ALBERT

Description: Acute myeloid leukaemia (AML) is a heterogeneous group of diseases with distinct clinicopathologic, cytogenetic and genetic characteristics. The heterogeneity has made unified and regimental approach unsuccessful for most patients, and outcome with standard chemotherapy and allogeneic haematopoietic stem cell transplantation (HSCT) was unsatisfactory with an overall cure rate of 30-40%. We hypothesised that an optimised in vitro drug screening platform for primary AML samples might help to identify the best personalised therapeutic agents for AML patients from whom the samples were obtained at real time. Primary mononuclear cells isolated from peripheral blood (PB) or bone marrow (BM) of AML patients at different stages of disease were seeded onto 96-well plates and treated with a panel of 25 selected drugs at 1000-fold concentration range for 3 days. The drugs included 17 tyrosine kinase inhibitors: axitinib, crizotinib, pazopanib, erlotinib, gefitinib, lapatinib, vandetanib, vemurafenib, sorafenib, quizartinib, ponatinib, lestaurtinib, nilotinib, dasatinib, ruxolitinib, TG101209, tofacitinib; 2 differentiation agents: arsenic trioxide, all-trans retinoic acid; a protein translation inhibitor: homoharringtonine; a proteasome inhibitor: bortezomib; a chemotherapy: cytarabine; a histone deacetylase inhibitor: vorinostat; a DNA methyltransferase inhibitor: azacitidine; and an mTOR inhibitor: rapamycin. The blast percentage in each sample was above 50% as confirmed by film review of the cytospin preparation. The inhibitory effect of each drug was evaluated by a high throughput PrestoBlue® fluorometric assay that was a measure of viable cell number. The results were expressed with reference to the vehicle control (0.1% DMSO) for each sample. There was significant cell death when primary AML cells were cultured in IMDM medium supplemented with 10% FBS (IMDM10) for 3 days (Annexin V+ cells: 53.8% ± 4.5% , n=33). To identify the optimal culture condition, multiple culture conditions were tested for each sample (n=53 samples). The 3-day post-culture survival of primary AML cells improved significantly in a 1:1 mix of IMDM10 and medium conditioned by a mixture of mouse fibroblast lines engineered to produce human G-CSF, SCF, IL-3 and Flt3L (50%CM) (47.1% ± 6.5% improvement in Annexin V/7AAD -/- cell count normalised to day 0 input compared to IMDM10, p