Abstract P1-18-25: Quantifying the effects of combination trastuzumab and radiation therapy in HER2 positive breast cancer under normoxic and hypoxic conditions
Authors: Bloom Meghan , Virostko Jack , Sorace Anna , Yankeelov Thomas 
Published: March 2020

Introduction: The purpose of this study is to quantify the effects of combination trastuzumab and radiation therapy in HER2+ breast cancer. Breast tumors can become hypoxic which increases radioresistance partly due to the upregulation of hypoxia inducible factors (HIFs). HIF-1α promotes cancer cell survival and has been found to be upregulated by phosphatidylinositol-3 kinase (P13K) signaling. Trastuzumab, a targeted therapy, has been used in combination with radiation in the adjuvant setting to treat HER2+ patients. Trastuzumab causes cell cycle arrest by inhibiting P13K pathways and been shown to increase tumor oxygenation in vivo. There is evidence that trastuzumab sensitizes HER2+ breast cancer to radiation therapy; however, longitudinal studies are limited and fail to quantify sensitization over time, reducing clinical relevance. There are three main goals to this study: 1) quantify the effects of combination trastuzumab and radiation therapy in normoxic and hypoxic conditions in vitro, 2) quantify the regulation of P13K when treated with trastuzumab in hypoxic conditions in vitro, and 3) test the hypothesis that trastuzumab sensitizes HER2+ breast cancer to radiation therapy in an in vivo model of HER2+ breast cancer.

Experimental Design: For in vitro studies, BT474-GFP expressing HER2+ breast cancer cells were plated in a 96 well plate. 24 hours later, cell media was changed to simulate hypoxic (50 or 100 μM CoCl2) or normoxic (0 μM CoCl2) conditions and then treated with trastuzumab and/or radiation (various dosing and timing) and imaged using the IncuCyte ZOOM System every 6 hours for 7 days. Cell number was automatically quantified and surviving fractions (SF) were calculated for each time point. An enzyme-linked immunosorbent assay (ELISA) was used to quantify HIF-1α and P13K protein levels in cells. For in vivo studies, 107 BT474 HER2+ breast cancer cells were implanted subcutaneously into athymic nude mice. After reaching 250 mm3, tumors were treated with trastuzumab (10 mg/kg, 2 doses), and/or radiation (5 or 10 Gy, single dose), or saline and were measured with calipers for six weeks. A non-parametric Wilcoxon rank sum test was used to determine statistical differences in tumor size between treatment groups. Degree of synergy over time in combination treatment groups both in vitro and in vivo was determined using a Bliss independence model.

Results and Discussion: The SF of cells 5 days after 10 Gy irradiation under hypoxic conditions was significantly higher at 51.2% (50 μM CoCl2, P &lt; 0.05) and 65.5% (100 μM CoCl2, P &lt; 0.01) than normoxic conditions with a SF of 44.8%. Cells treated with 3 μg/ml of trastuzumab 24 hours prior to 10 Gy irradiation had a significant decrease in SF 5 days post irradiation under both normoxic (SF 40.8%) and hypoxic conditions (100 μM CoCl2 - SF 58.8%) compared to either monotherapy (P &lt; 0.05). In vivo, treatment with trastuzumab and radiation results in a faster rate of tumor regression. Mice (n = 4) had a significant (P &lt; 0.05) 45% decrease in tumor size 7 days after 10 Gy irradiation when treated in combination with trastuzumab compared to control (n = 5) or radiation alone (n = 5). Mice treated with radiation alone did not have a significant decrease in tumor size until 14 days after radiation therapy.

Conclusion: Quantifying the synergy between trastuzumab and radiation over time will elucidate optimal combination regimens and has the potential to decrease the amount of therapy needed to achieve tumor regression. Systematically evaluating the radiosensitizing effect of trastuzumab in vitro under normoxic and hypoxic conditions may provide mechanistic insight on how to overcome radioresistance in HER2+ breast cancer.

We acknowledge the support of CPRIT RR160005, NCI R01CA186193, and ACS RSG-18-006-01-CCE.

Citation Format: Meghan Bloom, Jack Virostko, Anna Sorace, Thomas Yankeelov. Quantifying the effects of combination trastuzumab and radiation therapy in HER2 positive breast cancer under normoxic and hypoxic conditions [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-25.