Spatial profiling of ovarian clear cell carcinoma reveals immune-hot features

Abstract Introduction OCCC has high incidence in Asia with frequent occurrence at early stage but without sufficient data on molecular stratification for high-risk patients. Recently, immune-hot features have been proposed as an indicator for poor prognosis for early-stage OCCC. Specific patterns of intra-tumoral heterogeneity (ITH) associated with immune-hot features need to be defined. Methods Formalin-fixed paraffine embedded (FFPE) tumor sections from 10 early-stage OCCC patients were included. Digital Spatial Profiling (DSP) of 18 protein targets was conducted by using the nanoString GeoMx system to profile selected regions of interest (ROIs) based on the reference H&E staining morphology. Areas of illumination (AOIs) were defined according to ROI segmentation by the fluorescence signals of visualization markers pan-cytokeratin (PanCK), CD45, or DNA. Results Unsupervised hierarchical clustering of 252 AOIs from 229 ROIs showed that PanCK segments expressed different combinations of immune markers suggestive of immune mimicry features. Three immune-hot clusters were identified: granzyme B high (C1-a), immune signal high (C1-b) and immune-like cells (C1-c); two immune-cold clusters were identified: fibronectin-high (C2-a) and signal-cold (C2-b). Immune cells around C1-b and C1-c PanCK+ AOIs were tumor infiltrating immune cells (TIIs) with higher expression of CD68, while those around C1-a, C2-a and C2-b PanCK+ AOIs were non-TIIs with higher expression of SMA. C1-c and C2-a PanCK+ AOIs were associated with OCCC recurrence. TIIs had higher frequencies in C1-b and C1-c PanCK+ AOIs and were associated with OCCC recurrence. Correlating with morphology, tumor samples with recurrence showed higher frequency of papillary pattern. Plus, ROIs with papillary pattern had extremely high frequency of PanCK segments of C1-c feature, higher frequency of TIIs, and macrophage lineage immune mimicry with high intensity of CD68. Conclusions Spatial profiling of early-stage OCCC tumors revealed that immune mimicry of tumor cells, the presence of TIIs, and papillary pattern in morphology were associated with recurrence.