Advanced minimal residual disease monitoring for acute lymphoblastic leukemia with multiplex mediator probe PCR.

Abstract Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood. Minimal residual disease (MRD) monitoring is an important prognostic factor for treatment response and patient stratification. It 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, we 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. We developed personalized duplex MP PCR assays 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, we increased the multiplex level from duplex to 4-plex and still met the EuroMRD requirements for reliable quantification. In addition, our MRD-MP design guidelines and multiplex workflow facilitate and accelerate MP PCR assay development. This helps the standardization of personal diagnostics.