Background Ependymoma is the third most common brain tumor in children. At the moment, surgery and radiotherapy are the only effective treatments that can be offered, and despite this, a significant part of the patients relapse with no therapeutic salvage options. Therefore, new treatment modalities are needed. To develop immunotherapies for these children, knowledge of the tumor microenvironment is crucial. The current study aims to unravel the tumor immune microenvironment (TIME) of pediatric posterior fossa A (PFA) ependymomas.
Materials and Methods We used bulk RNA sequencing data of 22 pediatric ependymomas. We defined two groups, hereafter called PFA immune+ (PFAI+) and PFAI-, based on the RNA expression levels of the NanoString panel of Human PanCancer Immune Profiling genes. We performed gene set enrichment analysis and deconvoluted the bulk RNA samples with ependymoma-specific single-cell RNA sequencing datasets. To validate our findings on a protein level, we applied immunohistochemistry with antibodies recognizing tumor-infiltrating lymphocytes, tumor-associated macrophages and microglia.
Results Unsupervised hierarchical clustering of RNA expression of immune-related genes revealed two distinct PFA groups. Differential gene expression analysis showed that PFAI+ have a significantly higher expression of genes associated with immune functions, such as CD3E, CCR2, GZMA, CXCL9 and TRBC2. Accordingly, gene set enrichment analysis demonstrated that several immune pathways, including T-cell signalling, interferon-gamma response and TNFα signalling are enriched in PFAI+ ependymomas. RNA expression of immune checkpoints was also higher in PFAI+ tumors, indicating that these tumors might be more responsive to combinational therapies including immune checkpoint inhibitors. While immunohistochemistry showed low amounts of infiltrating CD3+, CD8+ and CD20+ cells, high numbers of CD163+ and HLA-DRA+ cells were detected. These cells were mainly located in regions of tumor necrosis. Increased amounts of CD4+ and CD8+ lymphocytes were present in PFAI+ tumors compared to PFAI- tumors. Deconvolution of the bulk RNA samples based on single-cell RNA sequencing data revealed an enrichment of myeloid cell populations, especially microglia and macrophages. Furthermore, PFAI+ tumors were found to contain significantly higher relative proportions of T-cells compared to PFAI- tumors (median of 3.76% for PFAI+ compared to 0.03% for PFAI-).
Conclusions We suggest that pediatric posterior fossa A ependymomas can be divided into two groups based on the expression of immune-related genes, in which PFAI+ ependymomas are characterized by higher RNA expression levels of these genes and greater amounts of tumor-infiltrating immune cells. Several techniques showed an enrichment of T-lymphocytes in the PFAI+ ependymomas relative to the PFAI- ependymomas.
Disclosure Information J. Lammers: None. F. Calkoen: None. M. Kranendonk: None. A. Federico: None. M. Kool: None. L. Kester: None. J. van der Lugt: None.