Digital droplet PCR for molecular monitoring of patients with chronic myeloid leukemia

With the BCR::ABL1 fusion as the sole disease-causing event, the therapy of chronic myeloid leukemia (CML) changed fundamentally with the advent of tyrosine kinase inhibitors (TKI). A large proportion of patients are now able to reach deep molecular remission with BCR::ABL1 only present at low level or even undetectable by quantitative real-time PCR. Good therapy responses encouraged the emergence of a new goal in CML therapy: treatment-free remission (TFR). As transcriptionally quiescent leukemic stem cells are believed to be the root of disease relapses, a genomic detection method is needed for the quantification of residual disease. In the present work a next generation sequencing (NGS)-based method was established to identify the exact breakpoint sequence by completely covering the genomic region of both genes involved in the fusion. This represented the optimal prerequisite for establishing patient-specific monitoring using genomic droplet digital PCR (gddPCR) for patients under TKI therapy as well as during TFR. Peripheral blood and bone marrow samples were analyzed prior to TKI discontinuation. Overall, a good agreement between BCR::ABL1 %IS levels and the percentage of leukemic cells was found. Differences in the BCR::ABL1 dynamics on RNA and DNA level could be seen in patients who relapsed during TFR. Bone marrow analyses performed before TKI cessation revealed that the presence of remaining leukemic cells was not necessarily followed by a disease relapse. A central finding of this study was that most patients, who were able to maintain TFR over a long period of time, showed BCR::ABL1-positive cells originating from the initial leukemic clone. Overall, an essential foundation for patient-specific CML monitoring was established. Those insights will help to more reliably identify those patients, who will be able to safely discontinue TKI therapy by refining the cessation criteria and providing as many patients as possible with the option of TFR.