Sequential bortezomib and temozolomide treatment promotes immunological responses in glioblastoma patients with positive clinical outcomes: A phase 1B study

Abstract

Background: Glioblastoma (GBM) is an aggressive malignant brain tumor where median survival is approximately 15 months after best available multimodal treatment. Recurrence is inevitable, largely due to O6 methylguanine DNA methyltransferase (MGMT) that renders the tumors resistant to temozolomide (TMZ). We hypothesized that pretreatment with bortezomib (BTZ) 48 hours prior to TMZ to deplete MGMT levels would be safe and tolerated by patients with recurrent GBM harboring unmethylated MGMT promoter. The secondary objective was to investigate whether 26S proteasome blockade may enhance differentiation of cytotoxic immune subsets to impact treatment responses measured by radiological criteria and clinical outcomes.

Methods: Ten patients received intravenous BTZ 1.3 mg/m2 on days 1, 4, and 7 during each 4th weekly TMZ-chemotherapy starting on day 3 and escalated from 150 mg/m2 per oral 5 days/wk via 175 to 200 mg/m2 in cycles 1, 2, and 3, respectively. Adverse events and quality of life were evaluated by CTCAE and EQ-5D-5L questionnaire, and immunological biomarkers evaluated by flow cytometry and Luminex enzyme-linked immunosorbent assay.

Results: Sequential BTZ + TMZ therapy was safe and well tolerated. Pain and performance of daily activities had greatest impact on patients' self-reported quality of life and were inversely correlated with Karnofsky performance status. Patients segregated a priori into three groups, where group 1 displayed stable clinical symptoms and/or slower magnetic resonance imaging radiological progression, expanded CD4+ effector T-cells that attenuated cytotoxic T-lymphocyte associated protein-4 and PD-1 expression and secreted interferon γ and tumor necrosis factor α in situ and ex vivo upon stimulation with PMA/ionomycin. In contrast, rapidly progressing group 2 patients exhibited tolerised T-cell phenotypes characterized by fourfold to sixfold higher interleukin 4 (IL-4) and IL-10 Th-2 cytokines after BTZ + TMZ treatment, where group 3 patients exhibited intermediate clinical/radiological responses.

Conclusion: Sequential BTZ + TMZ treatment is safe and promotes Th1-driven immunological responses in selected patients with improved clinical outcomes (Clinicaltrial.gov (NCT03643549)).

Keywords: MGMT; Th1/Th2 cytokine ratios; bortezomib; immune checkpoint; recurrent GBM; temozolomide.

Conflict of interest statement

The authors declare that there are no conflict of interests.

© 2020 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.

Figures

Figure 1

Schematic of trial schedule. A, Timeline showing BORTEM‐17 treatment regimen. Bortezomib administered intravenous at days 1, 4, and 7 (48 hours pretreatment to deplete MGMT protein) before target TMZ 200 mg/m2 dose for 5 days (from and including days 3‐7), repeated in six cycles. In n = 3 patients per each dose 150 vs 175 vs 200 mg/m2 TMZ in dose pathfinding, safety evaluation. Clinical chemistry for renal, hepatic, and bone marrow monitoring for toxicity based on CTCAE v. 4.03. MRI tumor monitoring radiological response assessment based on RANO criteria. Rationale for sequential treatment schedule based on preclinical data. B, sequential administration in BORTEM‐17 clinical trial vs (C) previous studies where BTZ on days 1, 4, 8, and 11 was given concomitantly with TMZ from day 1 to 5, when MGMT levels were high. Dashed boxes mark days when TMZ doses might be more effective, (B) all five doses vs (C) three doses every month. BTZ, bortezomib; CBC Diff, complete blood count with differential test; CMP, comprehensive metabolic panel; KPS, Karnofsky performance score; LFT, liver function test; MGMT, O6‐methyl guanine DNA methyltransferase; MRI, magnetic resonance imaging; NANO, neurologic assessment in neuro‐oncology; QoL, quality of life; TMZ, temozolomide

Figure 2

Clinical, radiological and quality of life response. A, Pharmacokinetic analysis of bortezomib concentration (ng/mL) in plasma of patients following five timepoints on day 1 and 7 of treatment. B, Thrombocyte counts during the first three treatment cycles. Data represent the mean ± SEM from n = 10 patients. C, Density scores for perception of overall health on EQ‐5D‐5L questionnaire. D, Correlation of self‐reported overall health and levels of pain on EQ‐5D‐5L. E, Swimmer plot of individual patients depicting treatment start/stop times of all trial patients, aligned according to their first surgery. Bars represent survival in months from first surgery, first and second MRI progression. Rightward arrow indicates that the patient was still alive at the time of final data collection

