Status of alternative angiogenic pathways in glioblastoma resected under and after bevacizumab treatment

Abstract

Glioblastoma multiforme (GBM) acquires resistance to bevacizumab (Bev) treatment. Bev affects angiogenic factors other than vascular endothelial growth factor (VEGF), which are poorly understood. We investigated changes in angiogenic factors under and after Bev therapy, including angiopoietin-1 (ANGPT1), angiopoietin-2 (ANGPT2), placental growth factor (PLGF), fibroblast growth factor 2, and ephrin A2 (EphA2). Fifty-four GBM tissues, including 28 specimens from 14 cases as paired specimens from the same patient obtained in three settings: initial tumor resection (naïve Bev), tumors resected following Bev therapy (effective Bev), and recurrent tumors after Bev therapy (refractory Bev). Immunohistochemistry assessed their expressions in tumor vessels and its correlation with recurrent MRI patterns. PLGF expression was higher in the effective Bev group than in the naïve Bev group (p = 0.024) and remained high in the refractory Bev group. ANGPT2 and EphA2 expressions were higher in the refractory Bev group than in the naïve Bev group (p = 0.047 and 0.028, respectively). PLGF expression was higher in the refractory Bev group compared with the naïve Bev group for paired specimens (p = 0.036). PLGF was more abundant in T2 diffuse/circumscribe patterns (p = 0.046). This is the first study to evaluate angiogenic factors other than VEGF during effective and refractory Bev therapy in patient-derived specimens.

Keywords: Alternative angiogenesis factor; Bevacizumab; Glioblastoma; Vascular endothelial growth factor.

Fig. 1

A Tissue sampling. Fifty-five tumor tissues obtained from three different settings: 15 tumor tissues were resected under neoadjuvant Bev (defined as “effective Bev”). Twenty-five tumor tissues were resected before Bev therapy (defined as “naïve Bev”). Fourteen tumor tissues were resected after Bev failure (defined as “refractory Bev”). Nine tumor tissues (seven from the naïve Bev group, and two from the effective Bev group) were obtained from salvage surgery. Five tumor tissues (four from the naïve Bev group, and one from the effective Bev group) were obtained from autopsy. B Treatment protocol of neoadjuvant Bev followed by surgery and postoperative adjuvant RT and TMZ combined with Bev. Surgery was performed in naïve, effective (just after preoperative neoadjuvant Bev), and refractory Bev periods. Bev bevacizumab, RT radiotherapy, TMZ temozolomide

Fig. 2

Levels of ANGPT1 (A), ANGPT2 (B), FGF2 (C), EphA2 (D) and PLGF (E) in tumor vessels. Immunohistochemistry photomicrographs shows naïve (upper left panel), effective (upper right panel) and refractory (bottom left panel). Numbers of positive vessels in tumor in five high power fields (5 HPF) were compared among naïve (blue), effective (orange), and refractory (green) Bev groups (bottom right panel)

Fig. 2

Levels of ANGPT1 (A), ANGPT2 (B), FGF2 (C), EphA2 (D) and PLGF (E) in tumor vessels. Immunohistochemistry photomicrographs shows naïve (upper left panel), effective (upper right panel) and refractory (bottom left panel). Numbers of positive vessels in tumor in five high power fields (5 HPF) were compared among naïve (blue), effective (orange), and refractory (green) Bev groups (bottom right panel)

Fig. 2

Levels of ANGPT1 (A), ANGPT2 (B), FGF2 (C), EphA2 (D) and PLGF (E) in tumor vessels. Immunohistochemistry photomicrographs shows naïve (upper left panel), effective (upper right panel) and refractory (bottom left panel). Numbers of positive vessels in tumor in five high power fields (5 HPF) were compared among naïve (blue), effective (orange), and refractory (green) Bev groups (bottom right panel)

Fig. 3

Paired comparisons of ANGPT1, ANGPT2, FGF2, EFNA2, and PLGF by immunoreactivity in tumor vessels between naïve (blue) and refractory (green) Bev groups. Numbers of positive vessels were quantitated from five HPFs

Fig. 4

A Images of recurrent MRI (cT1-flare up and T2 diffuse/T2 circumscribed). Left panel shows cT1-flare up, middle panel shows T2 diffuse and right panel shows T2 circumscribed images. B Numbers of positive vessels of each recurrent pattern were quantitated in five HPFs. Yellow shows cT1-flare up and Pink shows T2-diffuse/T2 circumscribed pattern

Fig. 5

Scheme representing changes in VEGF, CD34, and alternative angiogenic factors in tumor vessels at naïve, effective and refractory Bev stages. FGF2, EphA2, and PLGF levels increase in the effective and refractory Bev stages. ANGPT1 tends to decrease in the effective Bev stage compared with the naïve Bev stage, and increase in the refractory Bev stage again, while ANGPT2 tends to increase in the refractory stage compared with naïve and effective Bev stages