146 CD68 expression in tumor cells is associated with poor clinical outcomes following immunotherapy and immunotherapy plus chemotherapy combination therapies

<h3>Background</h3> CD68 is a glycoprotein expressed in lysosomes and on the cell surface of myeloid cells and frequently used as a macrophage marker. CD68 expression has also been described in epithelial tumor cells, but its function in tumor cell biology has not been described. <h3>Methods</h3> Using immunohistochemistry, multiplex immunofluorescence (mIF), in situ RNA hybridization, single-cell RNA sequencing (scRNAseq) and flow cytometry (FC), we analyzed CD68 expression in tumor cells using FFPE tumor biopsies collected in Phase 2 biliary tract cancer (BTC; NCT03875235) and hepatocellular carcinoma (HCC; NCT03298451) clinical trials, cell lines, and dissociated tumor cells (DTCs) from RCC (Renal Cell Carcinoma) commercial samples and evaluated association with clinical outcomes and expression of other genes/pathways. Potential mechanisms associated with CD68-positive tumor cells were explored using in vitro treatment of cell lines. <h3>Results</h3> In this study, CD68 expression in tumor cells was observed by mIF in 14/42 BTC and 42/129 HCC tumors and was associated with shorter Overall Survival (OS) and Progression-Free Survival in BTC (p=0.0013 and 0.014 respectively) and shorter OS in HCC (p=0.012) patients treated with immunotherapy +/- chemotherapy. In BTC, an inverse correlation between the number of CD68+ tumor cells and CD8+ T cells was observed by mIF. This observation was extended to renal cell carcinoma (RCC) in which an inverse relationship between the number of CD68+ and CD8+ cells was observed by FC on DTCs (R²=0.7188). To investigate the breadth of CD68+ tumor cells, we evaluated CD68 RNA expression using the Cancer Cell Line Encyclopedia (CCLE) database, finding that 377/968 cell lines representing multiple tumor types were positive. scRNAseq analysis of RCC DTCs showed one of the top 3 genes associated with CD68+ tumor cells when compared to CD68-/low tumor cells was TGF-beta induced (TGFBI), which is involved in epithelial-to-mesenchymal transition (EMT) and metastasis (2.5-fold change, p(adj)=8.12E-152). Further, treatment of solid tumor cell lines in vitro with TGFB1 upregulated CD68, TGFBI, and Vimentin, a marker of EMT. <h3>Conclusions</h3> These data suggest that ectopic expression of CD68 by tumor cells is associated with epithelial to myeloid transition and/or EMT and contributes to pro-metastatic and immune evasion mechanisms, resulting in worse outcome after treatment with immunotherapies. Understanding the mechanism underpinning CD68 expression in tumor cells may aid patient selection strategies and discovery of new therapeutic targets.