Prospective NGS-based monitoring of the kinetics of TKI-resistant low-level mutations (highlighted in bold and underlined) in 16 representative patients with warning and 4 representative patients with failure at the time of enrollment. Patient details can be found in supplemental Table 2. To define “optimal response,” failure and warning in patients receiving third-line TKI therapy and beyond, the same ELN 2013 criteria as for second-line had to be used. UPN138: after 3 months of bosutinib (to which the patient was switched before NGS results were made available), BCR-ABL1/ABL1^IS levels had increased (from 1.3% to 5.7%), yet according to ELN 2013, the response was formally definable as optimal. UPN141: after the switch to ponatinib 45 mg/d, the patient regained complete hematological response and showed a rapid decline of BCR-ABL1/ABL1^IS levels, so the dose was reduced to 15 mg/d for safety concerns. However, the 50% inhibitory concentration of the E255V mutant is higher than the average plasma concentration achievable with 15 mg/d, which explains the outgrowth of the E255V-positive clone. Similar E255V expansion under ponatinib 15 mg/d was observed in UPN003. L, line; PON, ponatinib.