FISH By Imaging Flow Cytometry in CLL for Diagnosis and MRD Assessment

Chronic lymphocytic leukaemia is a genetically heterogeneous disease with treatment and prognosis varying based on chromosomal abnormalities. These are detectable in up to 80% of cases when tested on the nuclei of interphase cells by fluorescence in situ hybridisation (FISH). Despite the clinical importance of FISH in management, as only up to 200 nuclei are generally assessed, it is not suitable for minimal residual disease (MRD) assessment. Since clinical decisions are based on detection thresholds of 10 ^-4, MRD assays are restricted to flow cytometry and molecular based assessment. Here we have explored the utility of a cutting-edge automated imaging flow cytometry method that incorporates cell immunophenotype and FISH ("immuno-flowFISH") to detect chromosomal abnormalities in CLL.

Aims: Our aim was to determine the capability of immuno-flowFISH using imaging flow cytometry to detect del(17p) and +12 in CLL, and, the lowest limit of detection. We hypothesized that this integrated automated immuno-flowFISH method would be suitable for MRD assessment of CLL.

Methods: Blood from 75 patients with CLL, at diagnosis or on therapy, was analysed. For MRD studies, cells from the CI cell line were spiked into normal blood at concentrations of 0.001 - 10%. After red cell lysis, samples were incubated with CD3, CD5 and CD19 fluorophore-conjugated antibodies (fluorophores: BV480, BV605, AF647). Following fixation and membrane permeabilization, DNA was denatured at 78 ^oC for 5 mins. FISH probes to 17p12 and centromeres of chromosomes 12 and 17 were added and hybridized for 24 hours at 42 ^oC. Nuclei were then stained with SYTOX AADvanced and up to 600,000 cells acquired on the Amnis ^® ImageStream ^®XMk II imaging flow cytometer. Digital images (x60 objective) and quantitative data derived from computational algorithms (IDEAS software) were used to assess FISH signals overlying cell nuclei. IDEAS was then used to assess the number and percent CD19/CD5-positive CLL cells with FISH abnormalities.

Results: Between 10,000 and 600,000 cells (mean 60,000) were acquired. CLL (CD19/CD5-positive) and T- (CD3/CD5-positive) cells could be clearly identified by their immunophenotype and assessed individually for probe signals. FISH signals were identifiable on the digital images as specific "spots" overlying the SYTOX AADvanced nuclear stain. The IDEAS software could enumerate the number of FISH spots per cell and this was confirmed by quantitative mean channel fluorescence intensity for each probe. A chromosome 12 or 17 abnormality was detected in 23 of the 75 CLL cases. Of these, 10 cases had only one 17p signal (but 2 for the centromere of chromosome 17), indicative of del(17p). Del(17p) was detected in 2-35% of CD19/CD5-positive cells (i.e. 0.4-23% or 270-35,441 of all cells), the lowest seen in a patient on cytoreductive therapy. In 13/75 cases, there were 3 FISH signals for CEP12, consistent with trisomy 12 (+12) in 0.1-46% of all cells analysed; the lowest number of 0.1% was when 26 out of 26,000 cells analysed were CD19/CD5-positive and had +12. We also performed multi-FISH, incorporating CEP12, CEP17 and 17p probes together with the CD3, CD5 and CD19 antibodies. This required 7-fluorophores (antibodies, probes and nucleus) and confirmed the ability to detect del(17p) and chromosome 12 copy number simultaneously in a single analysis. Spiking of CI CLL cells into normal blood demonstrated that +12 could be detected to a lowest limit of 10 ^-5. In all analyses, CLL cells had normal diploid spots for the control CEP17 probe, and the CD3/CD5-positive T cells had dual signals for CEP12, CEP17 and 17p12 probes on numerical analysis and on digital imagery.

Conclusion: This study of confirms that high-throughput automated imaging flow cytometry, integrating FISH and immunophenotyping, can detect chromosomal defects in CLL. The lowest limit of detection for a FISH-detectable abnormality was 10 ^-5. This high sensitivity and specificity exceeds current clinical practice (10 ^-4), and was achieved through the analysis of many thousands of cells and positive identification of CLL cells based on their phenotype. This immuno-flowFISH method does not require any prior cell separation and is automated. It adds a new dimension to chromosomal analysis in CLL, both at diagnosis and for MRD monitoring. The high precision and specificity of immuno-flowFISH illustrates that this has a real place as a new MRD assessment tool for CLL.