Table 2.
Description of the studies examining cell-free DNA (cfDNA) for acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS).
| Reference | Diseases | N | cfDNA Material | cfDNA Isolation | cfDNA Analysis | Genes Studied | Target | Clinically Relevant Findings | New Research Perspectives |
|---|---|---|---|---|---|---|---|---|---|
| Vasioukhin, V. 1994 [70] | AML and MDS | 10 | Plasma in 20–30 mL PB | Phenol–chloroform method | Dot-blot screening procedure | NRAS | SNV | N-ras mutant genes that were not found in peripheral blood or bone marrow were detected in plasma DNA | Further studies are needed to correlate plasma DNA with peripheral blood and bone marrow DNA |
| Rogers, A. 2004 [71] | AML and MDS | 45 | Plasma | N/A | PCR and capillary electrophoresis | 5q-, 7q-, +8, 17p-, 20q-, and X chromosome | LOH, X-chromosome clonality | Detection of LOH in the PB plasma of all 45 patients with cytogenetically documented chromosomal abnormalities | To test the possibility that PB can be an alternative tool to BM in assessing genetic abnormality |
| Gao, Y.J. 2010 [72] | AML and ALL | 60 | 2 mL of plasma | QIAamp DNA Blood Kit (Qiagen, Hilden, Germany) | qPCR | ACTB | Concentrations, integrity, CNVs | The cfDNA integrity index fluctuated in correlation with the dynamics of acute leukemia | The cfDNA integrity index may be useful for MRD monitoring in acute leukemia |
| Iriyama, C. 2012 [73] | MDS | 5 | 450 μL of serum | M inElute Virus Vacuum Kit (Qiagen, Hilden, Germany) | Pyrosequencing | methylation analysis (LINE-2), mutation detection (TET2) | Methylation analysis, SNV | Methylation rate decreases after azacytidine treatment in plasma DNA and the TET2 mutant gene is detectable in BM | Testing the possibility that circulating DNA from plasma better reflects DNA from MDS clones in the BM than DNA from intact cells |
| Quan, J. 2015 [74] | AML | 100 | 2 mL of plasma | QIAamp DNA Blood Kit (Qiagen, Hilden, Germany) | qPCR | NPM | Indel, CNVs | Copy number quantification of mutant genes established; copy number variation reflects clinical characteristics | Large-scale prospective studies to investigate the relationship between copy number of circulating NPM mutant genes and clinical outcomes are warranted |
| Albitar, F. 2016 [75] | MDS | 16 | Plasma | Nucli-SENS Easy MAG Automated Platform (BioMerieux, Marcy- l’E’ toile, France) | NGS (target sequence) | 14 target genes (ASXL1, ETV6, EZH2, IDH1, IDH2, NRAS, CBL, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, ZRSR2) | SNV/Indel | Confirmed the presence of a neoplastic abnormal MDS clone using cfDNA NGS | Further validation in advanced MDS patients |
| Suzuki, Y. 2016 [76] | MDS related disease | 33 | Plasma and serum | QIAamp MinElute Virus Vacuum Kit or QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) | Sanger sequence, SNP array analysis, and q-PCR or NGS (target sequence) | Sanger, SNP array, and q-PCR (IDH2, SETBP1, U2AF1, SRSF2, NRAS, TET2, and FLT3), NGS (39 targeted genes) | SNV/Indel | Detection of cfDNA that varies in correlation with disease state | Verification of the possibility that cfDNA reflects the disease status of MDS |
| Christenson, E.S. 2017 [90] | AML, MDS | 7 | Plasma in 10 mL PB | QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) | ddPCR | SF3B1 | SNV | Reported that SNPs in wild-type alleles affect allele frequencies detected by ddPCR | Validation of a companion diagnostic method using ctDNA ddPCR |
| Yeh, P. 2017 [96] | MDS | 12 | Plasma | QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) | NGS (target sequence) and/or ddPCR | 55 target genes | SNV/Indel | Prediction of treatment failure by tracking driver mutations and karyotype abnormalities using ctDNA and evidence that ctDNA dynamics reflects tumor burden in MDS | Application of ctDNA analysis as a non-invasive biomarker to complement existing monitoring strategies for MDS |
| Nakamura, S. 2018 [86] | AML, MDSMM, NHL | 17 | Serum | QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) | ddPCR | Genes whose mutations were detected by WES (STAG2, JAK3, NRAS, KRAS, TP53, DNMT3A, NPM1, GATA2, MYD88, B2M, SF3B1, U2AF1) | SNV/Indel | ctDNA monitoring facilitated the identification of patients with hematological cancers at risk of recurrence prior to established clinical parameters | Need to implement ctDNA monitoring of hematopoietic tumor patients on an even larger scale |
| Zhong, L. 2018 [89] | AML | 235 | Plasma | QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) | PCR and gel electrophoresis, qPCR | IGH and TCR | Ig-gene and TCRγ rearrangement | Detection of monoclonal IGH and TCR rearrangements in AML ctDNA | Application of monoclonal IGH and TCR rearrangements for MRD assessment in AML |
| Zhao, P. 2019 [77] | MDS | 26 | Plasma | N/A | NGS (target sequence) | 127 target genes | SNV/Indel | ctDNA reflects genetic variation in BM DNA and is useful for monitoring the pathogenesis of MDS | Application of ctDNA for prognosis prediction of MDS |
| Nakamura, S. 2019 [87] | AML/MDS | 53 | Serum | QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) | ddPCR | Genes whose mutations were detected by WES (DNMT3A, STAG2, SRSF2, SF3B1, WT1, GATA2, NPM1, CEBPA, IDH1, IDH2, TP53, U2AF1, BCORL1, ATRX, ASXL1, RUNX1, CEBPA, SH2B3, KIT, PTPN11, ETV6, RAD21, CSF3R, CTCF, ETNK1, KMT2D, BCOR, XPO7) | SNV/Indel | Prediction of relapse by MRD monitoring using ddPCR combined with NGS after hematopoietic stem cell transplantation | Conducting prospective tests |
| Short, N.J. 2020 [88] | AML | 22 | Plasma in 10 mL PB | QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) | NGS (target sequence) | 275 target genes | SNV/Indel | Detection of residual lesions in cfDNA specimens in remission by targeted sequencing | Evaluation the prognostic impact of MRD as detected by ctDNA sequencing |
| Zeidan, A.M. 2020 [95] | AML | 20 | Plasma | QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) | ddPCR | Genes whose mutations were detected by target sequence | SNV/Indel | Monitoring of ctDNA in a Phase Ib study of the PLK1 inhibitor, onvansertib, showed tumor burden during therapy | Evaluating the utility of serial ctDNA measurements as a predictor of clinical response |
BM, bone marrow; CNV, copy number variation; ddPCR, droplet digital PCR; LOH, loss of heterozygosity; MM, multiple myeloma; NGS, next-generation sequence; NHL, non-Hodgkin lymphoma; PB, peripheral blood; qPCR, quantitative PCR; SNP, single-nucleotide polymorphism; SNV, single-nucleotide variants; WES, whole-exome sequence.