Predictors of Response to Autologous Dendritic Cell Therapy in Glioblastoma Multiforme

Abstract

Background: Glioblastoma (GBM) is the most common and lethal primary malignant glioma in adults. Dendritic cell (DC) vaccines have demonstrated promising results in GBM clinical trials. However, some patients do not respond well to DC therapy, with survival rates similar to those of conventional therapy. We retrospectively analyzed clinical and laboratory data to evaluate the factors affecting vaccine treatment.

Methods: Forty-seven patients with de novo GBM were enrolled at China Medical University Hospital between 2005 and 2010 and divided into two subgroups. One subgroup of 27 patients received postsurgical adjuvant immunotherapy with autologous dendritic cell/tumor antigen vaccine (ADCTA) in conjunction with conventional treatment of concomitant chemoradiotherapy (CCRT) with temozolomide. The other 20 patients received only postsurgical conventional treatment without immunotherapy. Immunohistochemistry for CD45, CD4, CD8, programed death ligand 1 (PD-L1), and programed death 1 (PD-1) was performed on sections of surgical tumor specimens and peripheral blood mononuclear cells (PBMCs). Pearson's correlation, Cox proportional hazard model, and Kaplan-Meier analyses were performed to examine the correlations between the prognostic factors and survival rates.

Results: Younger age (<57 years), gross total resection, and CCRT and PD-1+ lymphocyte counts were significant prognostic factors of overall survival (OS) and progression-free survival (PFS) in the ADCTA group. Sex, CD45+ lymphocyte count, CD4+ or CD8+ lymphocyte count, tumor PD-L1 expression, isocitrate dehydrogenase 1 mutation, and O6 methylguanine-DNA methyltransferase promoter methylation status were not significant factors in both groups. In the ADCTA group, patients with tumor-infiltrating lymphocytes (TILs) with a lower PD-1+/CD8+ ratio (≤0.21) had longer OS and PFS (median OS 60.97 months, P < 0.001 and PFS 11.2 months, P < 0.008) compared to those with higher PD-1+/CD8+ ratio (>0.21) (median OS 20.07 months, P < 0.001 and PFS 4.43 months, P < 0.008). Similar results were observed in patients' PBMCs; lymphocyte counts with lower PD-1+/CD8+ ratio (≤0.197) had longer OS and PFS. There was a significant correlation of PD-1+/CD8+ ratio between TILs and PBMCs (Pearson's correlation R2 = 0.6002, P < 0.001). By contrast, CD4-, CD8-, but PD-1+, CD45+ tumor-infiltrating lymphocytes have no impact on OS and PFS (P = 0.073 and P = 0.249, respectively).

Conclusion: For patients receiving DC vaccine adjuvant therapy, better outcomes are predicted in patients with younger age, with TILs or PBMCs with lower PD-1+/CD8+ ratio, with gross tumor resection, and receiving CCRT.

Keywords: PD-1+/CD8+ ratio; autologous dendritic cell/tumor antigen; cytotoxic T-lymphocytes (CD8+); glioblastoma multiforme; immune checkpoints; peripheral blood mononuclear cell; programmed death protein 1 (PD-1+); tumor-infiltrating lymphocytes.

Figures

Figure 1

Treatment schema and vaccine preparation. Clinical schematic diagram (A). Subjects with primary GBM will be consent for operation and concomitant chemoradiotherapy (CCRT). Subjects assigned to the ADCTA group will be designated to receive dendritic cell (DC) vaccination ten times following the clinical trial schedule after operation. V: visits to hospital, numbers following indicates times of visit. DC vaccine manufacturing protocol (B). In China Medical University Hospital, the DC vaccine is produced in laboratories that meet the requirements of Good Tissue Practices and Good Manufacturing Practices. The final product is used in a clinical trial of autologous DC therapy for GBM patients between years 2005 and 2010.

Figure 2

Immunohistochemistry staining of CD4, CD8, programed death 1 (PD-1), and CD45 expression in tumor-infiltrating lymphocytes in glioblastoma (GBM) tissue and peripheral blood mononuclear cells. Representative photomicrographs showing staining for CD4 (A), CD8 (B), PD-1 (C), and CD45 (D) in GBM histological tissue sections (400× magnification) and CD4 (E), CD8 (F), PD-1 (G), and CD45 (H) in peripheral blood mononuclear cell cytoblocks (400× magnification).

