Page 265 - 2021_06-Haematologica-web

Letters to the Editor
Minimal residual disease monitoring in acute myeloid leukemia with non-A/B/D NPM1 mutations by digital polymerase chain reaction: feasibility and clinical use
In patients with acute myeloid leukemia (AML), treat- ment stratification is primarily based on pre-therapeutic factors, including cytogenetic and molecular aberrations and measurable/minimal residual disease (MRD) during treatment.1 Sequential MRD monitoring allows for assessment of the response to chemotherapy and early detection of relapses, possibly identifying patients who need pre-emptive or more intensive therapy.2 In clinical practice, MRD monitoring is based on molecular real- time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and/or flow cytometry.3 Currently available molecular markers are basically represented by fusion transcripts (especially CBFB-MYH11, RUNX1- RUNX1T1 and PML-RARA)4,5 or mutations, mainly NPM1 mutations.6 However, 60 to 70% of AML patients lack these leukemia-specific MRD targets and their samples are not informative for MRD detection by RT-qPCR.3 Additionally, RT-qPCR assays require the generation of standard curves covering the cycling threshold range of patients’ samples to ensure the linearity of the assay at the measured MRD level. This implies the maintenance of plasmid standards for each molecular target, limiting widespread use of this technique for rare markers in clin- ical practice. In this context, digital polymerase chain reaction (dPCR) is a promising approach to validate new MRD markers in AML patients. dPCR provides absolute quantification of nucleic acid target sequences with high sensitivity. Notably, it avoids the absolute quantification of plasmid standards and the pitfalls associated with vari- ations in reaction efficiencies (e.g., number of technical replicates performed, effect of the volume transferred throughout the dilution series).7 This makes dPCR more convenient for quantifying rare molecular markers and an
accurate alternative method for monitoring MRD. Briefly, the sample is divided into thousands of partitions (wells or droplets depending on the technology) containing amplification reagents in which the targets are randomly distributed. Each partition is analyzed and classified in a positive or negative category depending on the initial presence of the target. The absolute quantification is then estimated by modeling the measured number of positive fractions as a Poisson distribution model that estimates how many compartments contained one, two or more targets before amplification.
NPM1 mutations are one of the most frequent genetic abnormalities in adult AML, being detected in approxi- mately 35% of all patients with AML and in 50 to 60% of those with cytogenetically normal AML, in whom they are a major prognostic factor.1,8 Since their discovery in 2005, more than 50 different mutations located in exon 11 of NPM1 have been identified.9 Type A (c.860_863dupTCTG), B (c.863_864insCATG) and D (c.863_864insCCTG) mutations predominate in approxi- mately 90% of NPM1-mutated AML patients.10 While RT-qPCR could be effectively used to monitor all NPM1-mutated transcripts,11,12 in clinical practice, RT- qPCR analysis is mostly restricted to type A, B and D mutations for which commercial plasmid standards are available. Recently, in a study focused on NPM1-type A, B or D mutation quantification by RT-qPCR, the Acute Leukemia French Association (ALFA) group supported the strong prognostic significance of post-induction NPM1-based MRD on outcome, independently of addi- tional molecular or cytogenetic aberrations.6 Patients who did not achieve a 4-log reduction (poor responders) in NPM1-based MRD in peripheral blood were shown to have a higher cumulative incidence of relapse and shorter survival. Additionally, NPM1-based MRD was shown to be a good predictive factor for the indication of allogeneic stem cell transplantation in poor responders.
The purpose of the present study was to define the
Figure 1. Correlation between the quantification of levels of NPM1 type A mutation transcripts determined by real-time quantitative reverse transcriptase- polymerase chain reaction and droplet digital polymerase chain reaction. NPM1 type A mutation transcript levels were quantified in samples from 28 patients with acute myeloid leukemia using both real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) with a TaqMan chemistry assay and a droplet digital polymerase chain reaction (ddPCR) assay. The correlation between the ddPCR and RT-qPCR results was assessed using least squares regression after logarithmic transformation.
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