Imaging Flow Cytometric Identification of Chromosomal Defects in Paediatric Acute Lymphoblastic Leukaemia
Acute lymphoblastic leukaemia is the most common childhood malignancy that remains a leading cause of death in childhood. It may be characterised by multiple known recurrent genetic aberrations that inform prognosis, the most common being hyperdiploidy and t(12;21) <i>ETV6::RUNX1</i>. We...
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2025-01-01
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| author | Ana P. A. Simpson Carly E. George Henry Y. L. Hui Ravi Doddi Rishi S. Kotecha Kathy A. Fuller Wendy N. Erber |
| author_facet | Ana P. A. Simpson Carly E. George Henry Y. L. Hui Ravi Doddi Rishi S. Kotecha Kathy A. Fuller Wendy N. Erber |
| author_sort | Ana P. A. Simpson |
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| description | Acute lymphoblastic leukaemia is the most common childhood malignancy that remains a leading cause of death in childhood. It may be characterised by multiple known recurrent genetic aberrations that inform prognosis, the most common being hyperdiploidy and t(12;21) <i>ETV6::RUNX1</i>. We aimed to assess the applicability of a new imaging flow cytometry methodology that incorporates cell morphology, immunophenotype, and fluorescence in situ hybridisation (FISH) to identify aneuploidy of chromosomes 4 and 21 and the translocation <i>ETV6::RUNX1</i>. We evaluated this new “immuno-flowFISH” platform on 39 cases of paediatric ALL of B-lineage known to have aneuploidy of chromosomes 4 and 21 and the translocation <i>ETV6::RUNX1</i>. After identifying the leukaemic population based on immunophenotype (i.e., expression of CD34, CD10, and CD19 antigens), we assessed for copy numbers of loci for the centromeres of chromosomes 4 and 21 and the <i>ETV6</i> and <i>RUNX1</i> regions using fluorophore-labelled DNA probes in more than 1000 cells per sample. Trisomy 4 and 21, tetrasomy 21, and translocations of <i>ETV6::RUNX1,</i> as well as gains and losses of <i>ETV6</i> and <i>RUNX1,</i> could all be identified based on FISH spot counts and digital imagery. There was variability in clonal makeup in individual cases, suggesting the presence of sub-clones. Copy number alterations and translocations could be detected even when the cell population comprised less than 1% of cells and included cells with a mature B-cell phenotype, i.e., CD19-positive, lacking CD34 and CD10. In this proof-of-principle study of 39 cases, this sensitive and specific semi-automated high-throughput imaging flow cytometric immuno-flowFISH method has been able to show that alterations in ploidy and <i>ETV6::RUNX1</i> could be detected in the 39 cases of paediatric ALL. This imaging flow cytometric FISH method has potential applications for diagnosis and monitoring disease and marrow regeneration (i.e., distinguishing residual ALL from regenerating haematogones) following chemotherapy. |
| format | Article |
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| institution | Kabale University |
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| spelling | doaj-art-644eb5cd95064474ad928acddbed08072025-01-24T13:26:43ZengMDPI AGCells2073-44092025-01-0114211410.3390/cells14020114Imaging Flow Cytometric Identification of Chromosomal Defects in Paediatric Acute Lymphoblastic LeukaemiaAna P. A. Simpson0Carly E. George1Henry Y. L. Hui2Ravi Doddi3Rishi S. Kotecha4Kathy A. Fuller5Wendy N. Erber6School of Biomedical Sciences, The University of Western Australia, Crawley, WA 6009, AustraliaDepartment of Clinical Haematology, Oncology, Blood and Marrow Transplantation, Perth Children’s Hospital, Perth, WA 6009, AustraliaSchool of Biomedical Sciences, The University of Western Australia, Crawley, WA 6009, AustraliaSchool of Biomedical Sciences, The University of Western Australia, Crawley, WA 