Author: Erwin Brosens (Netherlands)
Co-authors: Anass Hajjaj, Koen van Overdam, Rogier Oldenburg, Rob Verdijk, Annelies de Klein, Emine Kiliç
Purpose
Retinal hemangioblastomas (RH) are are highly vascularized histologically benign neoplasms that can result in blindness due to detachment of the retina or massive exudation of the RH. Patients can either have sporadic mutations in the VHL gene or germline variants seen as part of autosomal dominant Von Hippel-Lindau disease. The VHL protein is part of the VCB-CR complex whit ubiquitin ligase activity. This complex can target proteins for degradation by the proteasome. An unstable VCB-CR complex is prone for degradation and deleterious mutations in VHL result in an unstable complex. In the CNS, the cells of the vasculature are VHL+/- whilst the neoplastic haemangioblast precursor like cells are VHL-/-. In contrast to retinal or central nervous system haemangioblastomas, malignant Clear Cell Renal Carcinoma’s (CCRC, VHL-/-) are extensively characterized, these tumour cells have additional somatic driver mutations. So far, no RH specific (somatic) mutational or copy number variation signatures have been described in literature. It is not known whether RH are distinct entities with other somatic mutations and differentfrom central nervous system hemangioblastomas. Future therapeutic strategies (eg gene therapy or small molecule inhibition) could depend on the presence or absence of additional changes.
Setting/Venue
This research presented here has been performed at the Erasmus MC Sophia Chlidren's hospital in collaboration with the Rotterdam Eye Hospital
Methods
Using whole genome sequencing, we compare the DNA of RH to the patient’s germline DNA. Using publicly available bulk-RNA expression profile datasets we identify cell population markers to be used in single cell sequencing experiments. Next, we selected the gene expression profiles of the stromal cell, pericyte cell and endothelial cell populations from previously published single cell experiments. These signatures are subsequent used to compare the expression profiles of central nervous system and retinal hemangioblastoma transcriptome profiles. Using immunohistochemistry, we evaluate VHL expression and biomarkers for the identification of pericytes, endothelial cells and stromal cells in different tumours of patients of which we determined the somatic mutation signature.
Results
We have isolated sufficient quantity and quality DNA from 4 retinal hemangioblastomas and corresponding germline DNA. The somatic changes in these samples -including Single Nucleotide Variants, small Insertions & Deletions and structural variants- have been assessed. To better appreciate the relevance of the detected damaging alterations, we determined which genes are expressed in normal retina and retinal stromal cells, endothelial cells and pericytes specifically. For this purpose, , we first evaluated publicly available bulk-RNA expression profile datasets of micro-vessels, endothelial cells, pericytes, mesenchymoangioblasts, mesenchymal stem cells, smooth muscle cells, retina and choroid too and identified cell population markers. Using these expression profiles, we selected the gene expression profiles of the stromal cell, pericyte cell and endothelial cell populations from previously published eye specific single cell experiments. We developed a core marker set that distinguishes these cells from pericytes, endothelial cells and stromal cells of other locations in the body. Additionally, we evaluated the gene expression signatures of these “retina micro-vessel genes” in central nervous system and retinal hemangioblastoma transcriptome profiles.
Conlusions
RH tumour material is very rare. We have characterized the mutational landscape of four RH and compared it to the other tumours present in four patients. Using a biomarker set we created from bulk-RNA transcriptome profiles of pericytes, endothelial cells and stromal cells, and we characterized retinal micro-vessel & RH transcriptome profiles. This biomarker set can be used in in-vitro induced pluripotent stem cell or direct transformation protocols. We evaluated the genomic landscape of RH using these expression profiles and are currently performing CRISPR/Cas9 gene editing experiments to confirm the results of our experiments.
Financial Disclosure
not applicable
Comments
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