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The semiquantitative grading scale used in these assays allows characterization of both intensity and location. By multiplying the intensity score by the distribution score, the product reflects an overall score for antigen amount in the section. We further wanted to document the differences among the perivascular ECM and the intratumoral parenchyma; therefore, the two components were scored separately. As noted in the results, perivascular reactivity for the two antigens varied, with perivascular immunoreactivity more commonly seen for TN and clear perivascular zones noted for EGFRwt reactivity.

Reactivity for both TN and EGFRwt clearly increased from the WDO to the AO, the biology of which is unknown but reflects the pattern noted in astrocytic gliomas. Clearly, from a therapeutic standpoint in which either antigen is utilized as an immunological target for radiolabeled MAbs, these observations establish the necessary background for pursuit of this treatment in humans with high-grade oligodendrogliomas. From a diagnostic standpoint, however, the intertumor variability is significant and cannot be depended on for grading purposes.

The TNs are an interesting family of large, oligomeric glycoproteins that are the subject of many reviews. Briefly, they are transiently expressed in the extracellular matrix in an organ/site-specific distribution and are the focus of active investigation with regard to structure, function, and distribution and, with regard to therapeutic implications, immunological targets. Three TN isoforms are best known—tenascin C, tenascin R, and tenascin X—and have been implicated in embryonal cell migration, adhesion, and angiogenesis. Structurally, the best documented, TN-C, is a six-armed structure attached at a central knob called a hexabrachion (Erickson and Inglesias 1984 ). All isoforms of TN exhibit repeat segments within the arms that exhibit homologies to the epidermal growth factor, to the Type III repeats of fibronectin, and to the ß- and {gamma}-chains of fibrinogen (Gulcher et al. 1991 ; Siri et al. 1991 ).

In the brain, TN is expressed predominantly during development (Crossin et al. 1986 ; Bartsch et al. 1992 , Bartsch et al. 1995 ; Derr et al. 1997 ), inflammation, and neoplasia. TN has been shown to inhibit both the adhesion and migration of oligodendroglial cells in culture (Frost et al. 1996 ; Kiernan et al. 1996 ). Normal brain exhibits minor (2–5 µg TN/mg extract protein) quantities of TN, in contrast to gliomas, which have levels up to 100-fold higher (Ventimiglia et al. 1992 ). Although this may be interpreted to indicate a contradiction to the use of anti-TN antibodies in therapeutic trials, the actual diffusion of the large MAb into surrounding tissues has been shown to be minimal, with the intent of the therapy to sterilize the surgical wall and its immediate 2–2.5-cm neighboring tissues of remaining tumor.

Zagzag and colleagues (Zagzag 1995 ; Zagzag et al. 1996 ; Jallo et al. 1997 ) have noted that the synthesis and immunoreactivity of TN correlates with tumor type, tumor grade, and angiogenesis in astrocytic gliomas. Whether TN is associated with angiogenesis or is a concomitant member of the ECM of new vessels is not clear. However, it is clear that malignant progression in astrocytic gliomas is associated with endothelial proliferation and with increased production of TN. Furthermore, immunohistochemical studies reveal specific localization of TN around vessels in astrocytic gliomas as well as peritumor cell localization in the highest grade glioma, glioblastoma. Although the effect of TN on angiogenesis in oligodendrogliomas was not addressed in this investigation, it appears to be an unlikely factor in this tumor. Although the observations that TN and angiogenesis increased with neoplastic progression were again confirmed, strong perivascular TN immunolocalization could be seen without concomitant endothelial proliferation, glomeruloid formations, or vascular tortuosity.

Previous studies have demonstrated the immunohistochemical localization of EGFRwt without concomitant genomic amplification in oligodendrogliomas. Reifenberger and colleagues 1996 , in a study of 33 oligodendrogliomas (including 13 WDOs and 20 AOs), found genomic amplification (with rearrangement) in only one tumor, elevated mRNA transcript (over normal brain) in 16 cases, and positive protein immunolocalization in 30 cases. These data appear to support elevated levels of transcription as well as decreased protein degradation in mediating EGFRwt immunolocalization. Similarly, Ekstrand et al. 1992 found EGFRwt in 5/5 WHO grade II tumor and 4/4 WHO grade III tumors, none of which was accompanied by genomic amplification. Studies by Torp et al. 1991 , Hawkins et al. 1991 , Diedrich et al. 1991 , and Broholm et al. 1999 lend further support to the finding of increased EGFR expression in a majority of oligodendroglial tumors.

Our previous study of 47 oligodendrogliomas, seven oligoastrocytomas, and four glioblastomas with predominant oligodendroglial histology revealed EGFR amplification in one anaplastic oligoastrocytoma and one glioblastoma. None of 47 oligodendroglial tumors revealed EGFR or EGFRvIII genomic amplification (Bigner et al. 1999 ). Forty of the 50 oligodendrogliomas reported herein were included in that study, lending further support to the previous observations of EGFRwt hyperexpression without amplification. Finally, we concur with Reifenberger's contention (1996) that the equal distribution of cases positive for EGFRwt hyperexpression among well-differentiated and anaplastic oligodendroglial tumors reflects an early event in tumorigenesis rather than in progression.

Received for publication December 14, 1999; accepted March 8, 2000.
  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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