Psammomatoid ossifying fibroma (PsOF), also known as juvenile PsOF, is a benign fibro-osseous neoplasm predominantly affecting the extragnathic bones, particularly the frontal and ethmoid bones,... Show morePsammomatoid ossifying fibroma (PsOF), also known as juvenile PsOF, is a benign fibro-osseous neoplasm predominantly affecting the extragnathic bones, particularly the frontal and ethmoid bones, with a preference for adolescents and young adults. The clinical and morphologic features of PsOF may overlap with those of other fibro-osseous lesions, and additional molecular markers would help increase diagnostic accuracy. Because identical chromosomal breakpoints at bands Xq26 and 2q33 have been described in 3 cases of PsOF located in the orbita, we aimed to identify the exact genes involved in these chromosomal breakpoints and determine their frequency in PsOF using transcriptome sequencing and fiuorescence in situ hybridization (FISH). We performed whole RNA transcriptome sequencing on frozen tissue in 2 PsOF index cases and identified a fusion transcript involving SATB2, located on chromosome 2q33.1, and AL513487.1, located on chromosome Xq26, in one of the cases. The fusion was validated using reverse transcription (RT)-PCR and SATB2 FISH. The fusion lead to a truncated protein product losing most of the functional domains. Subsequently, we analyzed an additional 24 juvenile PsOFs, 8 juvenile trabecular ossifying fibromas (JTOFs), and 11 cemento-ossifying fibromas (COFs) for SATB2 using FISH and found evidence of SATB2 gene rear-rangements in 58% (7 of 12) of the evaluable PsOF cases but not in any of the evaluable JTOF (n = 7) and COF (n = 7) cases. A combination of SATB2 immunofiuorescence and a 2-color SATB2 FISH in our index case revealed that most tumor cells harboring the rearrangement lacked SATB2 expression. Using immunohistochemistry, 65% of PsOF, 100% of JTOF, and 100% of COF cases showed moderate or strong staining for SATB2. In these cases, we observed a mosaic pattern of expression with >25% of the spindle cells in between the bone matrix, with osteoblasts and osteocytes being positive for SATB2. Interestingly, 35% (8 of 23) of PsOFs, in contrast to JTOFs and COFs, showed SATB2 expression in <5% of cells. To our knowledge, this is the first report that shows the involvement of SATB2 in the development of a neoplastic lesion. In this study, we have showed that SATB2 rearrangement is a recurrent molecular alteration that appears to be highly specific for PsOF. Our findings support that PsOF is not only morphologically and clinically but also genetically distinct from JTOF and COF. (c) 2022 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved. Show less
IJzendoorn, D.G.P. van; Matusiak, M.; Charville, G.W.; Spierenburg, G.; Varma, S.; Colburg, D.R.C.; ... ; Rijn, M. van de 2022
Purpose: A major component of cells in tenosynovial giant cell tumor (TGCT) consists of bystander macrophages responding to CSF1 that is overproduced by a small number of neoplastic cells with a... Show morePurpose: A major component of cells in tenosynovial giant cell tumor (TGCT) consists of bystander macrophages responding to CSF1 that is overproduced by a small number of neoplastic cells with a chromosomal translocation involving the CSF1 gene. An autocrine loop was postulated where the neoplastic cells would be stimulated through CSF1R expressed on their surface. Here, we use single-cell RNA sequencing (scRNA-seq) to investigate cellular interactions in TGCT. Experimental Design: A total of 18,788 single cells from three TGCT and two giant cell tumor of bone (GCTB) samples underwent scRNA-seq. The three TGCTs were additionally analyzed using long-read RNA sequencing. Immunofluorescence and IHC for a range of markers were used to validate and extend the scRNA-seq findings.Results: Two recurrent neoplastic cell populations were identi-fied in TGCT that are highly similar to nonneoplastic synoviocytes. We identified GFPT2 as a marker that highlights the neoplastic cells in TCGT. We show that the neoplastic cells themselves do not express CSF1R. We identified overlapping MAB features between the giant cells in TGCT and GCTB. Conclusions: The neoplastic cells in TGCT are highly similar to nonneoplastic synoviocytes. The lack of CSF1R on the neoplastic cells indicates they may be unaffected by current therapies. High expression of GFPT2 in the neoplastic cells is associated with activation of the YAP1/TAZ pathway. In addition, we identified expression of the platelet-derived growth factor receptor in the neoplastic cells. These findings suggest two additional pathways to target in this tumor. Show less
