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Comparison of RNA- and DNA-based methods for measurable residual disease analysis in NPM1-mutated acute myeloid leukemia

Pettersson, Louise LU orcid ; Johansson Alm, Sofie ; Almstedt, Alvar ; Chen, Yilun LU ; Orrsjö, Gustav ; Shah-Barkhordar, Giti ; Zhou, Li LU ; Kotarsky, Heike LU ; Vidovic, Karina LU and Asp, Julia , et al. (2021) In International Journal of Laboratory Hematology
Abstract

Introduction: Reverse transcriptase quantitative PCR (RT-qPCR) is considered the method of choice for measurable residual disease (MRD) assessment in NPM1-mutated acute myeloid leukemia (AML). MRD can also be determined with DNA-based methods offering certain advantages. We here compared the DNA-based methods quantitative PCR (qPCR), droplet digital PCR (ddPCR), and targeted deep sequencing (deep seq) with RT-qPCR. Methods: Of 110 follow-up samples from 30 patients with NPM1-mutated AML were analyzed by qPCR, ddPCR, deep seq, and RT-qPCR. To select DNA MRD cutoffs for bone marrow, we performed receiver operating characteristic analyses for each DNA method using prognostically relevant RT-qPCR cutoffs. Results: The DNA-based methods... (More)

Introduction: Reverse transcriptase quantitative PCR (RT-qPCR) is considered the method of choice for measurable residual disease (MRD) assessment in NPM1-mutated acute myeloid leukemia (AML). MRD can also be determined with DNA-based methods offering certain advantages. We here compared the DNA-based methods quantitative PCR (qPCR), droplet digital PCR (ddPCR), and targeted deep sequencing (deep seq) with RT-qPCR. Methods: Of 110 follow-up samples from 30 patients with NPM1-mutated AML were analyzed by qPCR, ddPCR, deep seq, and RT-qPCR. To select DNA MRD cutoffs for bone marrow, we performed receiver operating characteristic analyses for each DNA method using prognostically relevant RT-qPCR cutoffs. Results: The DNA-based methods showed strong intermethod correlation, but were less sensitive than RT-qPCR. A bone marrow cutoff at 0.1% leukemic DNA for qPCR or 0.05% variant allele frequency for ddPCR and deep seq offered optimal sensitivity and specificity with respect to 3 log10 reduction of NPM1 transcripts and/or 2% mutant NPM1/ABL. With these cutoffs, MRD results agreed in 95% (191/201) of the analyses. Although more sensitive, RT-qPCR failed to detect leukemic signals in 10% of samples with detectable leukemic DNA. Conclusion: DNA-based MRD techniques may complement RT-qPCR for assessment of residual leukemia. DNA-based methods offer high positive and negative predictive values with respect to residual leukemic NPM1 transcripts at levels of importance for response to treatment. However, moving to DNA-based MRD methods will miss a proportion of patients with residual leukemic RNA, but on the other hand some MRD samples with detectable leukemic DNA can be devoid of measurable leukemic RNA.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
deep sequencing, digital PCR, measurable residual disease, NPM1, qPCR, RT-qPCR
in
International Journal of Laboratory Hematology
pages
11 pages
publisher
Wiley-Blackwell
external identifiers
  • scopus:85106971473
  • pmid:34053184
ISSN
1751-5521
DOI
10.1111/ijlh.13608
language
English
LU publication?
yes
additional info
Publisher Copyright: © 2021 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.
id
e603d9a7-6a1b-4328-88c2-d7677ab30f9d
date added to LUP
2021-06-13 18:21:53
date last changed
2024-09-21 21:29:19
@article{e603d9a7-6a1b-4328-88c2-d7677ab30f9d,
  abstract     = {{<p>Introduction: Reverse transcriptase quantitative PCR (RT-qPCR) is considered the method of choice for measurable residual disease (MRD) assessment in NPM1-mutated acute myeloid leukemia (AML). MRD can also be determined with DNA-based methods offering certain advantages. We here compared the DNA-based methods quantitative PCR (qPCR), droplet digital PCR (ddPCR), and targeted deep sequencing (deep seq) with RT-qPCR. Methods: Of 110 follow-up samples from 30 patients with NPM1-mutated AML were analyzed by qPCR, ddPCR, deep seq, and RT-qPCR. To select DNA MRD cutoffs for bone marrow, we performed receiver operating characteristic analyses for each DNA method using prognostically relevant RT-qPCR cutoffs. Results: The DNA-based methods showed strong intermethod correlation, but were less sensitive than RT-qPCR. A bone marrow cutoff at 0.1% leukemic DNA for qPCR or 0.05% variant allele frequency for ddPCR and deep seq offered optimal sensitivity and specificity with respect to 3 log<sub>10</sub> reduction of NPM1 transcripts and/or 2% mutant NPM1/ABL. With these cutoffs, MRD results agreed in 95% (191/201) of the analyses. Although more sensitive, RT-qPCR failed to detect leukemic signals in 10% of samples with detectable leukemic DNA. Conclusion: DNA-based MRD techniques may complement RT-qPCR for assessment of residual leukemia. DNA-based methods offer high positive and negative predictive values with respect to residual leukemic NPM1 transcripts at levels of importance for response to treatment. However, moving to DNA-based MRD methods will miss a proportion of patients with residual leukemic RNA, but on the other hand some MRD samples with detectable leukemic DNA can be devoid of measurable leukemic RNA.</p>}},
  author       = {{Pettersson, Louise and Johansson Alm, Sofie and Almstedt, Alvar and Chen, Yilun and Orrsjö, Gustav and Shah-Barkhordar, Giti and Zhou, Li and Kotarsky, Heike and Vidovic, Karina and Asp, Julia and Lazarevic, Vladimir and Saal, Lao H. and Fogelstrand, Linda and Ehinger, Mats}},
  issn         = {{1751-5521}},
  keywords     = {{deep sequencing; digital PCR; measurable residual disease; NPM1; qPCR; RT-qPCR}},
  language     = {{eng}},
  publisher    = {{Wiley-Blackwell}},
  series       = {{International Journal of Laboratory Hematology}},
  title        = {{Comparison of RNA- and DNA-based methods for measurable residual disease analysis in NPM1-mutated acute myeloid leukemia}},
  url          = {{http://dx.doi.org/10.1111/ijlh.13608}},
  doi          = {{10.1111/ijlh.13608}},
  year         = {{2021}},
}