Clonal evolution in MPN patients carrying somatic mutations in epigenetic modifier genes. Single erythroid or granulocytic colonies (BFU-Es and CFU-G) grown in methylcellulose were individually picked and analyzed for the presence or absence of JAK2 V617F and other somatic mutations. (A) Examples of 3 patients who acquired an ASXL1 mutation before JAK2 V617F (left panel), after JAK2 V617F (middle panel), or in a clone separate from JAK2 V617F (right panel) are shown. Each dot represents a single colony that was genotyped and placed into the corresponding quadrant. (B) Summary of the temporal order of acquisition of mutations in relation to JAK2 V617F. Each dot represents 1 patient analyzed as shown in panel A and placed into the corresponding quadrant. Events in ET patients are depicted in yellow, PV patients in red, and PMF patients in brown. (C) Patterns of clonal evolution in 8 MPN patients carrying multiple somatic mutations. Dotted lines denote the time of analysis and the y-axis indicates the percentage of the colonies with or without the corresponding somatic mutations. %VF, JAK2-V617F mutant allele burden in purified granulocytes from peripheral blood. Although the order of events depicted can be deduced from the single-clone analysis (dotted line), the exact timing of the acquisition of the individual mutations and the time needed for the clonal expansion remains unknown and is shown only schematically. GRA, granulocytes.