Abstract A04: Profiling DNA damage repair and immunophenotypes in BRCA1/2 mutated high-grade serous ovarian cancers
Authors: Farkkila Anniina , Kochupurakkal Bose , Howitt Brooke E. , Strickland Kyle C. , Chowdhury Dipanjan , Shapiro Geoffrey , Gjini Evisa , Rodig Scott J. , Matulonis Ursula A. , Konstantinopoulos Panagiotis , D’Andrea Alan D. 
Published: August 2018

Abstract
High-grade serous ovarian cancers (HGSOCs) are characterized by chromosomal instability and defects in homologous recombination (HR) DNA repair pathway. HR deficient cancer cells rely on alternative, error-prone DNA repair pathways, leading to accumulation of new mutations and increased neoantigen load recognized by the immune system (Strickland et al., 2016). Importantly, preclinical evidence suggests a complex interplay between DNA damage response (DDR) and the antitumor immune response. DNA damage has been shown to increase the expression of immune-regulatory genes, such as MCH class I antigens, and interferons, which contribute to the response to DNA damaging agents. Further, HR-deficient tumors exhibit an increased response to immune checkpoint, and DDR-driven activation of interferon signaling has been shown to overcome resistance to anti-PD1/PD-L1 therapies (Wang et al., 2016). We hypothesize that HR-deficient, BRCA1/2 mutant cancers have evolved differential immune-escape mechanisms in the context of cytotoxic DNA damaging agents. To elaborate these mechanisms, we set out to characterize the DDR and immunophenotypes in BRCA1/2 mutated, HR-deficient HGSOCs.
We have collected a clinically annotated cohort of primary HGSOC tumor samples from 37 germline BRCA1/2 carriers and 17 DFCI-Oncopanel annotated, HR-proficient patients (Strickland et al., 2016). HR proficiency was assayed by excluding tumors with pathogenic mutations in HR-pathway genes (ATM, ATRX, BRCA1, BRCA2, BRIP, CHEK2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, NBN, PTEN, and U2AF1), or loss of BRCA1 protein expression in IHC. Clinical details, including progression-free survival and overall survival, and previously analyzed markers such as PD1/PDL1 expressions and tumor-infiltrating lymphocyte infiltration, are used as correlation of the DDR profiles and immunophenotypes.
FFPE embedded primary tumor samples are stained with multiplex immunofluorescence (IF) at the DFCI Center for Immuno-Oncology. We are in the process of characterizing the levels of endogenous DNA damage, cell cycle phase, and HR proficiency, e.g., markers for yH2AX, Geminin, and RAD51 foci. We have designed five panels for multiplex IF that characterize altogether twenty markers for DNA damage, T-cell activation, T-regulatory cell activation, macrophage/APC/NK cell activation, and inflammatory pathway activation. Multiplex IF staining allows us to quantitate and correlate activation of specific immune-cell subtypes, as well as regulatory and inflammatory pathways, to patient outcomes at the cellular level using also spatial resolution data within the tumor microenvironment. The morphologically profiled mechanisms between DDR and inflammatory pathways will be then complemented with further functional assays in BRCA1/2 mutated ovarian cancer cell lines.
HR-deficient HGSOCs exhibit a distinct immunoprofile, which is consistent with the preclinical findings on the complex interplay between DDR and immune system. Profiling the regulators of this crosstalk will form the biologic basis for effective combination of DNA-damaging agents with immune-checkpoint inhibitors.
Citation Format: Anniina Farkkila, Bose Kochupurakkal, Brooke E. Howitt, Kyle C. Strickland, Dipanjan Chowdhury, Geoffrey Shapiro, Evisa Gjini, Scott J. Rodig, Ursula A. Matulonis, Panagiotis Konstantinopoulos, Alan D. D’Andrea. Profiling DNA damage repair and immunophenotypes in BRCA1/2 mutated high-grade serous ovarian cancers. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A04.