6009, AustraliaDepartment of Clinical Haematology, Oncology, Blood and Marrow Transplantation, Perth Children’s Hospital, Perth, WA 6009, AustraliaSchool of Biomedical Sciences, The University of Western Australia, Crawley, WA 6009, AustraliaSchool of Biomedical Sciences, The University of Western Australia, Crawley, WA 6009, AustraliaAcute lymphoblastic leukaemia is the most common childhood malignancy that remains a leading cause of death in childhood. It may be characterised by multiple known recurrent genetic aberrations that inform prognosis, the most common being hyperdiploidy and t(12;21) <i>ETV6::RUNX1</i>. We aimed to assess the applicability of a new imaging flow cytometry methodology that incorporates cell morphology, immunophenotype, and fluorescence in situ hybridisation (FISH) to identify aneuploidy of chromosomes 4 and 21 and the translocation <i>ETV6::RUNX1</i>. We evaluated this new “immuno-flowFISH” platform on 39 cases of paediatric ALL of B-lineage known to have aneuploidy of chromosomes 4 and 21 and the translocation <i>ETV6::RUNX1</i>. After identifying the leukaemic population based on immunophenotype (i.e., expression of CD34, CD10, and CD19 antigens), we assessed for copy numbers of loci for the centromeres of chromosomes 4 and 21 and the <i>ETV6</i> and <i>RUNX1</i> regions using fluorophore-labelled DNA probes in more than 1000 cells per sample. Trisomy 4 and 21, tetrasomy 21, and translocations of <i>ETV6::RUNX1,</i> as well as gains and losses of <i>ETV6</i> and <i>RUNX1,</i> could all be identified based on FISH spot counts and digital imagery. There was variability in clonal makeup in individual cases, suggesting the presence of sub-clones. Copy number alterations and translocations could be detected even when the cell population comprised less than 1% of cells and included cells with a mature B-cell phenotype, i.e., CD19-positive, lacking CD34 and CD10. In this proof-of-principle study of 39 cases, this sensitive and specific semi-automated high-throughput imaging flow cytometric immuno-flowFISH method has been able to show that alterations in ploidy and <i>ETV6::RUNX1</i> could be detected in the 39 cases of paediatric ALL. This imaging flow cytometric FISH method has potential applications for diagnosis and monitoring disease and marrow regeneration (i.e., distinguishing residual ALL from regenerating haematogones) following chemotherapy.https://www.mdpi.com/2073-4409/14/2/114acute lymphoblastic leukaemiahyperdiploid<i>ETV6::RUNX1</i>imaging flow cytometryimmuno-flowFISH |
| spellingShingle | Ana P. A. Simpson Carly E. George Henry Y. L. Hui Ravi Doddi Rishi S. Kotecha Kathy A. Fuller Wendy N. Erber Imaging Flow Cytometric Identification of Chromosomal Defects in Paediatric Acute Lymphoblastic Leukaemia Cells acute lymphoblastic leukaemia hyperdiploid <i>ETV6::RUNX1</i> imaging flow cytometry immuno-flowFISH |
| title | Imaging Flow Cytometric Identification of Chromosomal Defects in Paediatric Acute Lymphoblastic Leukaemia |
| title_full | Imaging Flow Cytometric Identification of Chromosomal Defects in Paediatric Acute Lymphoblastic Leukaemia |
| title_fullStr | Imaging Flow Cytometric Identification of Chromosomal Defects in Paediatric Acute Lymphoblastic Leukaemia |
| title_full_unstemmed | Imaging Flow Cytometric Identification of Chromosomal Defects in Paediatric Acute Lymphoblastic Leukaemia |
| title_short | Imaging Flow Cytometric Identification of Chromosomal Defects in Paediatric Acute Lymphoblastic Leukaemia |
| title_sort | imaging flow cytometric identification of chromosomal defects in paediatric acute lymphoblastic leukaemia |
| topic | acute lymphoblastic leukaemia hyperdiploid <i>ETV6::RUNX1</i> imaging flow cytometry immuno-flowFISH |
| url | https://www.mdpi.com/2073-4409/14/2/114 |
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