Abstract A019: Single-cell analysis of tumor population diversity in synovial sarcoma

Synovial sarcoma (SyS) is a rare mesenchymal tumor, which constitutes around 10% of all soft tissue sarcoma cases. It is hard to detect due to slow initial growth, prone to metastasis, and has a high misdiagnosis rate due to diverse symptoms. Questions surrounding SyS include its cell of origin and the mechanisms that help it evade detection by the immune system. The objective of the study was to characterize the diversity of tumor populations in SyS at the single cell level with the potential aim of finding novel prognostic and therapeutic markers. Tumor samples from 16 patients with SyS were fixed and dissociated according to the Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling protocol (10x Genomics). Single cell transcriptome libraries were constructed according to the Chromium Fixed RNA protocol (10x Genomics) and sequenced on the Genolab M platform (GeneMind). The sequence files were analyzed using Cell Ranger 7.1.0, DoubletCollection, Seurat package, CytoTrace, SCEVAN, and CellChat packages. Five tumor clusters (TC) were identified in the integrated SyS tumor sample: HOXD11+ TC, NKD2+ TC, POSTN+ TC, MKI67+ TC, and EPCAM+ TC. The NKD2+ TC has the highest expression levels of the SS18-SSX oncogenic program among the five tumor clusters and a high differentiation state. It is enriched in genes related to extracellular matrix (ECM), WNT and Hippo signaling. The HOXD11+ TC has lower expression levels of the SS18-SSX oncogenic program. It is poorly differentiated and is enriched in genes related to oxidative phosphorylation and developmental processes, especially in the homeobox genes (HOXD8, HOXD9, HOXD10, HOX11). The POSTN+ TC lacks a significant level of SS18-SSX program expression, is poorly differentiated, and resembles fibroblasts with its extremely high levels of genes related to ECM remodeling. Finally, the MKI67+ TC contains poorly differentiated, highly proliferative tumor cells, while the EPCAM+ TC contains epithelial-like tumor cells. These last two tumor clusters express medium levels of the SS18-SSX oncogenic program. Patients with SyS that have the HOXD11+ TC as their prevalent tumor cluster have demonstrated the highest rates of local recurrence and metastasis in our study of 16 patients. HOXD11+ SyS tumors possess higher levels T cell activation and exhaustion signatures. According to the analysis of cell-cell communication, HOX11+ TC has a higher probability of employing MIF-CD74 signaling compared to other SyS TC. It can be hypothesized that the activation of the MIF-CD74 signaling axis between HOX11D11+ TC and T cells may lead to T cell exhaustion, which leads to worse prognosis for this group of patients. In conclusion, the presence of the HOXD11+ tumor cluster may have prognostic value for treatment of patients with SyS. The presence of this tumor cluster has been shown to correspond with higher rates of local recurrence and metastasis. The mechanism behind this phenomenon may involve T cell exhaustion through MIF-CD74 signaling. However, further studies are needed to provide a definitive answer.