Abstract 4987: Role of the PD-1/PDL-1 pathway in resistance of patients with metastatic breast cancer to treatment with radiotherapy and TGFβ neutralization
Authors: Demaria Sandra , Vanpouille-Box Claire , Hawtin Rachael E. , Fanton Christie , Conroy Andy , Evensen Erik , Dixit Neha , Barber Alan , Schaue Dorthe , McBride William H. , Barcellos-Hoff Mary Helen , Formenti Silvia C. 
Published: July 2016

Abstract
Experimental data and clinical observations indicate that local radiation therapy (RT) converts the irradiated tumor into an in situ vaccine and improves responses to immunotherapy. Preclinical breast cancer models show that TGFb neutralization was required to achieve CD8+ T cell priming specific for multiple tumor antigens, and rejection of the irradiated tumor and non-irradiated metastases (Vanpouille-Box et al., Cancer Res 2015). Tumor response and survival were improved by PD-1 blockade. We explored the role of the PD-1/PDL-1 pathway in resistance to combined RT + TGFβ neutralizing antibody (fresolimumab) in metastatic breast cancer patients (pts).
22 pts were treated in a prospective trial at NYU and UCLA (NCT01401062, supported by DOD MTA BC100481). Pts were randomly assigned to 2 doses of fresolimumab (freso) (1mg/kg and 10 mg/kg, q 3 weeks). Image guided RT was delivered to one metastasis on weeks 2 and 7, 7.5 GyX3. 1 pt achieved objective response, with 28% reduction of tumor burden. The response lasted 11 months, anthracycline-induced acute myelogenous leukemia developed. Pts randomized to the higher freso dose had a hazard ratio of 2.17 (95% CI: 0.753-6.272) for risk of death at 1 year follow up. Currently, at a median follow up of 2 years, 20/22 pts have died.
To explore reasons for the limited response, peripheral blood mononuclear cells collected at baseline, 5 and 15 weeks into treatment, were analyzed. Responses to survivin, as a tumor antigen, were assessed using tetramers. Single cell network profiling (SCNP) was used to evaluate expression of 6 immunomodulatory receptors plus immune signaling in T cells collected from 7 healthy donors (HD) and 15 breast cancer pts (6 at 1mg/kg, 9 at 10mg/kg fresolimumab). In vitro activation of p-AKT and p-Erk was quantified following in vitro T cell receptor (TCR) modulation with anti-CD3/αnti-CD28.
6/12 HLA-A2.1+ pts had pre-existing survivin-specific CD8+ T cells, and 3 showed an increase over time. 2 pts who were negative generated modest responses after RT and 10 mg/kg freso. Prior to treatment, PDL-1 expression was increased in monocytes (p = 0.01) and CD4+ T cells (p = 0.037) compared to HD. Elevated PD-1 (p = 0.054) and OX-40 (p = 0.014) expression were identified on CD4+ T cells of pts. Upon TCR modulation, CD4+ and CD8+ T cells from pts showed reduced signaling through p-AKT and, to a lesser extent, p-Erk, compared to HD. Signaling was lower in PD-1+ vs PD-1- CD8+ and CD4+ T cells. Addition of anti-PD-1 pembrolizumab partially restored TCR-mediated signaling through p-AKT and p-Erk in PD-1+ but not PD-1- T cells.
Overall, data indicate impaired T cell signaling in PD-1+ T cells of metastatic breast cancer pts, which may explain the inability to respond to RT + TGFβ blockade. In vitro addition of anti-PD-1 improved T cell signaling through p-AKT and p-Erk. Together with the preclinical data, this supports adding anti-PD-1 to the combination of RT +TGFβ blockade.
Citation Format: Sandra Demaria, Claire Vanpouille-Box, Rachael E. Hawtin, Christie Fanton, Andy Conroy, Erik Evensen, Neha Dixit, Alan Barber, Dorthe Schaue, William H. McBride, Mary Helen Barcellos-Hoff, Silvia C. Formenti. Role of the PD-1/PDL-1 pathway in resistance of patients with metastatic breast cancer to treatment with radiotherapy and TGFβ neutralization. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4987.