Combined effects of bevacizumab with erlotinib and irradiation: a preclinical study on a head and neck cancer orthotopic model

Abstract

Clinical benefit has been demonstrated in patients with head and neck tumours receiving an anti-epidermal growth factor receptor (EGFR) agent in combination with radiotherapy (RT). Recent preclinical and clinical studies suggest beneficial effects from combining anti-angiogenic drugs with RT. To investigate the effect of combining these approaches, we evaluated in vivo the anti-tumour efficacy of the anti-angiogenic compound bevacizumab, a highly specific monoclonal antibody directed against the vascular endothelial growth factor (VEGF), erlotinib, an EGFR tyrosine kinase inhibitor, and irradiation given alone and in combination. Investigations were performed using a VEGF-secreting human head and neck tumour cell line, CAL33, with a high EGFR content, injected as orthotopic xenografts into the mouth floor of nude mice. Three days after tumour cell injection, bevacizumab (5 mg kg(-1), 5 days a week, i.p.), erlotinib (100 mg kg(-1), 5 days a week, orally) and irradiation (6 Gy, 3 days a week) were administered alone and in combination for 10 days. As compared with the control, concomitant administration of drugs produced a marked and significant supra-additive decrease in tumour mass; the addition of irradiation almost completely abolished tumour growth. The drug association markedly reduced the number of metastatic nodes and the triple combination significantly reduced the total number of pathologically positive lymph nodes as compared with controls. The RT-induced proliferation, reflected by Ki67 labelling, was reduced to control level with the triple combination. Radiotherapy induced a strong and very significant increase in tumour angiogenesis, which was no longer observed when combined with erlotinib and bevacizumab. The efficacy of the combination of bevacizumab+erlotinib and RT may be of clinical importance in the management of head and neck cancer patients.

Figures

Figure 1

Primary tumour growth after 10 days of treatment with single agents and combinations (10 mice per treatment group). Bars denote s.d.; NS=nonsignificant (P>0.05); *P<0.05; **P<0.01; ***P<0.001. Values above the columns concern comparisons with the controls; other values concern comparisons between two following columns.

Figure 2

Impact of single agents and combinations on pathologically positive lymph nodes (10 mice per treatment group). The only significant differences were for node invasion status (bevacizumab+erlotinib vs control, P=0.03) and for total node number (bevacizumab+erlotinib+RT vs control, P<0.001). An illustrative example of an invaded node without capsular rupture (N+R−) is given.

Figure 3

Impact of the different treatments on Ki67 proliferation marker (10 mice per treatment group). The proportion of primary tumours with labelling less than 50% is shown in white (group 1), between 50 and 70% in grey (group 2) and more than 70% in black (group 3); NS=nonsignificant (P>0.05); *P<0.05; **P<0.01; ***P<0.001. Values above the columns concern comparisons with the controls; other values concern comparisons between two following columns. An illustrative example of each labelling group is given.

Figure 4

Impact of the different treatments on the number of tumour vessels labelled by the anti-VEGFR2 antibody measured at the centre. NS=nonsignificant (P>0.05); ***P<0.001. Indications above the columns concern statistical comparisons with the controls; other indications concern statistical comparisons between two following columns. An illustrative example of VEGFR2 labelling of the vessels is given.

Figure 5

Impact of the different treatments on VEGFR2 expression on tumour cells (10 mice per treatment group). NS=nonsignificant (P>0.05); *P<0.05; **P<0.01; ***P<0.001. Indications above the columns concern statistical comparisons with the controls; other indications concern statistical comparisons between two following columns. An illustrative example of strong and weak VEGFR2 labelling of tumour cells is given.