Clinical and histopathological analyses of VEGF receptors peptide vaccine in patients with primary glioblastoma - a case series

Abstract

Background: The expression of vascular endothelial growth factor (VEGF)-A/ VAGF receptors (VEGFRs) signaling plays a pivotal role in the tumor angiogenesis and the development of the immunosuppressive tumor microenvironment in glioblastomas. We have previously conducted exploratory clinical studies investigating VEGFRs peptide vaccination with and without multiple glioma oncoantigens in patients with recurrent high-grade gliomas. Recently, an exploratory clinical investigation of VEGFRs peptide vaccination was conducted in patients with progressive neurofibromatosis type 2. Those studies suggested that cytotoxic T lymphocytes (CTLs) induced by the vaccination can directly kill a wide variety of cells associated with tumor growth, including tumor vessels, tumor cells, and immunosuppressive cells expressing VEGFR1 and/or 2. In the present study, synergistic activity of the combination of VEGFRs peptide vaccination with chemotherapy was evaluated.

Methods: We performed the first clinical trial to assess VEGFR1 and 2 vaccination along with temozolomide (TMZ) -based chemoradiotherapy for the patients with primary glioblastomas. Furthermore, histopathological changes after the vaccination were evaluated using paired pre- and post- vaccination specimens.

Results: The disappearance of radiographically enhanced lesion was observed in 2 patients after the vaccination, including one in which the methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter was not observed. The histopathological findings of pre- and post-vaccination specimens demonstrated that tumor vessels showed negative or slight VEGFRs expressions after the vaccination and most endothelial cells were covered with PDGFR-β-positive pericytes. Notably, CTLs induced by VEGFRs peptide vaccination attacked not only tumor vessels but also tumor cells and regulatory T cells expressing VEGFRs even in recurrent tumors.

Conclusions: VEGFR1 and 2 vaccination may have a preliminary synergistic effect when administered with TMZ. The limitation of the present study was the paucity of the number of the samples. Further studies involving more patients are warranted to confirm the findings of this study.

Trial registration: This study was registered as UMIN000013381 (University Hospital Medical Information Network-Clinical Trial Registry: UMIN-CTR) on 5 March, 2014 and with the Japan Registry of Clinical Trials (jRCT) as jRCTs031180170 on 1 March, 2019.

Keywords: Bevacizumab; Glioblastoma; Peptide vaccine; VEGFR.

Fig. 1

The Protocol for this clinical trial. The scheme of the Protocol for this clinical trial is shown. VEGFR1 and R2 peptide were injected eight times every week and then six times monthly (a total of 14 times). Vaccination was synchronized with adjuvant TMZ

Fig. 2

Radiographic images of enrolled patients. Radiographic images of Case 1(a), Case 2 (b), Case 3 (c), Case 4 (d). a The enhanced lesion was decreased 15 months, and recurrent lesion was observed at the removal site 18 months after the first vaccination. b The enhanced lesion was decreased 12 months, and recurrent lesion was observed at the removal site 17 months after the first vaccination. c The enhanced lesion disappeared 12 months after the first vaccination. d The enhancement lesion disappeared 12 months after the first vaccination

Fig. 3

Clinical course of enrolled patients. a Overall survival of four patients. At the time of analysis, Case 3 and 4 still had CR. b Progression-free survival of four patients

Fig. 4

Histopathological images of the paired pre- and post-vaccination. a Histopathological analysis of VEGF-A, VEGFR1, VEGFR2, CD34 and PDGFR-β expressions in the tumor of pre- and post- vaccination. A square means tumor cells with positive VEGFR1 or VEGFR2 staining (T: tumor cell, V: vessel; original magnification, × 40; magnification bar, 100 μm). b Immunofluorescent analysis of VEGFR1, VEGFR2 and PDGFR-β expressions in tumor vessels in pre- and post- vaccination (original magnification, × 40; magnification bar, 100 μm). c Histopathological analysis of cleaved caspase3 expression on the tumor vessel of pre- and post- vaccination. (V: vessel; original magnification, × 40; magnification bar, 100 μm). d Immunofluorescent analysis of CD34, VEGFR1, and cleaved caspase3 expressions in the tumor of pre- and post- vaccination. Expression of cleaved caspase 3 was detected on the endothelial cells with slight VEGFR1 expression or without VEGFR1 expression (original magnification, × 40; magnification bar, 100 μm). e Histopathological analysis of Foxp3 expression in the tumor of pre- and post- vaccination (original magnification, × 40; magnification bar, 100 μm; black arrows, positive cells). f Immunofluorescent analysis of Foxp3 and cleaved caspase3 expressions in the tumor of pre- and post- vaccination (original magnification, × 40; magnification bar, 100 μm; white arrow, positive cells). g Relative gene expression of VEGF-A, VEGFR1, VEGFR2 and Foxp3 in the tumors of pre- and post- vaccination