Digital PCR can provide improved BCR-ABL1 detection in chronic myeloid leukemia patients in deep molecular response and sensitivity of standard quantitative methods using EAC assays

Dagmar Smitalova; Dana Dvorakova; Zdenek Racil; Marianna Romzova

doi:10.1016/j.plabm.2021.e00210

Digital PCR can provide improved BCR-ABL1 detection in chronic myeloid leukemia patients in deep molecular response and sensitivity of standard quantitative methods using EAC assays

Pract Lab Med. 2021 Mar 9:25:e00210. doi: 10.1016/j.plabm.2021.e00210. eCollection 2021 May.

Authors

Dagmar Smitalova^{1

2}, Dana Dvorakova³, Zdenek Racil^{4

5}, Marianna Romzova¹

Affiliations

¹ Department of Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
² Department of Internal Medicine, Hematology and Oncology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
³ Centre of Molecular Biology and Gene Therapy, University Hospital Brno, Czech Republic.
⁴ Internal Haematology and Oncology Clinic, University Hospital Brno, Czech Republic.
⁵ Institute of Hematology and Blood Transfusion, Prague, Czech Republic.

Abstract

BCR-ABL1 molecular detection using quantitative PCR (qPCR) methods is the golden standard of chronic myeloid leukemia (CML) monitoring. However, due to variable sensitivity of qPCR assays across laboratories, alternative methods are tested. Digital PCR (dPCR) has been suggested as a robust and reproducible option. Here we present a comparison of droplet dPCR with routinely used reverse-transcription qPCR (RT-qPCR) and automated GeneXpert systems. Detection limit of dPCR was above 3 BCR-ABL1 copies, although due to background amplification the resulting sensitivity was 0.01% BCR-ABL1 (MR4.0). Nevertheless, in comparison with GeneXpert, dPCR categorized more than 50% of the patients into different MR groups, showing a potential for improved BCR-ABL1 detection.

Keywords: BCR-ABL1 monitoring; CML, chronic myeloid leukemia; Chronic myeloid leukemia; Digital PCR; EAC, Europe Against Cancer; FPR, false positivity rate; GeneXpert BCR-ABL Monitor assay; IS, international scale; LOB, limit of blank; LOD, limit of detection; MR, molecular response; NTC, no template control; RT-qPCR; RT-qPCR, reverse-transcription quantitative PCR; TKI, tyrosine kinase inhibitors; cDNA, complementary DNA; dPCR, digital PCR; pDNA, plasmid DNA; qPCR, quantitative PCR.