Abstract
Monitoring minimal residual disease (MRD) provides important information during treatment of hematologic malignancies. Chimerism analysis also provides key information after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recent advances in next-generation sequencing (NGS) have enabled identification of various mutations and quantification of mutant allele burden. In this study, we developed a new analytic algorithm to monitor chimerism applicable to NGS multi-gene panel in use to identify mutations of myelodysplastic syndrome (MDS). We enrolled patients who were diagnosed with MDS and received allo-HSCT and their corresponding donors. Monitoring MRD by NGS assay was performed using 53 DNA samples by calculating mutant allele burden after treatment. For monitoring chimerism by NGS, we selected 121 single nucleotide polymorphisms (SNPs) after careful stepwise evaluation and calculated average donor allele burden. Data obtained from NGS were compared with bone marrow findings, chromosome analysis and short tandem repeat (STR)-based chimerism. SNP-based NGS chimerism analysis was accurate and even superior to conventional STR method by overcoming the various technical limitations of STR. In addition, simultaneous monitoring of mutation and chimerism using NGS could implement comprehensive pre- and post-HSCT monitoring of various clinical conditions such as complete donor chimerism, persistent mixed chimerism, early relapse, and even donor cell-derived diseases.
Highlights
Monitoring minimal residual disease (MRD) provides important information during treatment of hematologic malignancies
We developed a new analytic algorithm using a clinically used next-generation sequencing (NGS) myeloid panel that simultaneously monitors mutation and chimerism in myelodysplastic syndrome (MDS)
We examined 153 single nucleotide polymorphisms (SNPs) with a frequency of heterozygosity ranging from 0.2 to 0.8 in the Korean database and selected optimal SNPs for NGS chimerism analysis based on the following criteria: (1) >500 mean read depth, (2) ≤0.2% BE, and (3)
Summary
Monitoring minimal residual disease (MRD) provides important information during treatment of hematologic malignancies. Reverse transcription quantitative PCR can be used to measure fusion transcripts, but it cannot be applied in patients without gene fusion. Recurrent mutations such as those in the NPM1 gene are MRD markers [1]. Because recurrent gene fusions or mutations are rare in MDS, multi-gene targeting NGS is a suitable technique for MRD monitoring [5]. Real-time quantitative PCR of insertion/deletion markers showed higher sensitivity with a 0.1% threshold compared with that of STR analysis [7]. A custom NGS chimerism panel using single-nucleotide polymorphism (SNP) markers has been applied to chimerism and showed a high sensitivity of 0.5–1%, which was higher than that of STR analysis [8,9]. Chimerism was calculated from allele burden of SNPs included in the NGS panel after careful stepwise evaluation
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