Advanced Minimal Residual Disease Monitoring for Acute Lymphoblastic Leukemia with Multiplex Mediator Probe PCR

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood. Minimal residual disease (MRD) monitoring is an important prognostic factor for ALL treatment response and patient stratification. MRD monitoring uses personalized real-time PCR to measure the amount of cancer cells among normal cells. Due to clonal tumor evolution or secondary rearrangement processes, MRD markers can disappear during treatment, leading to false-negative MRD results and wrong decision-making in personalized treatments. Therefore, monitoring of multiple MRD markers per patient is required. For the first time, the authors present personalized multiplex mediator probe PCR (MP PCR) for MRD monitoring in ALL. These assays can precisely quantify more MRD markers in less sample material. Therefore, clinical outcomes will be less affected by clonal tumor evolution. Personalized duplex MP PCR assays were developed for different genomic MRD markers, including immunoglobulin/T-cell receptor gene rearrangements, gene fusions, and gene deletions. One duplex assay was successfully applied in a prospective patient case and compared with hydrolysis probes. Moreover, the authors increased the multiplex level from duplex to 4-plex and still met the EuroMRD requirements for reliable quantification. In addition, the authors' MRD-MP design guidelines and multiplex workflow facilitate and accelerate MP PCR assay development. This helps the standardization of personal diagnostics.