Variant-specific discrepancy when quantitating BCR-ABL1 e13a2 and e14a2 transcripts using the Europe Against Cancer qPCR assay

Lasse Kjaer*, Vibe Skov, Morten T. Andersen, Anni Aggerholm, Philippe Clair, Michal Gniot, Karen Soeby, Lene Udby, Mikkel H. Dorff, Hans Hasselbalch, Niels Pallisgaard

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Objective: Molecular monitoring of treatment response in patients with chronic myelogenous leukemia is performed using the Europe Against Cancer (EAC) qPCR assay using the International Scale (IS). The assay amplifies both e13a2 and e14a2 BCR-ABL1 transcript variants. Observing distinct variant-dependent amplification curves during qPCR, we aimed to determine if this affected quantitation of BCR-ABL1. Methods: We investigated the qPCR efficiency at three Danish diagnostic centers (Zealand University Hospital [ZUH], Aarhus University Hospital [AU], and Rigshospitalet [RH]) on cell lines expressing either the e13a2 or e14a2 BCR-ABL1 transcript variants and compared %IS values from 219 chronic myeloid leukemia patients from the centers with either the e13a2 (n = 113) or e14a2 (n = 106) transcript variants obtained by qPCR with absolute quantitation by droplet digital PCR (ddPCR). Results: Although no significant differences were found in amplification efficiencies of the transcript variants, Bland-Altman analysis of qPCR vs ddPCR values for patient samples revealed a significant average difference in the bias between variants (e3a2/e14a2) of 4.6-, 6.5-, and 1.8-fold for ZUH, AU, and RH, respectively. Furthermore, qPCR %IS values of diagnostic patient samples revealed a significant 4.7-fold difference between the e13a2 and e14a2 variants. Conclusion: Our findings suggest that the EAC qPCR assay may underestimate the e14a2 variant compared to the e13a2 variant.

Original languageEnglish
JournalEuropean Journal of Haematology
Volume103
Issue number1
Pages (from-to)26-34
Number of pages9
ISSN0902-4441
DOIs
Publication statusPublished - Jul 2019
Externally publishedYes

Keywords

  • BCR-ABL1
  • chronic myeloid leukemia
  • droplet digital PCR
  • molecular response
  • qPCR

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