Search

Immune checkpoint blockade has emerged as a new therapeutic approach for multiple cancers. The majority of high-grade serous ovarian, fallopian tube or peritoneal cancers (HGSCs) are deficient in homologous recombination (HR) DNA repair, typically due to mutations or hypermethylation of BRCA1 or BRCA2 genes. A detailed understanding how tumor genotypes affect the tumor microenvironment is unknown. We performed single-cell spatial analysis of the tumor microenvironment using a highly multiplex tissue cyclic immunofluorescence (t-CycIF) platform with a clinically annotated cohort of 31 patients with BRCA1/2 mutated (BRCAmut) tumors, and 13 patients with tumors without alterations in HR genes (HRwt) (Strickland et al, 2016). Using image analysis, we generated single-cell data for 24 markers in over 105 cells. We clustered the cells into tumor, immune, and stromal cells, and further divided the tumor cells into 7 metaclusters, immune cells into 10 subtypes and stromal cells into 9 metaclusters based on their functional states. Overall, the BRCAmut tumors showed a higher proportion of tumor cells over stromal cells as compared to HRwt tumors (p=0.031). Moreover, we found distinct functional states of tumor cell metaclusters with opposing prognostic roles in the tumor HR-genotypes. In immune profiling, the BRCAmut tumors exhibited an increased proportion of overall macrophages as compared to HRwt tumors (p=0.024). By contrast, HRwt tumors exhibited more CD11c expressing antigen-presenting cells than BRCAmut tumors (p=0.0013). Interestingly, the tumors with high CD163 expressing, M2 macrophages exhibited a lower overall immune diversity (Simpson's diversity index) in both BRCAmut tumors (p=8.9 × 10−6) and HRwt tumors (p=0.0076). However, the BRCAmut tumors with high immune diversity also had higher proportions of CD11c expressing macrophages (p=0.029). In survival analyses, CD4+ effector T-cells associated with a prolonged platinum free interval exclusively in the BRCAmut tumors (p=0.0011, HR 0.26, 95% CI 0.10-0.66). In correlative analyses of the tumor-immune-stromal populations, tumor metaclusters associated with distinct immune phenotypes in the BRCAmut tumors. By contrast, immune composition was shaped more by stromal metaclusters in HRwt tumors. These findings further support the differential regulation of tumor microenvironment composition in the tumor HR-genotypes. Further, our preliminary analyses on spatial arrangement of the single-cell subpopulations confirm the distinct microenvironment niches in BRCA1/2 vs HRwt HGSCs. In conclusion, our single-cell spatial t-CycIF analyses suggest functionally and spatially distinct microenvironments in BRCAmut tumors with the potential to accelerate the development of immunotherapeutic strategies to ultimately improve the treatment and outcomes of patients with ovarian cancer. Citation Format: Inga-Maria P. Launonen, Nuppu Lyytikäinen, Julia Casado, Ella A. Anttila, Connor A. Jacobson, Jia R. Lin, Zoltan Maliga, Sandro Santaga, Kevin M. Elias, Alan D. D'Andrea, Panagiotis A. Konstantinopoulos, Peter K. Sorger, Anniina Färkkilä. Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2747.

Introduction: TOPACIO/Keynote-162 is a multi-center, open-label, single-arm phase 1/2 trial of Poly-ADP Ribose Polymerase (PARP) inhibitor niraparib combined with an anti-PD-1 antibody pembrolizumab. One cohort enrolled patients with recurrent ovarian cancer (OC). As tumor BRCA1/2 mutation (tBRCA), Homologous Recombination Deficiency (HRD) test (Myriad), or PD-L1 testing by immunohistochemistry were unable to predict clinical responses, we set out to examine additional DNA repair and immune biomarkers for response. Methods: We analyzed pre-trial Formalin Fixed Paraffin Embedded (FFPE) tumor samples from 49 patients with recurrent OC. The samples were collected either at diagnosis, or a median of 4.3 months (range 1.5 - 91.1) after the diagnosis. The median time from diagnosis to trial entry was 35.8 months (range 11.4 - 136.5). To further assess HRD we performed BROCA sequencing of 69 DNA repair genes as previously reported. NanoString gene expression analysis was performed using an IO360 panel complemented with 30 DNA repair genes and analyzed with the NSolver software. Findings were correlated with clinical response evaluated based on RECIST v1.1. Results: Overall, the confirmed objective response (OR) rate was 18% with a clinical benefit (complete response + partial response + stable disease; CB) rate of 65%. HRD as assessed by BROCA sequencing did not associate with OR or CB. In the Nanostring mRNA expression analyses, the samples taken at the time of diagnosis (n=23) had significantly lower abundance score for several immune cell lineages, including cytotoxic cells, macrophages, dendritic cells, and NK-cells compared to samples collected after the diagnosis. In addition, in the at-diagnosis samples, lower abundance scores for overall immune cells (CD45), tumor-infiltrating lymphocytes, CD8+T-cells, and macrophages were associated with clinical benefit. Pathway analysis demonstrated that increased scores for Cytosolic DNA sensing- and interferon pathways significantly correlated with OR, particularly in the platinum-resistant patients. In contrast, the samples collected after diagnosis (n=21), had increased mRNA expression levels of PD-L1, PD-L2 and PD-1, and a higher score for exhausted CD8+ T-cells than the samples collected at diagnosis. In these samples, a low exhausted CD8+T cell/T-regulatory cell score ratio was associated with OR. Conclusions: Increased interferon signaling, and exhausted T/regulatory T-cell ratio are associated with response to the combination of niraparib and pembrolizumab. We are in the process of performing further profiling of the tumor microenviroment with a highly multiplexed tissue immunofluorescence (tCycIF) providing a comprehensive analysis of cell types, functional states, and spatial interactions at single-cell resolution. Citation Format: Anniina Farkkila, Jia R. Lin, Julia Casado, Huy Nguyen, Yinghui Zhou, Julie R. Graham, Bruce J. Dezube, Steven Waggoner, Pamela Munster, Gini F. Fleming, Sandro Santagata, Ursula A. Matulonis, Peter K. Sorger, Elizabeth M. Swisher, Alan D. D’Andrea, Panagiotis Konstantinopoulos. Association of the tumor-immune microenvironment with response to niraparib and pembrolizumab in relapsed, platinum-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4487.