Publication

Detection of Fusion Genes to Determine Minimal Residual Disease in Leukemia Using Next-Generation Sequencing

de Boer, E. N., Johansson, L. F., de Lange, K., Bosga-Brouwer, A. G., van den Berg, E., Sikkema-Raddatz, B. & van Diemen, C. C., Aug-2020, In : Clinical Chemistry. 66, 8, p. 1084-1092 9 p.

Research output: Contribution to journalArticleAcademicpeer-review

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 nonspecific 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.

Original languageEnglish
Pages (from-to)1084-1092
Number of pages9
JournalClinical Chemistry
Volume66
Issue number8
Early online date2-Jul-2020
Publication statusPublished - Aug-2020

    Keywords

  • ACUTE MYELOID-LEUKEMIA, DIAGNOSTICS

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