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Russell R. Lonser, Stuart Walbridge, Alexander O. Vortmeyer, Svetlana D. Pack, Tung T. Nguyen, Nitin Gogate, Jeffery J. Olson, Aytac Akbasak, R. Hunt Bobo, Thomas Goffman, Zhengping Zhuang and Edward H. Oldfield

Object. To determine the acute and long-term effects of a therapeutic dose of brain radiation in a primate model, the authors studied the clinical, laboratory, neuroimaging, molecular, and histological outcomes in rhesus monkeys that had received fractionated whole-brain radiation therapy (WBRT).

Methods. Twelve 3-year-old male primates (Macaca mulatta) underwent fractionated WBRT (350 cGy for 5 days/week for 2 weeks, total dose 3500 cGy). Animals were followed clinically and with laboratory studies and serial magnetic resonance (MR) imaging. They were killed when they developed medical problems or neurological symptoms, lesions appeared on MR imaging, or at study completion. Gross, histological, and molecular analyses were then performed.

Nine (82%) of 11 animals that underwent long-term follow up (> 2.5 years) developed neurological symptoms and/or enhancing lesions on MR imaging, which were defined as glioblastoma multiforme (GBM), 2.9 to 8.3 years after radiation therapy. The GBMs were categorized as either unifocal (three) or multifocal (six), and were located in the supratentorial (six), infratentorial (two), or both (one) cranial regions. Histological examination revealed distant, noncontiguous tumor invasion within the white matter of all nine animals harboring GBMs. Novel interspecies comparative genomic hybridization (three animals) uniformly showed deletions in the GBMs that corresponded to chromosome 9 in humans.

Conclusions. The high rate of GBM formation (82%) following a therapeutic dose of WBRT in nonhuman primates indicates that radioinduction of these neoplasms as a late complication of this therapy may occur more frequently than is currently recognized in human patients. The development of these tumors while monitoring the monkeys' conditions with clinical and serial MR imaging studies, and access to the tumor and the entire brain for histological and molecular analyses offers an opportunity to gather unique insights into the nature and development of GBMs.

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Martin A. Proescholdt, Marsha J. Merrill, Barbara Ikejiri, Stuart Walbridge, Aytac Akbasak, Steven Jacobson and Edward H. Oldfield

Object. Immunotherapy for glioblastoma has been uniformly ineffective. The immunological environment of the brain, with its low expression of major histocompatibility complex (MHC) molecules and limited access for inflammatory cells and humoral immune effectors due to the blood—brain barrier (BBB), may contribute to the failure of immunotherapy. The authors hypothesize that brain tumors are protected from immune surveillance by an intact BBB at early stages of development. To investigate the immunological characteristics of early tumor growth, the authors compared the host response to a glioma implanted into the brain and into subcutaneous tissue.

Methods. Samples of tumors growing in the brain or subcutaneously in rats were obtained for 7 consecutive days and were examined immunohistochemically for MHC Class I & II molecules, and for CD4 and CD8 lymphocyte markers. Additionally, B7-1 costimulatory molecule expression and lymphocyte-specific apoptosis were examined.

Conclusions. On Days 3 and 4 after implantation, brain tumors displayed significantly lower MHC Class II expression and lymphocytic infiltration (p < 0.05). After Day 5, however, no differences were detected. The MHC Class II expressing cells within the brain tumors appeared to be infiltrating microglia. Minimal B7-1 expression combined with lymphocyte-specific apoptosis were detected in both brain and subcutaneous tumors. Low MHC Class II expression and low lymphocytic infiltration at early time points indicate the importance of the immunologically privileged status of the brain during early tumor growth. These characteristics disappeared at later time points, possibly because the increasing perturbation of the BBB alters the specific immunological environment of the brain. The lack of B7-1 expression combined with lymphocyte apoptosis indicates clonal anergy of glioma-infiltrating lymphocytes regardless of implantation site.

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Douglas W. Laske, Orhan Ilercil, Aytac Akbasak, Richard J. Youle and Edward H. Oldfield

✓ Targeted protein toxins are a new class of reagents with the potential for great tumor selectivity and cytotoxic potency. Two such compounds were studied: 1) Tf-CRM107, a conjugate of human transferrin (Tf) and diphtheria toxin with a point mutation (CRM107); and 2) 454A12-rRA, a conjugate of a monoclonal antibody (454A12) to the human Tf receptor and recombinant ricin A chain (rRA). Both compounds are potent and specific in killing human glioblastoma cell lines in vitro. The authors investigated the activity of these reagents administered intratumorally against solid U251 MG human gliomas in vivo.

