Long-term Survival in Glioblastoma with Cytomegalovirus pp65-Targeted Vaccination

Abstract

Purpose: Patients with glioblastoma have less than 15-month median survival despite surgical resection, high-dose radiation, and chemotherapy with temozolomide. We previously demonstrated that targeting cytomegalovirus pp65 using dendritic cells (DC) can extend survival and, in a separate study, that dose-intensified temozolomide (DI-TMZ) and adjuvant granulocyte macrophage colony-stimulating factor (GM-CSF) potentiate tumor-specific immune responses in patients with glioblastoma. Here, we evaluated pp65-specific cellular responses following DI-TMZ with pp65-DCs and determined the effects on long-term progression-free survival (PFS) and overall survival (OS).Experimental Design: Following standard-of-care, 11 patients with newly diagnosed glioblastoma received DI-TMZ (100 mg/m²/d × 21 days per cycle) with at least three vaccines of pp65 lysosome-associated membrane glycoprotein mRNA-pulsed DCs admixed with GM-CSF on day 23 ± 1 of each cycle. Thereafter, monthly DI-TMZ cycles and pp65-DCs were continued if patients had not progressed.Results: Following DI-TMZ cycle 1 and three doses of pp65-DCs, pp65 cellular responses significantly increased. After DI-TMZ, both the proportion and proliferation of regulatory T cells (Tregs) increased and remained elevated with serial DI-TMZ cycles. Median PFS and OS were 25.3 months [95% confidence interval (CI), 11.0-∞] and 41.1 months (95% CI, 21.6-∞), exceeding survival using recursive partitioning analysis and matched historical controls. Four patients remained progression-free at 59 to 64 months from diagnosis. No known prognostic factors [age, Karnofsky performance status (KPS), IDH-1/2 mutation, and MGMT promoter methylation] predicted more favorable outcomes for the patients in this cohort.Conclusions: Despite increased Treg proportions following DI-TMZ, patients receiving pp65-DCs showed long-term PFS and OS, confirming prior studies targeting cytomegalovirus in glioblastoma. Clin Cancer Res; 23(8); 1898-909. ©2017 AACR.

Figure 1

Schema of ATTAC-GM trial. Following standard of care with gross total resection (> 90%), external beam radiation (RT) and temozolomide (TMZ), patients received DI-TMZ cycle 1 (100 mg/m²/d) for 21 days of a 28-day cycle. DC vaccines consisted of 2×10⁷ mature pp65- lysosome-associated membrane glycoprotein (LAMP) mRNA-pulsed DCs (pp65-DCs) admixed with 150 µg GM-CSF. Vaccination with pp65-DCs occurred on Day 23 ± 1 of the 28-day cycle with the first three DC vaccines administered two weeks apart. Following DI-TMZ cycle 2 and DC Vaccine-4, patients then received monthly DC vaccines administered on DI-TMZ cycle Day 23 ± 1 for a total of 10 vaccines in conjunction with monthly DI-TMZ cycles for a total of 6 to 12 cycles unless progression occurred. Patients were imaged bi-monthly without receiving any other prescribed anti-tumor therapy. For immune monitoring of pp65 responses, peripheral blood mononuclear cells were sampled at Pheresis-1 and Pheresis-2, along with blood draws just prior to vaccination with pp65-DCs.

Figure 2

Survival rates in patients receiving pp65-DCs and DI-TMZ compared to historical controls. A, PFS and B, OS of study patients (n = 11) with newly-diagnosed GBM receiving DI-TMZ conditioning and GM-CSF-containing pp65-DC vaccines compared to matched historical controls (n = 23) with newly diagnosed GBM treated with standard of care and additional therapies after disease progression. Kaplan Meier survival curves represent observed rates for DI-TMZ + pp65 DC patients who completed the predefined study therapy. Of all 11 patients, four had not progressed and were alive at the time of survival analysis (DI-TMZ + pp65-DCs median PFS = 25.3 months [CI₉₅: 11.0-∞] vs. Historical controls median PFS = 8.0 months [CI₉₅: 6.2–10.8], P = 0.0001; DI-TMZ + pp65-DCs median OS = 41.1 months [CI₉₅: 21.6-∞] vs. Historical controls median OS = 19.2 months [CI₉₅: 14.3–21.3], P = 0.0001, log-rank test).

