Successive acquisition of mutations. Five serial samples were available for patient CML#19. The clones with thick circles represent the mutations detected by direct sequencing. The number of sequenced clones for each sample is shown on the left. The number of each mutated clone is shown inside each clone unless it was detected only once. The unmutated or independent low-level mutant clones are not shown here. M351T and Y253F were detected by direct sequencing in the first sample under imatinib (IM) therapy (41 months). Cloning and sequencing confirmed M351T/Y253F as a compound mutation but also revealed other low-level clones presumably derived from M351T that were undetectable by direct sequencing. The red line indicates a 30-month gap between stoppage of imatinib therapy and start of nilotinib (NI) therapy, during which time the patient was treated with homoharringtonine. Direct sequencing in the next 3 samples taken after the start of nilotinib therapy detected only M351T. Similar to the first sample, cloning and sequencing revealed add-on compound mutants derived from M351T not detected by direct sequencing. An E255K/M351T compound mutation was first observed under dasatinib (DA) therapy. E255K/M351T was the only mutation detected in the last sample, and was evident by direct sequencing and the more sensitive cloning and sequencing method. M351T/E255K developed along with resistance to dasatinib in this patient. The patient's disease progressed to blast crisis a few months after detection of M351T/E255K and loss of response to dasatinib.