CLL cells from PB and BM are highly sensitive to venetoclax in vitro. (A) Fitted mean concentration-response curve (solid line) ± 95% prediction bands (thin lines) derived from data of 33 individual PB CLL samples from patients screened for the M12-175 trial. Circles and error bars represent the observed means ± standard deviations. CLL cells were incubated for 24 hours with varying concentrations of venetoclax (0.0128-1000 nM) or DMSO, and CD5⁺CD19⁺PI⁻ cells were enumerated. Percent CLL viability was determined by normalizing against the number of viable CLL cells in the corresponding DMSO-treated wells (mean viability 93% [range 68% to 99%]). The horizontal lines indicate 50% (gray, solid) and 90% (black, dotted) cell death. The estimated LC₅₀ was 1.9 nM, and the 90% lethal concentration was 105 nM. The blue shaded area indicates the range of steady-state plasma concentrations of venetoclax at a dose of 400 mg daily, ranging between the mean trough (dashed blue line) and peak postdose (solid blue line) concentrations.¹⁷  (B) CLL cells die rapidly in vitro. Mean fitted concentration-response curve (solid line) for 7 PB or BM CLL samples from 5 patients incubated for only 4 hours. The dashed green line (300 nM) represents the concentration approximating the observed peak plasma concentration after a single dose of 50 mg venetoclax in the M12-175 trial; the dashed red line (1.3 μM) represents the peak concentration observed after a single dose of 200 mg.¹⁷  (C) Correlation of individual LC₅₀ values for paired PB and BM CLL cells from the same patient at screening incubated in parallel for 24 hours (n = 32; P < .0001, R² = 0.83). (D) Correlation of mitochondrial depolarization as assessed by percentage cytochrome C loss induced by exposure to venetoclax or to BAD BH3 peptide in the BH3 profiling assay in screening PB samples from 13 CLL patients treated on the M12-175 trial (n = 13; P = .0001, R² = 0.75).