Discipline Molecular Medicine and Pathology College of Medicine and Public Health, Flinders University, Adelaide, South Australia, Australia.
Department of Immunology Allergy and Arthritis, Flinders Medical Centre, Adelaide, South Australia, Australia.
Cytometry A. 2017 Nov;91(11):1088-1095. doi: 10.1002/cyto.a.23264. Epub 2017 Oct 11.
Intra-tumor genetic heterogeneity is a hallmark of cancer. The ability to monitor and analyze these sub-clonal cell populations can be considered key to successful treatment, particularly in the modern era of targeted therapies. Although advances in sequencing technologies have significantly improved our ability to analyze the mutational landscape of tumors, this utility is reduced when considering small, but clinically significant sub-clones, that is, those representing <10% of the tumor burden. We have developed a high-throughput method that utilizes a 17-probe labeled bacterial artificial chromosome contig to quantify sub-clonal populations of cells based on deletion of a single locus. Chronic lymphocytic leukemia (CLL) cells harboring deletion of the short arm of chromosome 17 (del17p), an important prognostic marker for CLL were used to demonstrate the technique. Sub-clones of del17p cells were quantified and isolated from heterogeneous CLL populations using fluorescence in situ hybridization in suspension (FISH-IS) and the locus specific probe set. Using the combination of FISH-IS with the locus-specific probe set enables automated analysis of tens of thousands of cells, accurately quantifying and isolating cells carrying a del17p. Based on the fluorescence intensity of 17p probes, 17p (TP53) deleted cells were identified and sorted using flow cytometric techniques, and enrichment was demonstrated using single nucleotide polymorphism analysis. The ability to separate sub-clones of cells based on genetic heterogeneity, independent of the clone size, highlights the potential application of this method not only in the diagnostic and prognostic setting, but also as an unbiased approach to enable further detailed genetic analysis of the sub-clone with deep sequencing approaches. © 2017 International Society for Advancement of Cytometry.
肿瘤内遗传异质性是癌症的一个标志。监测和分析这些亚克隆细胞群体的能力可以被认为是成功治疗的关键，特别是在现代靶向治疗时代。尽管测序技术的进步显著提高了我们分析肿瘤突变景观的能力，但当考虑到小但具有临床意义的亚克隆时，这种用途就会降低，即那些代表肿瘤负担的<10%的亚克隆。我们开发了一种高通量方法，该方法利用 17 个探针标记的细菌人工染色体连续体，根据单个基因座的缺失来定量细胞的亚克隆群体。我们使用携带 17 号染色体短臂缺失（del17p）的慢性淋巴细胞白血病（CLL）细胞来证明该技术，del17p 是 CLL 的一个重要预后标志物。使用悬浮液中的荧光原位杂交（FISH-IS）和基因座特异性探针集，从异质 CLL 群体中定量和分离 del17p 细胞的亚克隆。使用 FISH-IS 与基因座特异性探针集的组合，可以自动分析数万甚至数十万个细胞，准确地定量和分离携带 del17p 的细胞。基于 17p 探针的荧光强度，使用流式细胞术技术鉴定和分选 del17p（TP53）缺失细胞，并通过单核苷酸多态性分析证明了富集。基于遗传异质性分离细胞亚克隆的能力，而与克隆大小无关，突出了该方法的潜在应用，不仅在诊断和预后设置中，而且作为一种无偏方法，还可以通过深度测序方法对亚克隆进行进一步详细的遗传分析。©2017 国际细胞分析协会。
