Development of locus specific sub-clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia.

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.

Trisomy 12 assessment by conventional fluorescence in-situ hybridization (FISH), FISH in suspension (FISH-IS) and laser scanning cytometry (LSC) in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has an extremely heterogeneous clinical course, and prognostication is based on common genetic abnormalities which are detected by standard cytogenetic methods. However, current methods are restricted by the low number of cells able to be analyzed, resulting in the potential to miss clinically relevant sub-clonal populations of cells. A novel high throughput methodology called fluorescence in situ hybridization in suspension (FISH-IS) incorporates a flow cytometry-based imaging approach with automated analysis of thousands of cells. Here we have demonstrated that the FISH-IS technique is applicable to aneuploidy detection in CLL samples for a range of chromosomes using appropriate centromere probes. This method is able to accurately differentiate between monosomy, disomy and trisomy with a sensitivity of 1% in CLL. An analysis comparing conventional FISH, FISH-IS and laser scanning cytometry (LSC) is presented.