Nude mice with established U251 MG flank tumors (0.5 to 1.0 cm in diameter) were randomly assigned to be treated with 100-µl intratumoral injections of Tf-CRM107 (10 µg) or 454A12-rRA (10 µg), equimolar doses of CRM107 (4.3 µg), 454A12 antibody (7.5 µg), or rRA (1.5 µg), or phosphate-buffered saline (PBS) every 2 days for a total of four doses. Tumor volume and animal weight were assessed by a blinded observer before each treatment and biweekly for 30 days after initiating therapy. With Tf-CRM107 administration, tumor regression of greater than 95% occurred by Day 14 (p < 0.01) and tumors did not recur by Day 30. Treatment with 454A12-rRA caused a 30% decrease in tumor volume by Day 14 (p < 0.01). Treatment with equimolar doses of the unconjugated targeted protein toxin components CRM107, 454A12, or rRA caused significant U251 MG tumor growth inhibition, but the effects were less potent than the antitumor effects of the conjugates.

This study also characterized the dose-response effect of Tf-CRM107 on tumor growth and tumor weight on Day 30. Nude mice with established U251 MG flank tumors (0.5 to 1.0 cm in diameter) were treated with 100-µl intratumoral injections of 10, 1.0, or 0.1 µg of Tf-CRM107 or PBS every 2 days for a total of four doses. All three doses of Tf-CRM107 significantly inhibited tumor growth by Day 14 (p < 0.01) and at Day 30 (p < 0.05), with a significant dose-response relationship.

This study demonstrated in vivo efficacy of the targeted toxins Tf-CRM107 and 454A12-rRA against a human glioma. With intratumoral administration, the effect of Tf-CRM107 was tumor-specific and in some animals curative. Regional therapy with these potent tumor-specific agents using direct intratumoral infusion should limit systemic toxicity and may be efficacious against brain tumors.

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Aytac Akbasak, Edward H. Oldfield and Stephen C. Saris

✓ Lysis of tumor cells by activated cytotoxic lymphocytes requires their recognition of antigens associated with major histocompatibility complex molecules. The authors studied the constitutive expression of Class I and Class II major histocompatibility complex antigens on mouse brain-tumor cells and the capacity of different cytokines and cytokine combinations to alter this expression in vitro. Cells from the murine glioma 26 (GL26), glioma 261 (GL261), and ependymoblastoma A (EpA) cell lines were established in monolayer culture and treated for 48 hours with either alpha interferon, gamma interferon, tumor necrosis factor alpha, tumor necrosis factor alpha plus gamma interferon, or interleukin-2. They were then analyzed by flow cytometry for baseline and cytokine-altered major histocompatibility complex expression.

All cell lines had a similar constitutive major histocompatibility complex pattern with low Class I antigen expression and no detectable Class II antigen expression. Alpha interferon substantially induced and up-regulated Class I antigen expression, but had no effect on Class II antigen expression. Gamma interferon also stimulated up-regulation of Class I antigen expression, generally doubling the anti-Class I antigen fluorescence of treated cells. Its effect on Class II antigen expression was more extensive. In the GL26 and GL261 cell lines, the expression of Class II antigen determinants increased to 12 × and 14 × control values and as many as 75% of cells that had no detectable constitutive expression of Class II antigen expressed this antigen after priming with gamma interferon. The addition of tumor necrosis factor alpha to gamma interferon further increased Class II antigen expression on EpA tumor cells only. Interleukin-2 and tumor necrosis factor alpha alone had no effect on Class I or Class II antigen expression of any cell lines.

It is concluded that Class I and Class II antigen expression in mouse glioma cell lines is induced and enhanced after treatment with certain cytokines in vitro. Use of these cell lines to create in situ primary brain tumors in C57BL/6 mice should provide an excellent animal system to study major histocompatibility complex modulation in brain tumor cells and to examine the potential impact of major histocompatibility complex up-regulation on the response of brain tumors to immunotherapy.