Figure 3

Tumour growth and population doubling time. A, Coregistered 3D T1 weighted gadolinium contrast enhanced serial MR image of patient‐03 (IDHwt; MGMT UM, 54 years male) treated for 6 months, time indicated in days and tumour volume in µL. B, Mean of 3D measured tumour volumes in mm3 from T1‐weighted MR images with contrast of all patients, and (C) tumor population doubling time (in days) for group 1, 2, and 3 patients

Figure 4

Immune mechanisms and augmented differentiation of CD4+ and CD8+ T cell subsets in patients with positive clinical responses. A, Cytokine concentrations in pg/mL in patients' plasma, (top panels) IL‐4 and IL‐10, and (bottom panels) the ratio between the average concentrations of IL‐5 and IL‐4 against IFNγ during treatment. B, Representative dot plots showing (from left to right) patient NK and T cell lymphocytes in CD56 vs CD3 gates, CD8+ vs CD4+ T cell subsets and CD3+CD4+ T cells expressing CTLA‐4. C, Representative dot plots showing CD3+CD4+ T cell subsets expressing CTLA‐4 and (D) Representative dot plots showing CD3+CD8+ T cell subsets expressing PD‐1. E, Mean ± SEM % of CD3+CD4+ T cells expression of PD‐1 and CTLA‐4 in group 1, 2, and 3 patients before, during and after treatment with BTZ and TMZ. F, Mean ± SEM % of CD3+CD8+ expressing PD‐1 and CTLA‐4 before and after treatment with BTZ and TMZ. G, Dot plots showing CCR7 vs CD45RO within CD3+CD4+ T cell subsets in group 1, 2, and 3 patients, before and during treatment with BTZ and TMZ. H, Mean ± SEM % expression of naïve (N), effector (E), central memory (CM) and effector memory (EM) CD4+ T‐cells, and (I) CD4+ T‐effector cells before, during and after treatment with BTZ and TMZ in group 1, 2, and 3 patients. *P < .05, **P < .01, ***P < .001, ****P < .0001, n = 10 patients. BTZ, bortezomib; IFNγ, interferon γ; IL, interleukin; NK, natural killer; TMZ, temozolomide

Figure 5

PBMCs from patients with positive clinical responses diminish CTLA‐4+ CD4+Th cells, attenuate IL‐10 secretion and have moderate intracellular IFNγ after bortezomib + temozolomide treatment. A, Representative dot plots showing CD4 vs CTLA‐4 in patient PBMCs unstimulated and stimulated, with PMA/ionomycin before or after exposure to BTZ or BTZ + TMZ treatment in situ. B, Mean ± SEM % of CD4+ CTLA‐4+ T cells at baseline or after treatment/stimulation conditions in group 1, 2, and 3 patients. C, Mean ± SEM % of CD8+ CTLA‐4+ at baseline or after treatment/stimulation conditions in group 1, 2, and 3 patients. D Representative dot plots showing intracellular IFNγ in CD8+T cells in patients' PBMCs unstimulated and stimulated with PMA/ionomycin in group 1 vs 2 patients. E, Mean ± SEM % of CD8+ IFNγ+ T cells at baseline or after treatment/stimulation conditions in group 1, 2, and 3 patients. Cytokines present in supernatants from patients' PBMCs after stimulation with PMA/ionomycin represented as mean ± SEM of Th2/Th1 ratio between (F) IL‐10 and IFNγ, (G) IL‐10 and TNF‐α, and (H) IL‐4 and TNF‐α in group 1, 2, and 3 patients at baseline or during treatment recovery. I, Mean ± SEM % expression of markers in patient‐02 tumour or PBMCs unstimulated and stimulated with PMA/ionomycin or P02‐tumour before or after exposure to bortezomib + temozolomide in situ. Cytokine concentrations in pg/mL from supernatants from patient‐02 tumour or PBMCs unstimulated or stimulated ex vivo with PMA + ionomycin or patient‐02 tumour or before and after exposure to bortezomib + temozolomide in situ of (J) IL‐2 and (K) TNF‐α. *P < .05; **P < .01; ***P < .001: ****P < .0001, n = 10 patients. BTZ, bortezomib; IFNγ, interferon γ; IL, interleukin; TMZ, temozolomide; TNF‐α, tumor necrosis factor α