Abstract P6-14-07: High miR-19a serum levels correlate with favorable prognosis in patients with metastatic HER2+ inflammatory breast cancer and may result from an effective antibody-dependent cell-mediated cytotoxicity induced by trastuzumab
Authors: Anfossi Simone , Giordano Antonio , Huo Lei , Alvarez Ricardo H , Valero Vicente , Hortobagyi Gabriel N , Woodward Wendy A , Ueno Naoto T , Calin George A , Reuben James M 
Published: May 2015

Abstract
Background: IBC is a rare but highly aggressive form of locally advanced breast cancer (5-year OS rate: 40.5% IBC vs 85% non-IBC patients) accounting for 10% of all breast cancer deaths. To date, no unique molecular diagnostic or prognostic biomarker has been identified for IBC. Increasing evidence supports the potential value of miRNA as prognostic and predictive serum biomarker in cancer. We found that IBC cells expressed high levels of miR-19a and patients with metastatic IBC HER2+ (MIBC HER2+) and high miR-19a serum levels had better prognosis than patients with MIBC HER2+ and low miR-19a serum levels. As one of the mechanisms of action of trastuzumab is the induction of antibody-dependent cell-mediated cytotoxicity (ADCC), we hypothesized that the increased miR-19a serum levels in MIBC HER2+ patients with favorable clinical outcome could result from an effective ADCC and be used as biomarker to monitor the response to trastuzumab.
Methods: Total RNA was isolated using the Total RNA Purification Kit (Invitrogen, Norgen Biotek). MiR-19a levels in tumor tissue, serum, cell lines and supernatants were evaluated by qRT-PCR (Applied Biosystems). ADCC was evaluated by Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen).
Results: Microarray was performed in IBC (n=23) and non-IBC (n=24) tumors and normal tissue (n=12). Microarray showed higher miR-19a expression in IBC compared with non-IBC (p=0.028) and normal tissue (p=0.0002). The two IBC cell lines SUM149 (triple receptor-negative) and KPL-4 (HER2-amplified) expressed higher levels of miR-19a compared with the non-IBC cell lines MDA-231 (triple receptor-negative), SKBR3 (HER2-amplified), and MCF-7 (HER2-non-amplified) (p&lt;0.001, p&lt;0.001, p&lt;0.001 and p&lt;0.05, p&lt;0.05, p&lt;0.01 respectively). To assess whether miR-19a could be released from IBC cells upon NK cell-mediated ADCC, we performed a NK cytotoxicity test using the NK-resistant KPL-4 cells and the NK-sensitive SKBR3 and the NK-sensitive MCF-7 cells as control. Co-incubation with trastuzumab induced increased MCF-7, SKBR3 and KPL-4 cell death (2.1-fold, 2.4-fold and 3.5-fold respectively) and accordingly increased miR-19a levels in their supernatants (MCF-7: 3-fold, p=0.017; SKBR3: 6-fold, p=0.005 and KPL-4: 8-fold, p=0.0001). The pattern of miR-19a levels in the supernatants correlated with that expressed at cellular levels (MCF-7&lt;SKBR3&lt;KPL-4). We measured miR-19a serum levels in patients with MIBC HER2+ (n=27) and metastatic nonIBC HER2+ (n=24). Patients with MIBC HER2+ and high miR-19a level had longer PFS (10.3 vs 3.2 months, p=0.022) and OS (median not reached vs 11.2 months, p=0.003) than patients with low miR-19a levels. Patients with metastatic nonIBC HER2+ and high miR-19a levels had longer OS (32.9 vs 13.3 months; p=0.015) than patients with low miR-19a levels (32.9 vs 13.3 months; p=0.015).
Conclusion: High miR-19a serum levels are associated with favorable prognosis in patients with MIBC HER2+ and could result from an effective NK cell-mediated ADCC. MiR-19a may represent a novel serum biomarker to monitor the response to trastuzumab therapy and predict clinical outcome in patients with MIBC HER2+.
Citation Format: Simone Anfossi, Antonio Giordano, Lei Huo, Ricardo H Alvarez, Vicente Valero, Gabriel N Hortobagyi, Wendy A Woodward, Naoto T Ueno, George A Calin, James M Reuben. High miR-19a serum levels correlate with favorable prognosis in patients with metastatic HER2+ inflammatory breast cancer and may result from an effective antibody-dependent cell-mediated cytotoxicity induced by trastuzumab [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-14-07.