GM-CSF–dependent pSTAT5 hypersensitivity is a feature of CMML. (A) As shown in a representative CMML sample and healthy control sample stimulated with increasing doses of GM-CSF, a distinct population of CMML primary cells becomes pSTAT5-positive at 0.1 ng/mL of GM-CSF, which does not occur in normal controls. The percentage of positive cells is indicated on the flow cytometry dot plot. (B) Bone marrow samples from 20 unique CMML patients (solid red line) were compared with 7 normal healthy controls (broken black line) after treatment with increasing doses of GM-CSF (0.01, 0.1, 1, and 10 ng/mL). All pSTAT5 flow cytometry data are expressed relative to the maximal cellular response. Data were normalized using square root transformation. P values are indicated where significant differences were detected using linear regression analysis. (Ci) Representative colonies from a CMML sample and normal control after treatment with 10 ng/mL of GM-CSF only. (Cii) Bar graph of colony numbers generated from CMML (n = 7) and healthy donors (n = 3) with GM-CSF (10 ng/mL) alone. As a positive control, GM-CSF (MethoCult H4034 Optimum), IL-6, IL-3, erythropoietin, and stem cell factor (GM-CSF+*) were added to demonstrate the capacity for colony formation by healthy control bone marrow. (Ciii) Spontaneous colonies without the addition of GM-CSF (0 ng/mL of GM-CSF) and the percent maximum colony-forming units in the presence of GM-CSF alone at increasing doses (0.1, 1, and 10 ng/mL) from CMML patients (solid red line) and controls (broken black line). Error bars represent standard error of the mean in each group. (D) Samples were placed in the signaling mutation group if a mutation in CBL (n = 5), JAK2 (n = 0), KRAS (n = 0), and/or NRAS (n = 2) was identified (n = 7). GM-CSF–dependent pSTAT5 response was compared with those without a signaling mutation (n = 11).