Abstract PR03: Prophylactic nodal irradiation abrogates the synergy of tumor radiotherapy and immune checkpoint blockade
Authors: Marciscano Ariel E. , Ghasemzadeh Ali , Nirschl Thomas R. , Francica Brian J. , Theodros Debebe , Velarde Esteban , Wong J , Thorek Daniel LJ , DeWeese Theodore L. , Drake Charles G. 
Published: March 2017

Abstract
Background: Prophylactic nodal irradiation (PNI) is a strategy used to treat early stage cancers with the potential to enhance local control and prevent metastatic spread. It remains unclear whether irradiation of tumor-associated draining lymph nodes (LN) influences anti-tumor T cell responses. Our aim was to investigate the in vivo effects of nodal irradiation on the phenotype and function of tumor infiltrating lymphocytes (TIL) and the impact of PNI on the anti-tumor effects of immunotherapy.
Methods: The Small Animal Radiation Research Platform (SARRP) delivered imaged-guided stereotactic radiation (RT) to tumor (T-only) or tumor and LN (T+LN). Syngeneic tumors (MC38 colon, B16 melanoma) were implanted in C57BL/6 mice and irradiated (12Gy x1) and/or treated with immune checkpoint blockade with αCTLA-4 IgG2a (depleting) or αPD-1 IgG1. The composition of the tumor microenvironment (TME) was assayed by flow cytometry. To query tumor-antigen specific T cell responses, adoptive transfer experiments were performed using OVA-specific CD8+ T-cells from Rag-/- OT-1 mice and OVA-MHC class I tetramer (SIINFEKL). Tumor lysate was also collected for chemokine analysis, and tumor outgrowth was quantified over time.
Results: T-only RT resulted in a significant increase (P&lt;0.01) in the proportion and absolute number of tumor infiltrating CD8+ effector T cells in comparison with T+LN RT. Immunosuppressive subsets (CD11b+ Gr-1hi MDSCs and FoxP3+ CD4+ Tregs) were also significantly increased in the T-only group. Ultimately, T-only RT significantly enhanced the CD8 effector:Treg ratio relative to untreated and T+LN treated tumors. Using the OVA-MHC class I tetramer to identify tumor-antigen specific CD8 T-cells, we observed that T-only RT significantly increased (P&lt;0.05) the number of tumor-specific CD8-T cells in the TME compared with T+LN RT. Interestingly, T-only RT resulted in a a significant expansion of non-OVA specific CD8 T cells which was not observed with T+LN RT, suggesting a polyclonal anti-tumor immune response. Functionally, a significant increase (P&lt;0.01) in the absolute number of IFNγ+ and TNFα+ antigen-specific TIL were noted with T-only RT. Mechanistically, a distinct chemokine signature correlated with robust TME immune infiltration and significantly elevated levels of CCL3/4/5 and CXCL10 (P&lt;0.05) were observed in tumor lysate collected from T-only RT tumors relative to T+LN RT samples. In an effort to understand the implications of the RT target on potential synergy with immune checkpoint blockade we performed survival experiments with T-only and T+LN RT in combination with αPD-1 or αCTLA-4. T-only RT in combination with αCTLA-4 yielded the best outcome with a 86% long-term survival (day 90 post-RT) compared with 30-36% long-term survival in mice treated with other combinations of RT + immune checkpoint blockade. Intriguingly, favorable CD8 effector:Treg ratio was able to predict treatment response and was dramatically higher (P&lt;0.001) among mice treated with T-only RT in combination with αCTLA-4.
Conclusions: We have successfully developed a SARRP-based early stage cancer model with the ability to target or spare the tumor-associated LN. Results to date demonstrate significant immunological differences that are contingent upon inclusion/exclusion of the LN. Long-term survival experiments suggest response and survival advantages with T-only RT, particularly in combination with αCTLA-4 blockade. Taken together, these data suggest that PNI may dampen anti-tumor immune responses and that Tumor-only RT might be a better strategy in combination immunotherapy regimens, although these results should be explored in carefully designed clinical trials.
This abstract is also being presented as Poster B41.
Citation Format: Ariel E. Marciscano, Ali Ghasemzadeh, Thomas R. Nirschl, Brian J. Francica, Debebe Theodros, Esteban Velarde, J Wong, Daniel LJ Thorek, Theodore L. DeWeese, Charles G. Drake. Prophylactic nodal irradiation abrogates the synergy of tumor radiotherapy and immune checkpoint blockade. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR03.