Effect of temozolomide on dendritic cell vaccine for glioblastoma.

Abstract

e14638 Background: Glioblastoma is an aggressive, malignant intracranial tumor with an average survival of 14.6 months. The standard therapeutic approach includes surgical resection followed with adjuvant chemotherapy. Recently there is a new immunotherapy: monocyte-derived dendritic cell (DC) vaccine. After exposure to patients' resected tumor sample, DC vaccine serves as antigen presenting cells to stimulate the immune system’s T cell response against patients' tumor. DC vaccine revealed an overall survival of 60 months in grade 4 patients (200% improvement) in a phase I/II clinical trial. However, in addition to DC vaccine, 70% of patients were also receiving chemotherapies including Temozolomide (TMZ), an alkylating agent. We conducted in vitro cell growth experiments to determine the effect and best timing of TMZ administration, before or after surgical resection, with DC vaccine to optimize care. Methods: Peripheral blood mononuclear cells were isolated from donors’ Leukoreduction Chambers via Ficoll-Paque gradient and cultured with GM-CSF and IL-4 for 5 days to differentiate into immature dendritic cells (iDC). Half of the iDC received TNF-a maturation stimulus for 2 days to become mature dendritic cells (mDC). The iDC and mDC were resuspended with U87 glioblastoma cell lines untreated and treated with TMZ, simulating potential TMZ before surgical resection. The treated and untreated mDC and iDC are harvested, irradiated, and used to stimulate a first round of lymphocyte expansion. After 7 days TMZ was added (to simulate TMZ post tumor resection) to half the samples for a second lymphocyte expansion. 8 treatment groups were analyzed with Flow Cytometry along with lymphocytes untreated (-) and treated with PHA (+). FloJo X software was used for CD25, CD3, CD4, and CD8 analysis and paired T tests calculated statistical significance of T cell induction. Results: First, untreated Glioblastoma (U87) cells allowed DC vaccine to induce more T cell proliferation. The iDC with untreated U87 cells induced statistically more T cells than iDC with TMZ treated U87 cells (p = .003). Notably, cell counts for CD4 is 11% and CD8 is 34% greater. The mDC with untreated U87 also induced statistically more T cells than mDC with TMZ treated U87 (p = .004). Notably, CD4 is 15% and CD8 is 27% greater. Next, a secondary lymphocyte stimulation of TMZ alongside DC vaccine induces more T cell proliferation. For lymphocytes exposed to TMZ treated iDC, a TMZ treatment induced statistically more T cells than without TMZ (p = .002). Notably, cell counts for CD4 is 13.7% and CD8 is 43.3% greater. For lymphocytes exposed to untreated iDC, a TMZ treatment also induced statistically more T cells than without TMZ (p = .01). Notably, CD4 is 43.7% and CD8 is 117% greater. Conclusions: The optimal induction of T cells is first surgical resection of the untreated glioblastoma tumor to create the DC vaccine followed by administration of DC vaccine with TMZ to elicit a secondary lymphocyte challenge.