Abstract P5-14-20: Mechanisms of radiation-induced brain edema alone and in combination with T-DM1 in Her2+ brain metastases
Authors: Contreras-Zárate María J , Lai Steven , Stumpf Priscilla , Fisher Christine , Ormond Ryan , Karam Sana D , Kabos Peter , Cittelly Diana M 
Published: March 2020

Introduction: Brain metastases (BM) occurs in ~40% of breast cancer patients with Her2+ tumors, which are typically treated with the Her-2 targeting drugs ado-trastuzumab emtansine (T-DM1) and trastuzumab. Radiotherapy (RTx), including stereotactic radiosurgery (SRS), is often utilized to treat BM. A subset of BM patients treated with SRS develops brain edema and radionecrosis (ERN), causing neurological deterioration, functional loss, and rarely death. We have reported an increased rate of ERN in patients treated with T-DM1 and SRS (39.1%), compared to those receiving SRS alone (4.5%). Thus, defining the mechanisms underlying ERN alone or in combination with T-DM1, is critical to reduce life-threatening side effects. While brain edema associated with BM and RTx is often attributed to blood-brain-barrier (BBB) disruption (vasogenic edema), recent studies now identify cytotoxic edema (osmotic cell expansion) as the initial andreversible step in a sequence of changes that leads to ionic edema, vasogenic edema, and ultimately brain swelling and necrosis. Here, we test the hypothesis that RTx alone and in combination with T-DM1 induces cytotoxic edema in astrocytes via upregulation of the water channel Aquaporin 4 (AQP4) and that blockage of cytotoxic edema will decrease ERN. Results: A cohort of BM from Her2+ patients treated with T-DM1/SRS showed enlarged reactive astrocytes expressing high levels of AQP4. To assess the role of astrocytic swelling in ERN, human astrocytes (THV) were treated with increasing doses of RTx and astrocytic cell size was measured 24 h later. A single 8 Gy RTx-dose increased cell size by 4.5 times compared to non-treated astrocytes (1248 ± 760.8 µm2 vs 5685 ± 4475 µm2, P&lt;0.0001). Consistent with a role for AQP4 in mediating astrocytic swelling, western blot (WB) showed increased AQP4 levels in RTx treated cells. To assess whether Her2-targeting (or directed) therapies impacted AQP4 expression and astrocytic survival/swelling, astrocytes were treated with increasing doses of RTx alone (0, 2, 4, 8 Gy), with trastuzumab or T-DM1 (1 μg/mL). WBs showed that only T-DM1 further exacerbated RTx-induced AQP4 upregulation and increased PARP cleavage. The combination of 4 Gy-RTx and T-DM1 showed a synergistic effect in reducing astrocytic confluence (32.1%), compared with astrocytes treated with 4 Gy-RTx alone (32.8 ± 3 vs 64.9 ± 13.3% respectively, P=0.0001, at 5 days). Moreover, T-DM1/RTx increased astrocytic size (P = 0.004) to a greater extent than equivalent RTx dose in combination with trastuzumab (P = 0.005). Other chemotherapeutic agents (paclitaxel and cisplatin) did not affect AQP4 protein levels, astrocytic survival or astrocytic swelling. TGN-020, a novel AQP4 inhibitor, and topiramate (TPM), an FDA-approved anticonvulsant/antiepileptic able to inhibit AQP4 activity, were evaluated for their potential role in reducing astrocytic swelling. Pretreatment with 100 µM TPM reduced RTx-induced astrocytic swelling by 36 % (7153 ± 4522 µm2 vs 4627 ± 3999 µm2, P&lt;0.0001). TPM pretreatment did not prevent 8 Gy-RTx-induced cell death (24.96 ± 5.7 % vs 23.47 ± 4.7 %) nor prevent DNA double stranded breaks measured by IF of p-γH2AX (89.17 ± 13.89 % vs 97.32 ± 2.94 %). Ongoing studies are assessing the effect of TPM on blocking astrocytic swelling and decreasing brain edema in vivo. Conclusions: These results suggest that astrocytic swelling (cytotoxic edema) is involved in the pathophysiology of ERN during BM treatment. In vitro, AQP4-inhibitors decreased astrocytic swelling without affecting astrocyte survival, suggesting that blockage of AQP4 could reduce brain edema without rescuing lethally-damaged astrocytes. Thus, a combination of drugs preventing vasogenic dysfunction (i.e bevacizumab) and AQP4-inhibitors (i.e TPM) could diminish ERN by targeting both, vasogenic and cytotoxic edema.

Citation Format: María J Contreras-Zárate, Steven Lai, Priscilla Stumpf, Christine Fisher, Ryan Ormond, Sana D Karam, Peter Kabos, Diana M Cittelly. Mechanisms of radiation-induced brain edema alone and in combination with T-DM1 in Her2+ brain metastases [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 P5-14-20.