Immune-modulating effects of bevacizumab in metastatic non-small-cell lung cancer patients

Abstract

The mPEBev is an anticancer regimen which combines a chemotherapy doublet, based on cisplatin and oral etoposide (mPE), with bevacizumab (mPEBev), a mAb targeting the vasculo-endothelial growth factor (VEGF). In previous studies, this regimen showed powerful anti-angiogenetic effects and significant antitumor activity in metastatic non-small-cell lung cancer (mNSCLC) patients. We also recorded the best benefit in patients exhibiting low-systemic inflammatory profile at baseline. On these bases, we hypothesized that mPEBev antitumor activity could be partially related to bevacizumab-associated immunological effects. For this reason, we performed an immunological monitoring in 59 out of 120 stage IIIb-IV NSCLC patients enrolled in the BEVA2007 phase II trial, who received fractioned cisplatin (30 mg/sqm days 1-3q21) and oral etoposide (50 mg, days 1-15q21) (mPE doublet) ±bevacizumab. In this group of patients, 12 received the mPE doublet alone and 47 the doublet in combination with bevacizumab (5 mg/kg on the day 3q21; mPEBev regimen). Blood cell counts, serum analysis, multiplex cytokine assay and immunocytofluorimetric analysis, performed on baseline and post-treatment on blood samples from these patients, revealed that bevacizumab addition to the doublet decreased levels of pro-angiogenic (VEGF, Angiostatin-1 and Follistatin) and inflammatory cytokines (interferon (IFN)γ, IL4 and IL17), improved in vivo and in vitro cytotoxic T-lymphocytes (CTL) response and promoted dendritic cell activation. These results suggest that the mPEBev regimen improve the micro-environmental conditions for an efficient antigen-specific CTL response, making it a feasible candidate regimen to be assessed in combination with immune-checkpoint inhibitors in NSCLC patients.

Figure 1

A multiplex analysis concerning the dosage of multiple pro-angiogenetic factors in the serum of mNSCLC patients enrolled in the BEVA2007 trial who have received the mPE doublet alone () or combined with bevacizumab (). Results are expressed as fold induction relative to baseline indicated as 1 (±S.E.). Asterisks represent statistical significance to the correspondent baseline values (*P<0.05; **P<0.01).

Figure 2

A multiplex analysis concerning the dosage of T_h1 (IFNɣ, TNFα and IL12), T_h2 (IL4 and IL10), T_h17 and inflammatory cytokines (IL8 and G-CSF) in the serum of mNSCLC patients enrolled in the BEVA2007 trial who have received the mPE doublet alone () or combined with bevacizumab (). Results are expressed as fold induction relative to baseline indicated as 1 (±S.E.). Asterisk represents statistical significance to the correspondent baseline values (*P<0.05).

Figure 3

A flow cytometric analysis concerning the expression of (a) different T-cell subsets and (b) myeloid derivative cells (activated DCs and MDICs) in the PBMCs of mNSCLC patients enrolled in the BEVA2007 trial who have received the mPE doublet alone () or combined with bevacizumab (). Results are expressed as fold induction relative to baseline indicated as 1 (±S.E.). Asterisks represent statistical significance to the correspondent baseline values (*P<0.05; **P<0.01).

Figure 4

Flow cytometry dot plots of two representative patient PBMCs: activated DCs, isolated at the baseline (a and b) and after four treatment courses with mPE doublet and bevacizumab (c and d).

Figure 5

(a) A flow cytometric analysis concerning the expression of CD8⁺Ki67⁺ cells in T-cell cultures undergone multiple in vitro stimulation with IRIV and derived from the PBMCs of normal donors (ND) or mNSCLC patients isolated at the baseline () and after four treatment courses () with the mPE doublet alone or combined with bevacizumab (mPEBev). Further panels describe the ELISA dosage of T_h1 (IFNɣ, TNFα ) and T_h2 (IL4 and IL10) cytokines in the supernatant of T-cell cultures in vitro stimulated with SEB (b), IRIV (c) and TSPP (d), and derived from the PBMCs of normal donors (ND) or mNSCLC patients and isolated at the baseline () or after four treatment courses () with the doublet alone (mPE) or combined with bevacizumab (mPEBev). Results are expressed as fold induction relative to baseline indicated as 1 (±S.E.). Asterisk represents statistical significance to the correspondent baseline values (*P<0.05).