Figure 3

Immunohistochemistry staining pattern of positive and negative PD-L1 expression in GBM tumor cells. Representative photomicrographs showing diffuse strong cell membrane staining for PD-L1 (A), and complete negative staining for PD-L1 (B) in GBM histological tissue sections (400× magnification).

Figure 4

Overall survival (OS) in ADCTA versus reference group glioblastoma patients. Kaplan–Meier survival plots of the OS of 27 ADCTA patients compared to 20 reference patients.

Figure 5

Overall survival (OS) (A) and progression-free survival (PFS) (B) by high or low PD-1+/CD8+ ratio tumor-infiltrating lymphocytes in the ADCTA group. Kaplan–Meier survival plots of OS and PFS of 14 patients with high PD-1+/CD8+ ratio compared to 13 patients with low PD-1+/CD8+ ratio patients.

Figure 6

PD-1+/CD8+ ratios of individual patients’ tumor-infiltrating lymphocytes and overall survival (OS) and progression-free survival (PFS) in the ADCTA group. Pearson’s correlation of each patient’s PD1+/CD8+ ratio value and their OS (A) and PFS time (B) after natural logarithm transformation.

Figure 7

Correlation between tumor-infiltrating lymphocytes and peripheral blood mononuclear cells (PBMCs) in the ADCTA group. Pearson’s correlation between tumor-infiltrating lymphocytes and PBMCs from ADCTA patients (18 patients).

Figure 8

Overall survival (OS) (A) and progression-free survival (PFS) (B) according to high or low PD-1+/CD8+ ratio in peripheral blood mononuclear cells in the ADCTA group. Kaplan–Meier survival plots of OS and PFS of 10 high PD-1+/CD8+ ratio patients compared to 8 low PD-1+/CD8+ ratio patients.

Figure 9

Co-expression of programed death 1 (PD-1) and CD8 in PD-1-positive and CD8-positive tumor-infiltrating lymphocytes and peripheral blood mononuclear cells (PBMCs). Paired GBM tissue sections and PBMC cytoblocks from a representative patient with a high PD-1+/CD8+ ratio were selected for immunofluorescence (IF) analysis of PD-1 and CD8 co-expression. CD8 and PD-1 expression in the TIL of GBM tissue [(A), upper panel] and reconstruction of single slices of the xz and the yz planes in the z-axis stacked image [(A), lower panel]. CD8 and PD-1 expression in PBMCs derived from the same patient [(B), upper panel] and reconstruction of the same immunofluorescent slide [(B), lower panel]. Paired GBM tissue sections and PBMC cytoblocks from a representative patient with a low PD-1+/CD8+ ratio were selected for IF analysis of PD-1 and CD8 co-expression. CD8 and PD-1 expression in the TIL of GBM tissue [(C), upper panel] and reconstruction of single slices of the xz and yz planes in the z-axis stacked image [(C), lower panel]. CD8 and PD-1 expression in PBMCs derived from the same patient [(D), upper panel] and reconstruction of the same immunofluorescent slide [(D), lower panel].

Figure 10

Flow cytometry of peripheral blood mononuclear cells (PBMCs) of representative patients. PBMCs (1 × 105) were collected and stained with antibodies against programed death 1 (PD-1), CD8, and CD3. The percpCy5.5-labeled CD3+ T cells (middle panel) were gated form the size and granularity dot-plot (left panel), then these CD3+ T cells were analyzed by the PE-Cy7 and FITC dot-plot to identify the populations of CD8 and PD-1-expressing cells. Patient 21 had a low PD-1+/CD8+ ratio in the PBMC cytoblock (A). Patient 41 was with a high PD-1+/CD8+ ratio in PBMC cytoblock (B).

Figure 11

Schematic representation of dendritic cell vaccine treatment PD-1+ or PD-1− cytotoxic T cells in the peripheral blood arrive at lymph nodes for antigen activation after vaccination. After reaching the tumor microenvironment, the efficiency of tumor cell killing by cytotoxic T cells is determined by the proportion of PD-1+ cytotoxic T cells.