Bio-O2-02 - Improved Sézary cell detection and novel insights into immunophenotypic and molecular heterogeneity in Sézary syndrome

Abstract

Introduction: Sézary syndrome (SS) is an aggressive leukemic form of cutaneous T-cell lymphoma with neoplastic CD4+ T cells present in skin, lymph nodes, and blood. The detection of these so-called Sézary cells and the assessment of tumor burden in blood mostly relies on the recognition of immunophenotypic aberrancies by flow cytometry (FC). Nevertheless, despite the extensive use in research and widespread implementation in clinical practice, FC protocols lack standardization and is further hampered by the profound diversity in Sézary cell immunophenotypic characteristics. Exploring heterogeneity is critical for the overall knowledge of SS pathogenesis, tumor evolution, and resistance to therapy. In turn, this is essential for the discovery of Sézary-specific targets and biomarkers. Methods: We applied highly sensitive and standardized EuroFlow-based multiparameter FC (MFC) methods and tools on 37 SS samples for the in-depth immunophenotypic characterization and classification of Sézary cells according to their T-helper differentiation and maturation profiles. We combined MFC with fluorescence-activated cell sorting (FACS) and RNA sequencing (RNA-seq) on purified immunophenotypically distinct Sézary and matched normal CD4+ T-cell subsets of the same patients and healthy controls. With this approach, we aimed to determine the clonality status of FACS-cell sorted populations, study the transcriptome of immunophenotypically distinct Sézary cell subsets, and discover novel Sézary-specific markers using gene expression data. Results: By applying MFC, we accurately identified, quantified, and characterized Sézary cells in all SS samples. Comparative immunophenotypic analyses between Sézary cells and their normal CD4+ T-cell counterpart revealed substantial inter-and intra-patient heterogeneity in the expression of different markers and immunophenotypic changes over time. Sézary cells exhibited phenotypes corresponding with classical and non-classical T-helper subsets with different maturation phenotypes. Our RNA-seq data confirmed pure mono-clonality of isolated immunophenotypically aberrant CD4+ T-cell subsets and revealed the transcriptional profiles of different FACS-sorted CD4+ T-cell populations thereby identifying novel Sézary-specific signature genes that were consistently and exclusively perturbed across Sézary cell subsets. Conclusion: Our in-depth analyses provided a thorough overview of the immunophenotypic and transcriptional profiles in SS and further unraveled the heterogeneity of Sézary cell subpopulations between and within patients. Incorporating additional resolutive SS-specific markers could prove diagnostic and prognostic utility. Together, these new data will support improved diagnosis, disease monitoring, therapy, and prognosis. Sézary syndrome (SS) is an aggressive leukemic form of cutaneous T-cell lymphoma with neoplastic CD4+ T cells present in skin, lymph nodes, and blood. The detection of these so-called Sézary cells and the assessment of tumor burden in blood mostly relies on the recognition of immunophenotypic aberrancies by flow cytometry (FC). Nevertheless, despite the extensive use in research and widespread implementation in clinical practice, FC protocols lack standardization and is further hampered by the profound diversity in Sézary cell immunophenotypic characteristics. Exploring heterogeneity is critical for the overall knowledge of SS pathogenesis, tumor evolution, and resistance to therapy. In turn, this is essential for the discovery of Sézary-specific targets and biomarkers. We applied highly sensitive and standardized EuroFlow-based multiparameter FC (MFC) methods and tools on 37 SS samples for the in-depth immunophenotypic characterization and classification of Sézary cells according to their T-helper differentiation and maturation profiles. We combined MFC with fluorescence-activated cell sorting (FACS) and RNA sequencing (RNA-seq) on purified immunophenotypically distinct Sézary and matched normal CD4+ T-cell subsets of the same patients and healthy controls. With this approach, we aimed to determine the clonality status of FACS-cell sorted populations, study the transcriptome of immunophenotypically distinct Sézary cell subsets, and discover novel Sézary-specific markers using gene expression data. By applying MFC, we accurately identified, quantified, and characterized Sézary cells in all SS samples. Comparative immunophenotypic analyses between Sézary cells and their normal CD4+ T-cell counterpart revealed substantial inter-and intra-patient heterogeneity in the expression of different markers and immunophenotypic changes over time. Sézary cells exhibited phenotypes corresponding with classical and non-classical T-helper subsets with different maturation phenotypes. Our RNA-seq data confirmed pure mono-clonality of isolated immunophenotypically aberrant CD4+ T-cell subsets and revealed the transcriptional profiles of different FACS-sorted CD4+ T-cell populations thereby identifying novel Sézary-specific signature genes that were consistently and exclusively perturbed across Sézary cell subsets. Our in-depth analyses provided a thorough overview of the immunophenotypic and transcriptional profiles in SS and further unraveled the heterogeneity of Sézary cell subpopulations between and within patients. Incorporating additional resolutive SS-specific markers could prove diagnostic and prognostic utility. Together, these new data will support improved diagnosis, disease monitoring, therapy, and prognosis.