Dataset Information

High Specificity of BCL11B and GLG1 for EWSR1-FLI1 and EWSR1-ERG Positive Ewing Sarcoma.

ABSTRACT: Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for EWSR1-FLI1-positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including EWSR1-ERG-positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by EWSR1-ERG-positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.

SUBMITTER: Orth MF

PROVIDER: S-EPMC7139395 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Publications

High Specificity of BCL11B and GLG1 for EWSR1-FLI1 and EWSR1-ERG Positive Ewing Sarcoma.

Orth Martin F MF Hölting Tilman L B TLB Dallmayer Marlene M Wehweck Fabienne S FS Paul Tanja T Musa Julian J Baldauf Michaela C MC Surdez Didier D Delattre Olivier O Knott Maximilian M L MML Romero-Pérez Laura L Kasan Merve M Cidre-Aranaz Florencia F Gerke Julia S JS Ohmura Shunya S Li Jing J Marchetto Aruna A Henssen Anton G AG Özen Özlem Ö Sugita Shintaro S Hasegawa Tadashi T Kanaseki Takayuki T Bertram Stefanie S Dirksen Uta U Hartmann Wolfgang W Kirchner Thomas T Grünewald Thomas G P TGP

Cancers 20200310 3

Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for <i>EWSR1-FLI1</i>-positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including <i>EWSR1 ...[more]

PMID: 32164354

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Project description:BackgroundThe EWSR1/FLI1 gene fusion is the most common rearrangement leading to cell transformation in Ewing sarcoma (ES). Previous studies have indicated that expression at the cellular level is heterogeneous, and that levels of expression may oscillate, conferring different cellular characteristics. In ES the role of EWSR1/FLI1 in regulating subpopulation dynamics is currently unknown.MethodsWe used siRNA to transiently suppress EWSR1/FLI1 expression and followed population dynamics using both single cell expression profiling, CyTOF and functional assays to define characteristics of exponentially growing ES cells and of ES cells in which EWSR1/FLI1 had been downregulated. Novel transcriptional states with distinct features were assigned using random forest feature selection in combination with machine learning. Cells isolated from ES xenografts in immune-deficient mice were interrogated to determine whether characteristics of specific subpopulations of cells in vitro could be identified. Stem-like characteristics were assessed by primary and secondary spheroid formation in vitro, and invasion/motility was determined for each identified subpopulation. Autophagy was determined by expression profiling, cell sorting and immunohistochemical staining.ResultsWe defined a workflow to study EWSR1/FLI1 driven transcriptional states and phenotypes. We tracked EWSR1/FLI1 dependent proliferative activity over time to discover sources of intra-tumoral diversity. Single-cell RNA profiling was used to compare expression profiles in exponentially growing populations (si-Control) or in two dormant populations (D1, D2) in which EWSR1/FLI1 had been suppressed. Three distinct transcriptional states were uncovered contributing to ES intra-heterogeneity. Our predictive model identified ~1% cells in a dormant-like state and ~ 2-4% cells with stem-like and neural stem-like features in an exponentially proliferating ES cell line and in ES xenografts. Following EWSR1/FLI1 knockdown, cells re-entering the proliferative cycle exhibited greater stem-like properties, whereas for those cells remaining quiescent, FAM134B-dependent dormancy may provide a survival mechanism.ConclusionsWe show that time-dependent changes induced by suppression of oncogenic EWSR1/FLI1 expression induces dormancy, with different subpopulation dynamics. Cells re-entering the proliferative cycle show enhanced stem-like characteristics, whereas those remaining dormant for prolonged periods appear to survive through autophagy. Cells with these characteristics identified in exponentially growing cell populations and in tumor xenografts may confer drug resistance and could potentially contribute to metastasis.

Dataset Information

High Specificity of BCL11B and GLG1 for EWSR1-FLI1 and EWSR1-ERG Positive Ewing Sarcoma.

Publications

High Specificity of BCL11B and GLG1 for EWSR1-FLI1 and EWSR1-ERG Positive Ewing Sarcoma.

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