Targeted RNA-sequencing for the quantification of measurable residual disease in acute myeloid leukemia

Despite the achievement of a complete remission (CR) following therapy, acute myeloid leukemia (AML) patients remain at risk of relapse due to persistence of disease not evident by conventional cytomorphological methods

1. Risk stratification of such patients is possible based on tests designed to detect this residual leukemic burden (termed minimal, or measurable, residual disease; MRD)

2. A variety of technologies, such as real-time quantitative PCR (qPCR) or flow cytometry, have been developed focusing on the identification of recurrent molecular abnormalities or leukemia-associated immune phenotypes

3. Despite these advances, AML is a heterogeneous disease, making it difficult to develop a single MRD detection assay which is highly reproducible, has a high sensitivity limit of detection, limited operator dependence, and the ability to simultaneously examine multiple abnormalities.

To overcome these limitations, we developed a multi-gene, targeted RNA-sequencing (RNA-seq)-based method for the sensitive detection and quantitation of known AML genetic signatures. This AML RNA-seq panel utilizes a pool of target-specific primers containing 12 nucleotide (nt) unique molecular indices (UMIs) which capture and individually tag RNA molecules of interest during reverse transcription, followed by targeted PCR of the barcoded cDNA, and then library construction.