27MO - Integrated digital pathology and single-cell analysis identify the spatial and temporal evolution of immune cells networks in epithelial ovarian cancer

Background

Immune checkpoint blockade (ICB) failed to demonstrate activity in epithelial ovarian cancer (EOC) so far. What controls the complex spatial distribution of tumor-infiltrating lymphocytes (TILs) and immune cells in EOC is poorly understood and it remains challenging to systematically define tumor-immune phenotype due to its highly heterogeneous nature.

Methods

We profiled two large cohorts of primary-recurrent EOCs FFPE samples (N=180) by multi-immunofluorescence (mIF). We used a targeted gene panel for homologous recombination deficiency (HRD) to link genomic determinants with immune phenotypes. We established syngeneic mouse models to characterize TIL infiltration and single-cell T-cell and TME states based on BRCA mutational status and in response to chemotherapy (ChT).

Results

Applying digital pathology imaging we established a new algorithm to quantify the quantity and spatial distribution of CD8⁺ TILs while taking into consideration intra-tumoral heterogeneity. We defined four CD8⁺ immune-categories in our cohorts: purely-inflamed, mixed-inflamed, excluded and desert EOCs. Overall survival (OS) was significantly longer in inflamed EOCs compared to excluded and desert ones (p=0.021). Results were validated in a third independent cohort (N=62, p=0.001). By interrogating cellular neighborhoods and leukocytes networks on a whole-tissue basis we captured immune homotypic niches (T-cell to T-cell and macrophages to antigen-presenting cells [APC]) and heterotypic ones. TILs-APC niches were significantly enriched in purely inflamed tumors at baseline and were maintained at recurrence. Single-cell RNAseq analysis of mouse models confirmed higher CD8⁺ and TILs-APC infiltration in recurrent Brca1^mut tumors concomitant with enhanced TILs exhaustion program characterized by PD1, TIM3, TIGIT and cytotoxic signatures.

Conclusions

We provide a new immune-classification for EOC which is relevant for OS and demonstrate the key role of TIL-APC niches to drive TILs inflammation at recurrence. Our findings suggest that next-generation tissue-based biomarkers for ICB should include the simultaneous analysis of multiple immune cell type and their spatial interactions.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

G. Coukos: Financial Interests, Personal, Research Grant: Celgene; Financial Interests, Personal, Research Grant: Boehringer-Ingelheim; Financial Interests, Personal, Research Grant: Roche; Financial Interests, Personal, Research Grant: Bristol Myers Squibb; Financial Interests, Personal, Research Grant: Iovance Therapeutics; Financial Interests, Personal, Research Grant: Kite Pharma. All other authors have declared no conflicts of interest.