Detection of Fusion Genes to Determine Minimal Residual Disease in Leukemia Using Next-Generation Sequencing.
BACKGROUND: Measuring minimal residual disease (MRD), the persistence of leukemic cells after treatment, is important for monitoring leukemia recurrence. The current methods for monitoring MRD are flow cytometry, to assess aberrant immune phenotypes, and digital droplet PCR (ddPCR), to target geneti...
| Publicado en: | Clinical Chemistry Vol. 66; no. 8; pp. 1084 - 1093 |
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| Autores principales: | , , , , , , |
| Formato: | Journal Article |
| Publicado: |
Oxford University Press / USA
Aug2020
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=148684218&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 148684218 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00099147 10CS jtl: Clinical Chemistry issn: 00099147 maglogo: N pubinfo: dt: Aug2020 vid: 66 iid: 8 pid: 622 pub: Oxford University Press / USA artinfo: ui: 148684218 10.1093/clinchem/hvaa119 148684218 ppf: 1084 ppct: 9 formats: fmt: @attributes: type: P tig: atl: Detection of Fusion Genes to Determine Minimal Residual Disease in Leukemia Using Next-Generation Sequencing. aug: au: de Boer, Eddy N. Johansson, Lennart F. Lange, Kim de Bosga-Brouwer, Anneke G. den Berg, Eva van Sikkema-Raddatz, Birgit van Diemen, Cleo C. affil: Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands. sug: ab: BACKGROUND: Measuring minimal residual disease (MRD), the persistence of leukemic cells after treatment, is important for monitoring leukemia recurrence. The current methods for monitoring MRD are flow cytometry, to assess aberrant immune phenotypes, and digital droplet PCR (ddPCR), to target genetic aberrations such as single-nucleotide variants and gene fusions. We present the performance of an RNA-based next-generation sequencing (NGS) method for MRD gene fusion detection compared with ddPCR. This method may have advantages, including the capacity to analyze different genetic aberrations and patients in 1 experiment. In particular, detection at the RNA level may be highly sensitive if the genetic aberration is highly expressed. METHODS: We designed a probe-based NGS panel targeting the breakpoints of 11 fusion genes previously identified in clinical patients and 2 fusion genes present in cell lines. Blocking probes were added to prevent non-specific enrichment. Each patient RNA sample was diluted in background RNA, depleted for rRNA and globin mRNA, converted to cDNA, and prepared for sequencing. Unique sequence reads, identified by unique molecular identifiers, were aligned directly to reference transcripts. The same patient and cell-line samples were also analyzed with ddPCR for direct comparison. RESULTS: Our NGS method reached a maximum sensitivity of 1 aberrant cell in 10 000 cells and was mostly within a factor of 10 compared with ddPCR. CONCLUSIONS: Our detection limit was below the threshold of 1:1000 recommended by European Leukemia Net. Further optimizations are easy to implement and are expected to boost the sensitivity of our method to diagnostically obtained ddPCR thresholds. pubtype: Academic Journal doctype: Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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