Gene expression profile correlates with T-cell infiltration and relative survival in glioblastoma patients vaccinated with dendritic cell immunotherapy

Abstract

Purpose: To assess the feasibility, safety, and toxicity of autologous tumor lysate-pulsed dendritic cell (DC) vaccination and toll-like receptor (TLR) agonists in patients with newly diagnosed and recurrent glioblastoma. Clinical and immune responses were monitored and correlated with tumor gene expression profiles.

Experimental design: Twenty-three patients with glioblastoma (WHO grade IV) were enrolled in this dose-escalation study and received three biweekly injections of glioma lysate-pulsed DCs followed by booster vaccinations with either imiquimod or poly-ICLC adjuvant every 3 months until tumor progression. Gene expression profiling, immunohistochemistry, FACS, and cytokine bead arrays were performed on patient tumors and peripheral blood mononuclear cells.

Results: DC vaccinations are safe and not associated with any dose-limiting toxicity. The median overall survival from the time of initial surgical diagnosis of glioblastoma was 31.4 months, with a 1-, 2-, and 3-year survival rate of 91%, 55%, and 47%, respectively. Patients whose tumors had mesenchymal gene expression signatures exhibited increased survival following DC vaccination compared with historic controls of the same genetic subtype. Tumor samples with a mesenchymal gene expression signature had a higher number of CD3(+) and CD8(+) tumor-infiltrating lymphocytes compared with glioblastomas of other gene expression signatures (P = 0.006).

Conclusion: Autologous tumor lysate-pulsed DC vaccination in conjunction with TLR agonists is safe as adjuvant therapy in newly diagnosed and recurrent glioblastoma patients. Our results suggest that the mesenchymal gene expression profile may identify an immunogenic subgroup of glioblastoma that may be more responsive to immune-based therapies.

Figure 1

MRI changes after DC vaccination. Transient increase in MRI T2/FLAIR lesions (A) and contrast enhancement (B) observed in a primary, newly diagnosed glioblastoma patient following DC vaccination (patient GBM5-4). Axial T2/FLAIR (A) and T1/contrast (B) MRI scans taken at 2 weeks pre-vaccination, 2 weeks post-vaccination, and 4 months later.

Figure 2

Peripheral blood immune monitoring data. (A) PBMC’s from normal volunteers and DC trial patient pre-vaccination timepoints were thawed and stained for the expression of CD3, CD4 and CD25, followed by the intracellular labeling of Foxp3. Stained cells were acquired on a BD FacsCalibur flow cytometer and analyzed using FloJo software. The frequencies of CD3⁺CD4⁺Foxp3⁺ and CD3⁺CD4⁺CD25⁺Foxp3⁺ PBMC’s between normal volunteers and glioblastoma patients enrolled in this trial are compared. (*p=0.04; **p=0.01) (B,C) Serum cytokine responses, measured pre- and day 14 post-vaccination, after the initial course of DC vaccination (B) or after booster DC vaccinations with either 5% imiquimod or poly ICLC (C). Serum from patients enrolled on this clinical trial was thawed, labeled with cytometric bead array (CBA) antibody-coated beads, washed and subjected to analysis on a BD FacsCalibur flow cytometer together with cytokine standards. Quantitative assessment of cytokine levels was accomplished with a Microsoft Excel-based CBA software program. (D) Th1/Th2 cytokine ratios. Raw cytokine data for serum TNF-α and IL-10 at each timepoint were divided to generate a Th1:Th2 ratio.

Figure 3

Microarray-based, expression profiling of pre-treatment glioblastoma samples from DC vaccine patients. Total RNA was isolated from frozen, surgically-resected tumors and subjected to global gene expression classification using Affymetrix human U133 Plus 2.0 microarray chips. Sufficient fresh-frozen tissue was available for extraction of high-quality RNA (without amplification) in 17 of the cases. Proneural (HC1, yellow legend), Proliferative (HC2A, blue legend), and Mesenchymal (HC2B, red legend) gene expression signatures were identified using probesets previously published (21). Heat maps were created using the dChip microarray software program.

Figure 4

Extended survival in DC vaccinated patients with mesenchymal gene expression signatures, but not in patients with a proneural signature. The overall survival time of DC vaccine patients expressing a (A) Proneural (PN) gene signature (n=5) or (B) Mesenchymal (Mes) gene signature (n=9) was compared with the survival generated from a control, multi-institutional dataset of PN (n=60) or Mes glioblastomas (n=82; solid lines) previously published by our group (21). To accurately account for the potential bias associated with the time delay needed to generate the DC vaccine, we omitted control patients that experienced early progression (<250 days). PN comparison: p=0.664 (not statistically different, ns); Mes comparison (p=0.0046) by the Log-rank (Mantel-Cox) Test calculated in GraphPad software.

Figure 5

Increased density of CD3⁺ and CD8⁺ lymphocytes in Mes gene expression groups compared with PN tumor sections. (A) 3 µm paraffin-embedded adjacent tissue sections from DC vaccinated patients were stained separately with CD3 and CD8 antibodies and scored in a blinded fashion by a neuropathologist (WHY). The IHC scores were compared between samples known to be PN (n=5) vs. Mes tumor samples (n=9). *p=0.006 by two-tailed t test calculated in GraphPad software. (B) Representative hematoxylin & eosin staining and CD8 IHC staining (pre- and post-DC vaccination) of a PN and Mes glioblastoma showing increased CD8⁺ TILs in the Mes glioblastoma. Original magnification: × 400.