Correlation between a 10-colour flow cytometric minimal residual disease (MRD) analysis and molecular MRD in adult acute lymphoblastic leukaemia (ALL).

Background: MRD monitoring in ALL is a strong predictive factor and a stratification tool for treatment intensification. The currently accepted standard of molecular monitoring with either immunoglobin heavy or kappa chain (IG) or T-cell receptor (TCR) quantitative PCR (qPCR) in Philadelphia negative (Ph-) ALL offers high sensitivity, but accessibility is limited by expertise, cost and turnaround time. Flow cytometric assays are increasingly utilised and improved sensitivity is seen with multi-parameter flow cytometry at 8 or more colours. Method(s): We developed a 10-colour single tube flow cytometry assay (Figure 1). Samples were subject to bulk ammonium chloride lysis to maximise cell yields with a target of 1 x 106 events. Once normal maturation patterns were established, patient samples were analysed in parallel to standard molecular monitoring with either IG/TCR qPCR in Ph-disease or BCR-ABL qRT-PCR in Ph+ disease. Statistical analysis was performed in Graphpad Prism v7.0. Result(s): Flow cytometry was performed on 47 samples from 16 patients. 13 samples were at diagnosis or morphologic relapse. An informative immunophenotype was identifiable in all patients; however a molecular assay could not able to be developed in one patient. 38 samples were tested for MRD by flow cytometry (Figure 2). In 2 samples, flow cyto-metric MRD was detected despite blinatumomab (anti-CD19) therapy. 27 samples were tested concurrently for MRD by both molecular and flow cytometric methods (Figure 3A and 3B). There was a strong correlation between molecular and flow cytometric MRD (R2=0.909, p<0.001; Figure 3B). Correlation was strong with both IG/TCR-based (n=16; R2=0.955, p<0.001) and BCR-ABL-based (n= 11; R2=0.957, p<0.001) (Figure Presented) assays. The cost was significantly lower than IG/TCR qPCR (eg. Cost for four time-points per patient approximately $1200 vs $3700). Conclusion(s): Our 10-colour flow cytometric MRD assay attained sensitivity of <=0.01% in 87% of samples, and