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Howard Broder, Andrea Anderson, Sylvia K. Odesa, Thomas J. Kremen and Linda M. Liau

Object

Dendritic cells (DCs) are potent antigen-presenting cells that have been shown to play a critical role in the initiation of host immune responses against tumor antigens. In this study, a recombinant adenovirus vector encoding the melanoma-associated antigen, MART-1, was used to transduce murine DCs, which were then tested for their ability to activate cytotoxic T lymphocytes (CTLs) and induce protective immunity against B16 melanoma tumor cells implanted intracranially.

Methods

Genetic modification of murine bone marrrow–derived DCs to express MART-1 was achieved through the use of an E1-deficient, recombinant adenovirus vector (AdVMART1). Sixty-two C57BL/6 mice were immunized by subcutaneous injection of AdVMART-1-transduced DCs (23 mice), untransduced DCs (17 mice), or sterile saline (22 mice). Using the B16 murine melanoma, which naturally expresses the MART-1 antigen, all the mice were then challenged intracranially with viable, unmodified syngeneic B16 tumor cells 7 days later. Splenocytes obtained from representative animals in each group were harvested for standard cytotoxicity and enzyme-linked immunospot assays. The remaining mice were followed for survival.

Immunization of C57BL/6 mice with DCs transduced with AdVMART1-DC elicited the development of antigen-specific CTL responses. As evidenced by a prolonged survival curve when compared with control-immunized mice harboring intracranial B16 tumors, AdMART1-DC vaccination was able to elicit partial protection against central nervous system (CNS) tumor challenge in vivo. However, this CNS antitumor immunity was weaker than that previously demonstrated against subcutaneous B16 tumors in which the same vaccination strategy was used.

Conclusions

These data suggest that immune responses generated against CNS tumors by DC-based vaccines may be different from those obtained against subcutaneous tumors.

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Isaac Yang, Thomas J. Kremen, Adrian J. Giovannone, Elena Paik, Sylvia K. Odesa, Robert M. Prins and Linda M. Liau

Object

Little is known about the quantitative modulation of major histocompatibility complex (MHC) Class I expression on human gliomas that is effected by interferons; even less is known about the immunogenic peptides that are accommodated in the peptide-binding motifs of MHC Class I alleles in these brain tumors. In this article the authors investigated the ability of interferon (IFN)α and IFNγ to upregulate MHC Class I expression and to modulate acid-eluted Class I—bound peptides on human glioblastoma multiforme (GBM) cells in vitro.

Methods

Early-passage primary human GBM cell cultures and U87MG GBM cells were incubated with varying doses of INFα or IFNγ ranging between 0 and 2000 U/ml. Upregulation of MHC Class I expression was assayed by immunocytochemical analysis, flow cytometry, and Western blot analysis. Modulation of acid-eluted MHC Class I—bound peptides from the IFN-treated GBM cells was examined with the aid of mass spectroscopy.

The in vitro expression of the MHC Class I molecule was upregulated by both IFNα and IFNγ in a dose-dependent manner. Interferon-γ exhibited a more potent effect on MHC Class I upregulation, peaking at 10 U/ml; whereas the effect of IFNα was less marked, reaching a plateau at 500 U/ml. In addition, a native peptide eluted from MHC Class I molecules of human GBM cells was identified and found to be consistently upregulated by IFN treatment.

Conclusions

Interferon-α and IFNγ can significantly upregulate the MHC Class I molecules that are expressed on the cell surface of human GBM cells as well as the potentially immunogenic peptides bound to the MHC. These results may help explain the molecular basis for increased immunogenicity with IFN treatment of human GBMs and might provide added insight into the design of future antitumor vaccines for human brain tumors.

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Linda M. Liau, Keith L. Black, Neil A. Martin, Steven N. Sykes, Jeff M. Bronstein, Lisa Jouben-Steele, Paul S. Mischel, Arie Belldegrun and Timothy F. Cloughesy

Dendritic cells (DCs) are antigen-presenting cells that play a central role in the initiation and modulation of antitumor immune responses. In this pilot study, we investigated the ability of autologous DCs pulsed ex vivo with allogeneic major histocompatibility complex class I–matched glioblastoma peptides to stimulate host antitumor immune responses when injected as a vaccine.

A patient with recurrent brainstem glioblastoma multiforme (GBM) received a series of three intradermal immunizations of antigen-pulsed DCs on an outpatient basis following surgical debulking of her posterior fossa tumor. Dendritic cell vaccination was well tolerated, and no clinical signs of autoimmunity or experimental allergic en-cephalomyelitis were detected. She developed a measurable cellular immune response against the allogeneic glioblastoma peptides used in her vaccine preparation, as demonstrated by in vitro T-cell proliferation assays. In addition, increased T-cell infiltration was noted within the intracranial tumor site in the biopsy sample obtained following DC vaccination. An objective clinical response, however, was not evident, and this patient eventually died 21 months after her disease was diagnosed.

To our knowledge, this is the first patient with brain cancer ever to be treated with DC-based immunotherapy. This case illustrates that vaccination with DCs pulsed with acid-eluted glioblastoma peptides is feasible and can induce systemic antigen-specific immunity in a patient with recurrent GBM. Additional studies are necessary to determine the optimum DC doses and antigen loading conditions that may translate into clinical effectiveness and survival benefit for patients with brain tumors. Phase I trials for malignant glioma are currently underway.

