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

Abstract

Great effort is spent on developing therapies to improve the dire outcomes of those diagnosed with acute myeloid leukemia, while the methods for quantifying response to therapeutic intervention have lacked sensitivity. As a result, those patients achieving a complete remission remain at risk of subsequent relapse due to disease persistence not evident by conventional cytomorphological methods. Improved risk stratification is possible based on tests designed to detect this residual leukemic burden (measurable residual disease). However, acute myeloid leukemia is a genetically diverse set of diseases, which has made it difficult to develop a single, highly reproducible, and sensitive detection assay for measurable residual disease. To overcome these limitations, we present the development of a digital targeted RNA-sequencing-based approach which detects all newly approved European Leukemia Network molecular targets for measurable residual disease in acute myeloid leukemia in a single standardized assay. Iterative modifications and novel bioinformatics approaches resulted in a greater than one hundred-fold increase in performance compared with commercially available targeted RNA-sequencing approaches and a limit of detection as low as 1 in 100,000 cells comparable with quantitative PCR, the current gold standard for measurable residual disease detection. This assay, which is customizable and expandable, is the first demonstrated use of high-sensitivity RNA-sequencing for measurable residual disease detection in acute myeloid leukemia and thus could serve as a broadly applicable standardized tool.

Measurable Residual Disease in Acute Myeloid Leukemia Using Flow Cytometry: A Review of Where We Are and Where We Are Going

“…Newer molecular methods being evaluated include digital droplet PCR, currently used in the diagnostic molecular hematopathology space, which is highly sensitive, specific, and allows for an absolute quantitation (as opposed to qPCR which is relative); and targeted RNA sequencing which can have a sensitivity of 1 in 10 5 , although there is significant inter-individual differences in RNA expression limiting sensitivity [ 35 , 36 ].…”

NARASIMHA: Novel Assay based on Targeted RNA Sequencing to Identify ChiMeric Gene Fusions in Hematological Malignancies

“…This enables us to perform absolute cDNA molecule counting and reduction of PCR bias as every molecule of cDNA is marked uniquely by a random oligonucleotide. Recently, Dillon et al described a method for ultrasensitive MRD monitoring of CGF 10 . They performed multiplexed cDNA synthesis of a limited number of targets and incorporated a UMI using PCR after the cDNA synthesis stage.…”

Sequencing-Based Measurable Residual Disease Testing in Acute Myeloid Leukemia

“…One of the biggest technical challenges for AML MRD assays has been making them broadly applicable to most or all AML patients. Sensitive PCR-based AML MRD assays have been used for some time to monitor both recurrent translocations including core binding factor leukemias (Yin et al, 2012;Pigazzi et al, 2015;Ouyang et al, 2016;Schumacher et al, 2017) inv(16) and t(8;21) RUNX1-RUNX1T1 and t(15;17) PML-RARA (Burnett et al, 2015;Brunetti et al, 2017) from RNA and can now be detected using multiplex RNAseq NGS-based approaches (Dillon et al, 2019). Specific recurrent gene mutations can also be monitored including NPM1 (Salipante et al, 2014;Ivey et al, 2016) and FLT3 (Bibault et al, 2015;Levis et al, 2018) from RNA or DNA.…”