Figure 3

Patient pp65 ELISpot responses following DI-TMZ and sequential pp65-DC vaccination. A, pp65 antigen-specific T-cell responses as measured by IFN-γ ELISpot ex vivo. Before-and-after pp65 ELISpot following three vaccinations of pp65-DCs from Vaccine-1 to Pheresis-2 (mean ± sem spot-forming cells (SFC) per 10⁶ PBMC) in all patients (n = 11) are shown after stimulation with 138 15-mer peptides overlapping by 11 amino acids spanning the entire pp65 gene (P = 0.019 Wilcoxon signed rank). B, Kinetics of pp65 ELISpot throughout continuous pp65-DC vaccination and intervening DI-TMZ cycles. Timing of DI-TMZ cycles are shown as detached lines. C, Fold changes in functional pp65 ELISpot from baseline pp65 reactivity prior to Vaccine-1. Fold increases stratified by patient OS > 40 months (n = 6) and OS < 40 months (n = 5). Post Vaccine-1: mean 1.51 vs. 3.75 (P = 0.031), Post Vaccine-2: mean 2.20 vs. 5.14 (P = 0.031), Post Vaccine-3: mean 3.45 vs. 9.79 (P = 0.031 Wilcoxon signed rank). ELISpot 0 values normalized to [0 + 1] for calculation of fold change from baseline.

Figure 4

MRI changes in long-term survivors with sequential pp65-DC vaccination. A, Sequential MRI scans (FLAIR and T1-weighted with contrast) of four long term survivors receiving pp65-DCs and DI-TMZ (Patient 2, Patient 3, Patient 4, and Patient 5). Repeat MRI scans demonstrate steadily decreasing FLAIR hyperintensity and stable or decreasing contrast enhancement with collapse of the resection cavity. B, Satellite FLAIR hyperintense lesions appearing after several vaccinations with pp65-DCs in two patients with prolonged OS (Patient 4 and Patient 7). These lesions were not originally present at the post-XRT/TMZ scan and were calculated to be outside the range of XRT high-dose radiation fields. Presentation of these lesions was first detected after Vaccine-4 and Vaccine-7, and their signal persisted through Vaccine-10.

Figure 4

MRI changes in long-term survivors with sequential pp65-DC vaccination. A, Sequential MRI scans (FLAIR and T1-weighted with contrast) of four long term survivors receiving pp65-DCs and DI-TMZ (Patient 2, Patient 3, Patient 4, and Patient 5). Repeat MRI scans demonstrate steadily decreasing FLAIR hyperintensity and stable or decreasing contrast enhancement with collapse of the resection cavity. B, Satellite FLAIR hyperintense lesions appearing after several vaccinations with pp65-DCs in two patients with prolonged OS (Patient 4 and Patient 7). These lesions were not originally present at the post-XRT/TMZ scan and were calculated to be outside the range of XRT high-dose radiation fields. Presentation of these lesions was first detected after Vaccine-4 and Vaccine-7, and their signal persisted through Vaccine-10.

Figure 5

T_Reg responses following DI-TMZ. A, T_Reg proportions increase from Pheresis-1 (7.3% ± 0.96, range 3.4–13.1) to Vaccine-1 (13.1% ± 1.3, range 6.9–22.2) following DI-TMZ cycle 1 (mean ± sem, P = 0.001 Wilcoxon signed rank). B, Repeated vaccination and reintroduction of DI-TMZ cycles are compounded by steadily increasing T_Reg proportions from Pheresis-2 to Vaccine-7). C, T_Reg proliferation by Ki67 from Pheresis-1 (18.3% ± 4.4, range 2.96–56.2) to Vaccine-1 (40.8% ± 3.7, range 27.2–69.9) following DI-TMZ cycle 1 (mean ± sem, P = 0.002 Wilcoxon signed rank). D, T_Reg proliferation initially increases following DI-TMZ cycle 1 but remain steady in proliferative capacity following continuous DI-TMZ cycles.

Figure 6

Sequential pp65 DC vaccination expands peripheral CD8⁺ T cells and CD8 : T_Reg ratios but does not affect conventional CD4⁺ counts or CD4 : T_Reg ratios. A, CD8⁺ T cell counts in the peripheral blood of patients decrease following DI-TMZ cycle 1 from Pheresis-1 to Vaccine-1 (P = 0.020) and steadily increase with sequential vaccination of pp65-DCs from Vaccine-1 to Vaccine-3 (P = 0.012). B, CD8 : T_Reg ratios steadily increase following sequential pp65-DCs (Vaccine-1 to Vaccine-3, P = 0.004) C, Conventional CD4⁺ T cell counts in the peripheral blood of patients dramatically diminish following DI-TMZ cycle 1 (P = 0.037) and do not increase following sequential pp65 DC vaccination. D, CD4 : T_Reg ratios decrease following DI-TMZ cycle 1 (P = 0.002) but are not affected by sequential pp65-DCs (A-D, mean ± sem, Wilcoxon signed rank).