Impact of BCR::ABL1transcript type on RT-qPCR amplification performance and molecular response to therapy

Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1have 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::ABL1reverse 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/GUSBwere 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::ABL1transcript type; these differences are small but may need to be considered for optimal patient management.