There are no validated housekeeping genes in induced pluripotent stem cells (iPSC) and derived endothelial iPSC (iPSC-EC). Thus a comparison of gene expression levels is less reliable, especially... Show moreThere are no validated housekeeping genes in induced pluripotent stem cells (iPSC) and derived endothelial iPSC (iPSC-EC). Thus a comparison of gene expression levels is less reliable, especially during drug treatments. Here, we utilized transcriptome sequencing data of iPSC and iPSC-EC with or without CRISPR-Cas9 induced translocation to identify a panel of 15 candidate housekeeping genes. For comparison, five commonly used housekeeping genes (B2M, GAPDH, GUSB, HMBS, and HPRT1) were included in the study. The panel of 20 candidate genes were investigated for their stability as reference genes. This panel was analyzed and ranked based on stability using five algorithms, delta-Ct, bestkeeper, geNorm, Normfinder, and Reffinder. Based on the comprehensive ranking of Reffinder, the stability of the top two genes-RPL36AL and TMBIM6, and the bottom two genes-UBA1 and B2M, were further studied in iPSC-EC with and without genetic manipulation, and after treatment with telatinib. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), it was shown that gene expression of the top two housekeeping genes, RPL36AL and TMBIM6, remained stable during drug treatment. We identified a panel of housekeeping genes that could be utilized in various conditions using iPSC and iPSC-derived endothelial cells as well as genetically modified iPSC for drug treatment. Show less
IJzendoorn, D.G.P. van; Salvatori, D.C.F.; Cao, X.; Hil, F. van den; Briaire-de Bruijn, I.H.; Jong, D. de; ... ; Orlova, V.V. 2020
Chromosomal translocations are prevalent among soft tissue tumors, including those of the vasculature such as pseudomyogenic hemangioendothelioma (PHE). PHE shows endothelial cell (EC) features and... Show moreChromosomal translocations are prevalent among soft tissue tumors, including those of the vasculature such as pseudomyogenic hemangioendothelioma (PHE). PHE shows endothelial cell (EC) features and has a tumor-specific t(7;19)(q22;q13) SERPINE1-FOSB translocation, but is difficult to study as no primary tumor cell lines have yet been derived. Here, we engineer the PHE chromosomal translocation into human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 and differentiate these into ECs (hiPSC-ECs) to address this. Comparison of parental with PHE hiPSC-ECs shows (1) elevated expression of FOSB, (2) higher proliferation and more tube formation but lower endothelial barrier function, (3) invasive growth and abnormal vessel formation in mice after transplantation, and (4) specific transcriptome alterations reflecting PHE and indicating PI3K-Akt and MAPK signaling pathways as possible therapeutic targets. The modified hiPSC-ECs thus recapitulate functional features of PHE and demonstrate how these translocation models can be used to understand tumorigenic mechanisms and identify therapeutic targets. Show less
This thesis contributes to understanding vascular tumors and other soft-tissue sarcomas. For epithelioid hemangioma a new translocation is described leading to truncation of the FOS protein. It was... Show moreThis thesis contributes to understanding vascular tumors and other soft-tissue sarcomas. For epithelioid hemangioma a new translocation is described leading to truncation of the FOS protein. It was found that this truncation leads to a longer half-life of the FOS protein, which could explain the tumorigenesis of epithelioid hemangioma. For patients with pseudomyogenic hemangioendothelioma the mechanism of action for treatment with telatinib is elucidated. For pseudomyogenic hemangioendothelioma a new cell-line based model was developed. With CRISPR/Cas9 the characterizing translocation could be inserted into the DNA of endothelial cells, thereby recapitulating multiple characteristics of pseudomyogenic hemangioendothelioma. Throughout this thesis computational biology was used to study vascular tumors and other soft-tissue sarcomas to gain new insights and find potential new treatment options. Show less