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Linda M. Liau, Keith L. Black, Robert M. Prins, Steven N. Sykes, Pier-Luigi DiPatre, Timothy F. Cloughesy, Donald P. Becker and Jeff M. Bronstein

Object. An approach toward the treatment of intracranial gliomas was developed in a rat experimental model. The authors investigated the ability of “professional” antigen-presenting cells (dendritic cells) to enhance host antitumor immune responses when injected as a vaccine into tumor-bearing animals.

Methods. Dendritic cells, the most potent antigen-presenting cells in the body, were isolated from rat bone marrow precursors stimulated in vitro with granulocyte—macrophage colony-stimulating factor (GM-CSF) and interleukin-4. Cultured cell populations were confirmed to be functional antigen-presenting cells on the basis of expressed major histocompatibility molecules, as analyzed by fluorescence-activated cell sorter cytofluorography. These dendritic cells were then pulsed (cocultured) ex vivo with acid-eluted tumor antigens from 9L glioma cells. Thirty-eight adult female Fischer 344 rats harboring 7-day-old intracranial 9L tumors were treated with three weekly subcutaneous injections of either control media (10 animals), unpulsed dendritic cells (six animals), dendritic cells pulsed with peptides extracted from normal rat astrocytes (10 animals), or 9L tumor antigen—pulsed dendritic cells (12 animals). The animals were followed for survival. At necropsy, the rat brains were removed and examined histologically, and spleens were harvested for cell-mediated cytotoxicity assays.

The results indicate that tumor peptide-pulsed dendritic cell therapy led to prolonged survival in rats with established intracranial 9L tumors implanted 7 days prior to the initiation of vaccine therapy in vivo. Immunohistochemical analyses were used to document a significantly increased perilesional and intratumoral infiltration of CD8+ and CD4+ T cells in the groups treated with tumor antigen—pulsed dendritic cells compared with the control groups. In addition, the results of in vitro cytotoxicity assays suggest that vaccination with these peptide-pulsed dendritic cells can induce specific cytotoxic T lymphocytes against 9L tumor cells.

Conclusions. Based on these results, dendritic antigen-presenting cells pulsed with acid-eluted peptides derived from autologous tumors represent a promising approach to the immunotherapy of established intracranial gliomas, which may serve as a basis for designing clinical trials in patients with brain tumors.

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Edward F. Chang, Aaron Clark, Randy L. Jensen, Mark Bernstein, Abhijit Guha, Giorgio Carrabba, Debabrata Mukhopadhyay, Won Kim, Linda M. Liau, Susan M. Chang, Justin S. Smith, Mitchel S. Berger and Michael W. McDermott

Object

Medical and surgical management of low-grade gliomas (LGGs) is complicated by a highly variable clinical course. The authors recently developed a preoperative scoring system to prognosticate outcomes of progression and survival in a cohort of patients treated at a single institution (University of California, San Francisco [UCSF]). The objective of this study was to validate the scoring system in a large patient group drawn from multiple external institutions.

Methods

Clinical data from 3 outside institutions (University of Utah, Toronto Western Hospital, and University of California, Los Angeles) were collected for 256 patients (external validation set). Patients were assigned a prognostic score based upon the sum of points assigned to the presence of each of the 4 following factors: 1) location of tumor in presumed eloquent cortex, 2) Karnofsky Performance Scale (KPS) Score ≤ 80, 3) age > 50 years, and 4) maximum diameter > 4 cm. A chi-square analysis was used to analyze categorical differences between the institutions; Cox proportional hazard modeling was used to confirm that the individual factors were associated with shorter overall survival (OS) and progression-free survival (PFS); and Kaplan–Meier curves estimated OS and PFS for the score groups. Differences between score groups were analyzed by the log-rank test.

Results

The median OS duration was 120 months, and there was no significant difference in survival between the institutions. Cox proportional hazard modeling confirmed that the 4 components of the UCSF Low-Grade Glioma Scoring System were associated with lower OS in the external validation set; presumed eloquent location (hazard ratio [HR] 2.04, 95% CI 1.28–2.56), KPS score ≤ 80 (HR 5.88, 95% CI 2.44–13.7), age > 50 years (HR 1.82, 95% CI 1.02–3.23), and maximum tumor diameter > 4 cm (HR 2.63, 95% CI 1.58–4.35). The stratification of patients based on scores generated groups (0–4) with statistically different OS and PFS estimates (p < 0.0001, log-rank test). Lastly, the UCSF patient group (construction set) was combined with the external validation set (total of 537 patients) and analyzed for OS and PFS. For all patients, the 5-year survival probability was 0.79; the 5-year cumulative OS probabilities stratified by score group were: score of 0, 0.98; score of 1, 0.90; score of 2, 0.81; score of 3, 0.53; and score of 4, 0.46.

Conclusions

The UCSF scoring system accurately predicted OS and PFS in an external large, multiinstitutional population of patients with LGGs. The strengths of this system include ease of use and ability to be applied preoperatively, with the eventual goal of aiding in the design of individualized treatment plans for patients with LGG at diagnosis.