Next-Generation Sequencing Technology to Identify Minimal Residual Disease in Lymphoid Malignancies
Next-generation sequencing (NGS) of immunoglobulin (IG) and T cell receptor (TR) rearrangements represents a modern alternative to classical RQ-PCR-based minimal residual disease (MRD) detection. The same primer sets and conditions can be used for all pat
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Introduction Immunoglobulin (IG) and/or T cell receptor (TR) genes are rearranged in the vast majority of immature lymphoid neoplasms and virtually all mature B and T cell malignancies, and thus form highly specific molecular targets for minimal residual disease (MRD) detection. IG/TR-based MRD assessment is part of standard diagnostic care in acute lymphoblastic leukemia (ALL) where it forms a fundamental marker for treatment stratification. Also in chronic lymphocytic leukemia, as well as in multiple myeloma, MRD gains in importance thanks to the development of new and effective therapies that require more sensitive monitoring strategies. So far, DNA-based allele-specific real-time quantitative (RQ)-PCR is the gold standard for sensitive IG/TR-based MRD monitoring, but
Ce´sar Cobaleda and Isidro Sa´nchez-Garcı´a (eds.), Leukemia Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 2185, https://doi.org/10.1007/978-1-0716-0810-4_7, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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Michaela Kotrova et al.
next-generation sequencing (NGS) can deliver highly sensitive MRD assessment in lymphoid malignancies [1, 2]. The situation is considerably different in myeloid malignancies where IG/TR genes are usually not rearranged. Depending on the respective disease category, different molecular aberrations may serve as MRD markers. In CML, the fusion gene transcript BCRABL1 is a universal MRD marker being used for RQ-PCR-based MRD tracking since decades. In acute myeloid leukemia (AML) no “one size fits all” approach for MRD detection is achievable. Fusion genes PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 are present in 13%, 7%, and 5% of AML patients, respectively, and NPM1 mutations (typically 4-nucleotide frameshift insertions in exon 12, present in about 30% of AML patients) are also stable between diagnosis and relapse and are therefore reliable markers for RQ-PCR-based MRD assessment [3]. Recently, NGS was introduced to track MRD in AML with the theoretical advantage of a broader applicability to all genetic aberrations. On the other hand, mutations are often subclonal and can disappear at the time of relapse. Because of frequent losses or gains of certain mutations at relapse, the European LeukemiaNet MRD Working Party does not recommend the use of mutations in FLT3-ITD, FLT3-TKD, NRAS, KRAS, IDH1, IDH2, MLL-PTD, and expression levels of EVI1 as single markers of MRD [4]. Other mutations like DNMT3A, TET2, and ASXL1, which are among the most frequently mutated genes in AML, are also acquired during aging—a process known as “clonal hematopoiesis of indeterminate potential”—and often represent persisting pre-leukemic populations [5– 7]. These founder clones may persist at significant levels during remission, but the detection of these may not reliably represent residual AML and may not be of prognostic significance. In addition, experimental and bioinformatics conditions have to be optimized for each single molecular marker, and combinations of different markers should be u
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