Abstract Background and Objectives: Central nervous system germinomas are characterized by a massive immune cell infiltrate. This inflammatory component can be so extensive that tumor diagnostics is challenging because of masking of the malignant germ cell tumor (GCT) component. Similarely, it is often difficult to detect other malignant components in mixed germ cell tumors that dictate the biology and treatment stratification. In this study we employed recent comprehensive technologies to study the composition and spatial distribution of malignant as well as inflammatory components of the tumor microenvironment (TME). Methods: We systematically characterized immune cells in a cohort of germinomas by immunophenotyping and image analysis of formalin-fixed, paraffin-embedded (FFPE) tumor samples. Bulk mRNA expression data was generated by Nanostring technology and high-resolution spatial RNA expression data by Visium-HD technology. With the latter method we studied 10 germinomas and 3 mixed germ cell tumors. Individual candidate transcripts and proteins were validated by in-situ RNA hybridization and immunohistochemistry. Results: Spatial transcriptomics by Visium-HD allowed the sensitive detection of different malignant germ cell tumor components including germinoma, yolk sac tumor, embryonal carcinoma and choriocarcinoma components, as well as the spatial distribution of immune cells. Tumor infiltrating lymphocytes in germinomas were abundant and predominated by CD3-positive T cells, including CD4-positive T-helper cells, CD8-positive cytotoxic T cells and interspersed FoxP3-positive regulatory T cells. B lymphocytes outnumbered plasma cells. Some germinomas and mixed GCT showed tertiary lymphoid structures. Tumor-associated macrophages (TAM) consisted of clusters of activated PD-L1-positive macrophages and interspersed anti-inflammatory macrophages expressing CD163. While germinoma cells did not express PD-L1 on both the RNA and protein level, a high expression of BIRC5 (survivin) as anti-apoptotic gene was identified. Spatial mRNA expression analysis indicated specific ligand-receptor interactions between malignant GCT cells and TME components. In particular, germinoma cells produced high levels of macrophage migration inhibitory factor (MIF), and its receptor CD74 was found expressed in adjacent inflammatory cells. Conclusions: In conclusion, spatial high resolution transcriptomics was able to sensitively identify different malignant germ cell tumor components by their signature. The strong immune reaction observed in germinomas involved various inflammatory as well as suppressive mechanisms. Interaction analysis between GCT and TME cells indicated paracrine mechanisms in the control of growth and survival of intracranial GCT cells. Citation Format: Torsten Pietsch, Verena Dreschmann, Pia Zapka, Evelyn Dörner, Christian Vokuhl. Dissecting the complex ecosystem of intracranial germ cell tumors by spatial RNA and protein analysis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 636.
Pietsch et al. (Fri,) studied this question.