1 DEFINING DISEASE RISK GROUPS THROUGH THE QUANTIFICATION OF GENETIC HETEROGENEITY ACROSS SINGLE LEUKAEMIA CELLS

Chronic myeloid leukaemia (CML) is typified by the BCR-ABL fusion gene. Primitive CML cells are less responsive to treatment and have high BCR-ABL mRNA and protein expression. BCR-ABL may also be required for cell adhesion, which may possess increased resistance. Previous studies have analysed bulk cell populations but the significance of BCR-ABL expression heterogeneity at the single cell level is unknown. In this study, the K562 CML cell line was used. Surface-adherent (K562/Adh) and non-adherent (K562/NonAdh) cell populations from standard suspensions were generated through 4 months of passages. Isolated K562/Adh and K562/NonAdh were used for the detection and quantification of BCR-ABL DNA, mRNA and protein levels across single and bulk cell populations, using fluorescent in situ hybridisation (FISH), RT-qPCR, flow cytometry and proximal ligation assay (PLA). Cell viability was measured for both Adh and NonAdh cells grown in the presence of a tyrosine kinase inhibitor (Imatinib) using the Cell Proliferation Kit (XTT).The passage of K562 cells demonstrated a small fraction (5% + 2.1%) of K562/Adh cells adhering to the plastic surface of the culture flask. Genomic BCR-ABL gene amplification was present in K562 cells, but displayed no significant difference between K562/Adh and K562/NonAdh cell types (P=0.8227). RT-qPCR showed up-regulation of BCR-ABL mRNA in K562/Adh cells, compared to K562/NonAdh in single and bulk cells (p