Abstract P2-15-01: Integrated immunologic assessment of tumor infiltrating lymphocytes (TILs) and peripheral blood to assess synergy of cryoablation (cryo) plus ipilimumab (ipi) in early stage breast cancer (ESBC) patients (pts)
Authors: Page David , Yuan Jianda , Diab Adi , Dong Zhiwan , Ginsberg Arielle , Wong Phillip , Emerson Ryan , Redmond David , Blum Brian , Mu Zhenyu , Zhao Chunjun , Comstock Christopher , Morris Elizabeth , Comen Elizabeth , Kotin Alan , Sung Janice , Brogi Edi , Morrow Monica , Solomon Stephen , Sacchini Virgilio , Maybody Majid , Neville Deirdre , Robins Harlan , Patil Sujata , Wolchok Jedd , Hudis Clifford , Norton Larry , Allison James , Sharma Padmanee , McArthur Heather 
Published: May 2015

Abstract
Background: In pts with ESBC, cryo combined with cytotoxic T-lymphocyte antigen 4 blockade was well tolerated and did not delay standard-of-care mastectomy. As observed in mice, cryo+ipi may liberate tumor-associated antigens, synergistically activate tumor-reactive T-cells, and confer long-term anti-tumor immunity. Because singular biomarkers of response to immunotherapy have not been well defined, we conducted an integrated immunologic assessment to explore potential predictors of immune activation and response.
Methods: Serial blood and pre-/post-treatment tumor tissue were collected from 18 pts treated with cryo (6 pts), single-dose ipi at 10mg/kg (6 pts), or cryo + ipi (6 pts). A Meso Scale Discovery platform was used to measure plasma cytokine interferon gamma (IFNγ). Multiparameter flow cytometry was used to evaluate peripheral and intratumoral T-cell and myeloid cell quantity, T-cell phenotype (effector versus regulatory), proliferation state (Ki67), and activation state (inducible costimulator [ICOS] expression). Finally, a DNA deep sequencing platform was used to conduct T-cell repertoire analysis of peripheral T-cells and TILs.
Results: Sustained &gt;2-fold elevations (1 month post-treatment) in plasma IFNγ were observed in the majority (4/6) of pts receiving cryo/ipi (median 6-fold increase), but in the minority of pts receiving cryo (0/6, median 0-fold) or ipi (2/6, median 0-fold). Similarly, sustained &gt;2 fold elevations in ICOS expression in peripheral CD3+CD4+ T-cells, a known pharmacodynamic marker of ipi, were observed in the majority (5/6) of pts receiving cryo/ipi (median 4-fold increase), but in the minority of pts receiving cryo (0/6, median 0-fold) or ipi (2/6 ipi; median 1-fold). No trends were observed in peripheral myeloid derived suppressor cells. Analysis of TILs by flow cytometry identified increased numbers of proliferating CD8+ T-cells (CD8+Ki67+) in ipi and cryo/ipi groups relative to cryo alone; furthermore, the ratio of proliferating (CD8+Ki67+) to regulatory (CD4+CD25+FoxP3+) cells was enhanced in the cryo/ipi group. Finally, analysis of T-cell repertoire in TILs demonstrated that cryo/ipi generated an influx of novel T-cell clones, with select clones surging dramatically in predominance and circulating within the periphery.
Conclusions: Utilizing an integrated assessment, we identified evidence of immunologic synergy with combination cryo/ipi versus either therapy alone. Of the tested parameters, peripheral CD4+ ICOS expression, plasma IFNγ, Ki67-gated TIL effector/regulatory ratios, and clonal repertoire analysis were identified as promising biomarkers of immune activation. These findings will inform a prospective assessment of potential immunologic biomarkers of immune response and clinical benefit in a phase 2 study of cryo-immunotherapy in ESBC.
Citation Format: David Page, Jianda Yuan, Adi Diab, Zhiwan Dong, Arielle Ginsberg, Phillip Wong, Ryan Emerson, David Redmond, Brian Blum, Zhenyu Mu, Chunjun Zhao, Christopher Comstock, Elizabeth Morris, Elizabeth Comen, Alan Kotin, Janice Sung, Edi Brogi, Monica Morrow, Stephen Solomon, Virgilio Sacchini, Majid Maybody, Deirdre Neville, Harlan Robins, Sujata Patil, Jedd Wolchok, Clifford Hudis, Larry Norton, James Allison, Padmanee Sharma, Heather McArthur. Integrated immunologic assessment of tumor infiltrating lymphocytes (TILs) and peripheral blood to assess synergy of cryoablation (cryo) plus ipilimumab (ipi) in early stage breast cancer (ESBC) patients (pts) [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 P2-15-01.