Simpson Ana P A, George Carly E, Hui Henry Y L, Doddi Ravi, Kotecha Rishi S, Fuller Kathy A, Erber Wendy N
School of Biomedical Sciences, The University of Western Australia, Crawley, WA 6009, Australia.
Department of Clinical Haematology, Oncology, Blood and Marrow Transplantation, Perth Children's Hospital, Perth, WA 6009, Australia.
Cells. 2025 Jan 14;14(2):114. doi: 10.3390/cells14020114.
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) . 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 . 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 . 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 and regions using fluorophore-labelled DNA probes in more than 1000 cells per sample. Trisomy 4 and 21, tetrasomy 21, and translocations of as well as gains and losses of and 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 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.
急性淋巴细胞白血病是最常见的儿童恶性肿瘤，仍是儿童死亡的主要原因。它可能具有多种已知的复发性基因畸变，这些畸变可用于判断预后，最常见的是超二倍体和t(12;21)。我们旨在评估一种新的成像流式细胞术方法的适用性，该方法结合了细胞形态学、免疫表型和荧光原位杂交（FISH）来识别4号和21号染色体的非整倍性以及易位。我们在39例已知有4号和21号染色体非整倍性及易位的B系小儿急性淋巴细胞白血病病例中评估了这种新的“免疫流式FISH”平台。在根据免疫表型（即CD34、CD10和CD19抗原的表达）识别白血病细胞群体后，我们使用荧光团标记的DNA探针在每个样本的1000多个细胞中评估4号和21号染色体着丝粒以及 和 区域的基因座拷贝数。4号和21号染色体三体、21号染色体四体、 的易位以及 和 的增减都可以根据FISH斑点计数和数字图像识别出来。个别病例的克隆组成存在差异，表明存在亚克隆。即使细胞群体占细胞总数不到1%，并且包括具有成熟B细胞表型（即CD19阳性，缺乏CD34和CD10）的细胞，也能检测到拷贝数改变和易位。在这项对39例病例的原理验证研究中，这种灵敏且特异的半自动高通量成像流式细胞术免疫流式FISH方法能够表明，在39例小儿急性淋巴细胞白血病病例中可以检测到倍性和 的改变。这种成像流式细胞术FISH方法在化疗后疾病诊断和监测以及骨髓再生（即区分残留的急性淋巴细胞白血病与再生的造血细胞）方面具有潜在应用。
