Abstract 2747: Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer
Authors: Launonen Inga-Maria P. , Lyytikäinen Nuppu , Casado Julia , Anttila Ella A. , Jacobson Connor A. , Lin Jia R. , Maliga Zoltan , Santaga Sandro , Elias Kevin M. , D'Andrea Alan D. , Konstantinopoulos Panagiotis A. , Sorger Peter K. , Färkkilä Anniina 
Published: July 2021

Abstract
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.