IL-15 and IL-2 improve Cetuximab mediated cytotoxicity EGFR-expressing triple negative breast cancer cell lines

ROBERTI, MARÍA PAULA; BARRIO, MARÍA MARCELA; BRAVO, ALICIA INÉS; ROCCA, YAMILA SOL; ARRIAGA, JUAN MARTÍN; BIANCHINI, MICHELE; MORDOH, JOSÉ; LEVY, ESTRELLA MARIEL

Orlando, Florida

Congreso; Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011

Approximately 10 - 15% of Breast Cancers (BC) are known to be ‘triple-negative (TN) receptor’, not expressing Estrogen receptor, Progesterone receptor and not exhibiting overexpression and/or gene amplification of HER2-neu. TNBC is associated with a more aggressive clinical course and worse clinical outcome than non-TNBC. As patients with TNBC are not likely to benefit from anti-estrogen or anti-HER2 therapy, first-line treatment usually consists of conventional cytotoxic chemotherapy. Taking into account the limited treatment possibilities in TNBC, in the present work we studied a potential therapy based on Cetuximab-mediated immune activity by Natural Killer (NK) cells. Results: We performed in vitro studies on a human cell line, IIB-BR-G, previously established in our laboratory and its metastatic in vivo variant, IIB-BR-G MT. The immunohistochemical analysis of both cell lines showed a TNBC phenotype with high expression of EGFR, the target for Cetuximab antibody. We also determined by DNA sequencing that both cell lines have a mutated K-RAS status (38 G>A at codon 12) which is the main Cetuximab predictive lack-of-response marker. Consequent to that, Cetuximab did not inhibit cellular proliferation or induced apoptosis, suggesting that it does not interfere with the EGFR signaling pathway, even at a concentration of 200 ìg/ml (p>0.05). To investigate the immune mechanisms triggered by Cetuximab, we performed in vitro cytotoxicity assays. K-RAS mutated cell lines treated with 1 ìg/ml Cetuximab were susceptible to antibody dependent cellular cytotoxicity activity (ADCC), reaching a lytic activity of 40 ± 2.5% using as effectors peripheral blood mononuclear cells, and 50 ± 10% using enriched NK cells (p<0.001). Furthermore, IL-2 or IL-15 pretreatment of PBMC increased the effect of Cetuximab-mediated ADCC, reaching a cellular lysis of 75 ± 3 % in IIB-BR-G and 80 ± 3.5 % in IIB-BR-G MT (p<0.001), consistent with the upregulation of NK activating receptors CD16 and NKG2D and the enhancement of IFN-ã production in cocultures (p<0.001). Conclusions: EGFR-expressing TNBC cell lines were not affected by Cetuximab inhibition of its tyrosine kinase signaling, in concordance with their mutated K-RAS status. However, they could be killed by Cetuximab-mediated ADCC at clinically achievable concentrations. IL-15 could replace IL-2 in most of its immunologic activities, stimulating the cytotoxicity of NK cells and its ability to produce IFN-ã, paralleling the upregulation of activating receptors. This in vitro study shows the involvement of an antibody-mediated immune response rather than the blockade of intracellular signaling in the antitumor activity of Cetuximab in K-RAS mutated TNBC. The combination of Cetuximab with IL-2 or IL-15 and NK cells may be considered an attractive therapeutic approach to enhance the clinical efficacy of Cetuximab in TNBC.