Abstract AP22: DNA DAMAGE RESPONSES AND IMMUNE PROFILING THROUGH HIGHLY MULTIPLEXED TISSUE IMMUNOFLUORESCENCE (T-CYCIF) IN HIGH-GRADE SEROUS OVARIAN CANCER
Authors: Färkkilä Anniina , Lin Jia-Ren , Maliga Zoltan , Chopra Sameer S. , Koruchupakkal Bose , Howitt Brooke E. , Strickland Kyle C. , Santagata Sandro , Swisher Elizabeth M. , Matulonis Ursula A. , Guerriero Jennifer. L. , Elias Kevin , Konstantinopoulos Panagiotis , Sorger Peter K. , D'Andrea Alan D. 
Published: June 2021

Abstract
INTRODUCTION: Immune checkpoint blockade (ICB) has emerged as a new therapeutic approach for multiple cancers, however, the responses to single-agent ICBs have been modest in high-grade serous ovarian cancer (HGSOC). Preclinical and early clinical data indicate promising efficacy of combination with DNA damaging agents and immunotherapy, however lack of functional- and tissue geographical knowledge on the interplay between DNA repair and immune activation has hampered the future development of these strategies. The majority of HGSOC are deficient in homologous recombination (HR) DNA repair, and this deficiency is associated with increased immune recognition and potentially increased response to ICBs. Compelling evidence has shown that DNA damaging agents increase the expression of immune-regulatory genes, such as interferons, which can potentially overcome resistance to ICB. There is a critical need for a deeper understanding of the dynamics between DNA damage in cancer cells and anti-tumor immune responses in HGSOC in order to find rational combinations and predictive biomarkers for DNA damaging agents and immunotherapy.
RESULTS: We are employing a novel, high-multiplex tissue cyclic immunofluorescence (t-CycIF) platform allowing for the simultaneous detection of up to 60 different antigens at single cell resolution. To reveal the effects of between intrinsic and treatment-induced DNA damage in HGSOC, we are profiling the microenvironments in HGSOCs with inherent DNA repair deficiencies, and after DNA damaging therapy. We collected clinically annotated cohorts of 37 BRCA1/2 mutated and 17 HR wild-type patients (Strickland et al, 2016), as well as six paired pre- and post-treatment and 18 post-treatment tumor samples from patients undergoing neoadjuvant chemotherapy (NACT). Using image analysis we generated highly multiplexed single cell data for over 106 cells. Through supervised clustering, we evidenced distinct cell compositions in the tumor microenvironment of BRCA1/2 mutated and HR-wild type HGSOCs. Consistent with the role of immune-suppression in HGSOC progression, we found that high infiltration of CD4/FOXP3+ regulatory T-cells associated with more actively proliferating cancer cells. Interestingly, tumors with high expression of PD1/PD-L1 were found to have high infiltration of CD1c+ dendritic cells potentially indicating active suppression of antigen presenting pathways in these tumors. Further, tumors with high levels of DNA damage show active interferon signaling, which associated with significantly higher CD8+ cytotoxic T-cell infiltration. In addition, our preliminary evidence suggests heterogenous DNA damage response- and immune profiles in samples collected after NACT.
CONCLUSIONS: BRCA1/2 mutated tumors have a distinct microenvironment compared to HR-wt HGSOC. In support of earlier findings, FOXP3+ T-cells contribute to immune suppression in HGSOC. The high infiltration of dendritic cells and PD1/PD-L1 expression indicates a subgroup of HGSOC that are likely sensitive to ICBs. Further, increased DNA damage and interferon pathway activation delineated a more immunogenic subset of HGSOC. We conclude that t-CycIF could accelerate the development of rational strategies for combining DNA damaging agents with immunotherapy to ultimately improve the treatment and outcomes of patients with ovarian cancer.
Citation Format: Anniina Färkkilä, Jia-Ren Lin, Zoltan Maliga, Sameer S. Chopra, Bose Koruchupakkal, Brooke E. Howitt, Kyle C. Strickland, Sandro Santagata, Elizabeth M. Swisher, Ursula A. Matulonis, Jennifer. L. Guerriero, Kevin Elias, Panagiotis Konstantinopoulos, Peter K. Sorger, and Alan D. D'Andrea. DNA DAMAGE RESPONSES AND IMMUNE PROFILING THROUGH HIGHLY MULTIPLEXED TISSUE IMMUNOFLUORESCENCE (T-CYCIF) IN HIGH-GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP22.