Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (n = 10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (p = 0.015), and the amplification efficiency was 2% greater (P = 0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (n = 67) compared to e14a2 (n = 78) when analysed by RT-qPCR (P = 0.0005) but not ddPCR (P = 0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management.

Abteilung Hämatologie Onkologie Klinik für Innere Medizin 2 University of Jena Jena Germany

Center of Molecular Biology and Gene Therapy Internal Hematology and Oncology Clinic Faculty Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

CLIP Dept of Paediatric Haematology and Oncology 2nd Faculty of Medicine Charles University and University Hospital Motol Prague Czech Republic

Department of Molecular Medicine and Medical Biotechnology University 'Federico II' and CEINGE Advanced Biotechnologies Naples Italy

Faculty of Medicine University of Southampton Southampton UK

Fundeni Clinical Institute Hematology Department Bucharest Romania

Hematology Department Faculty of Medicine University of Medicine and Pharmacy Carol Davila Bucharest Romania

Institute for Medical Informatics and Biometry Carl Gustav Carus Faculty of Medicine TU Dresden Dresden Germany

Institute of Hematology and Blood Transfusion Prague Czech Republic

Internal Hematology and Oncology Clinic Faculty Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

IRCSS Azienda Ospedaliero Universitaria di Bologna Istituto di Ematologia Seràgnoli Bologna Italy

Labdia Labordiagnostik St Anna Children´s Cancer Research Institute Vienna Austria

Laboratory of Chemical and Clinical Analysis Area 3 A O U San Luigi Gonzaga Orbassano Turin Italy

National Center for Tumor Diseases Dresden Germany

Pathology Department Hospital Clinic Institut d' Investigacions Biomèdiques August Pi i Sunyer CIBERONC Barcelona Spain

Portuguese Oncology Institute of Porto Porto Portugal

The University Hospital in Krakow Krakow Poland

University of Catania Department of Clinical and Experimental Medicine Center of Experimental Oncology and Hematology Catania Italy

University of Leipzig Medical Center Department for Hematology Cellular Therapies and Hemostaseology Leipzig Germany

Wessex Regional Genetics Laboratory Salisbury NHS Foundation Trust Salisbury UK

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