JAK2 allele burden analysis in MPN patients and the expression pattern of total JAK2 with myeloid differentiation. (A) Scatter graphs of the JAK2 V617F allele burden are shown for PV, ET, and MF patients in 4 separate compartments of myeloid differentiation; the Lin⁻/CD34⁺/CD38⁻ HSC compartment, CMP compartment, CD34⁻/CD71⁺/GPA⁺ erythrocyte compartment, and CD15⁺ PB granulocytes. For the HSC and CMP compartments, on average, 60 colonies were picked from methylcellulose and individually genotyped by pyrosequencing. JAK2 V617F allele burden in later nonclonogenic compartments was also analyzed by pyrosequencing. The average mutant allele burden was higher in the later granulocyte and erythroid compartments of both PV and MF patients. However, although heterogeneity was noted in both diseases, the growth kinetics of the mutant clone differs. In MF patients, the average mutant allele burden is already high in the stem cell–enriched fraction and remained, on average, stable throughout differentiation. For PV patients, the allele burden increased significantly during myeloid maturation (P = .02 by 1-sided analysis of variance test). In ET patients, the mutant burden was relatively low in the stem cell compartment and remained stable throughout myeloid ontogeny. (B) A comparison between the contribution of homozygous (left column) and heterozygous (right column) clones to hematopoiesis at early (Lin⁻/CD34⁺/CD38⁻ HSC compartment) and 2 later differentiation compartments (row above, the CD34⁻/CD71⁺/GPA⁺ erythroid and below the CD15⁺ granulocyte compartment) for 6 patients with PV. The scatter graphs show the linked values at the 2 time points for individual patients. In both differentiation to mature erythroid and granulocytic lineages, there is a significant expansion of the homozygous clone (P = .005 for erythroid and P = .01 for granulocytic), although the heterozygous clone size does not alter significantly (both nonsignificant). (C) Similar paired analysis for 6 PV patients who lack a homozygous clone, comparing the V617F allele burden in the HSC-enriched (Lin⁻/CD34⁺/CD38⁻) with the CD34⁻/CD71⁺/GPA⁺ erythroid (left panel) and the CD15⁺ granulocyte compartment (right panel). No significant clonal expansion is seen during differentiation from the HSC-enriched to later compartments. (D) Expression of total JAK2 mRNA was determined by quantitative real-time PCR in purified flow-sorted cells from healthy controls (n = 4) and patients with JAK2 V617F-positive MPN (n = 8, 4 PV and 4 MF patients). Expression values were normalized to the expression levels of the control gene ABL1 mRNA and are shown on a logarithmic scale. Expression levels were seen to increase on myeloid differentiation. Experiments were performed in duplicate for each patient (mean ± SEM). In addition, a statistically significant increase in total JAK2 levels was seen in the Lin⁻/CD34⁺/CD38⁻ compartment between PV and MF patients and normal controls (P = .003 and .02, respectively), and in the CD71⁺/GPA⁺ compartment, between PV patients and normal controls (P = .03). Taken together, this suggests that JAK2 expression may be higher for MPN patients compared with normal controls. Similar results were found with another housekeeping gene, GUSB (data not shown).