G417(P) Droplet digital polymerase chain reaction (ddpcr) assessment of deep molecular response in chronic myeloid leukaemia (cml)

Abstract

Molecular monitoring of BCR-ABL1 transcript levels is central to the effective clinical management of patients with CML. Standardised Real time quantitative PCR (RT-qPCR) is currently used to measure the depth and durability of molecular response and forms the basis of the European Leukaemia Net (ELN) treatment recommendations. Close monitoring of patients who come off treatment using maximum sensitivity techniques, is vital in order to ensure early therapeutic intervention, should a BCR-ABL1 clone re-emerge. However, RT-qPCR is susceptible to fluctuations at the limits of its sensitivity, thus limiting its accuracy in assessing complete molecular response. Droplet Digital PCR technology is a new digital PCR technique where the test sample into many individual real-time PCR reactions, where some reactions contain the target molecule (positive) while others (negative) do not. The proportion of negative reactions is used to calculate an absolute count of the number of target molecules in the sample, without reference to external standards. This method should therefore enable increased accuracy and absolute quantification of target molecules, in comparison to the RT-qPCR technique. The aim of this study was to assess the potential of Droplet digital PCR (ddPCR) to improve the sensitivity and precision of the assessment of deep molecular response in patients with CML. A series of 21 patients previously determined to have achieved major molecular response (MMR) using RT-qPCR, were retrospectively monitored for BCR-ABL1 transcripts using ddPCR. A comparison of BCR-ABL1 to ABL1 ratios obtained by ddPCR and RT-qPCR suggested that in 63% of patients ddPCR was more sensitive than RT-qPCR, while in 28% of patients BCR-ABL1 transcript levels were better detected by RT-qPCR. This study demonstrates that ddPCR has the potential to precisely measure changes in minimal residual disease at the limits of detection of current RT-qPCR assays. Assessment of deep molecular response using ddPCR will help to accurately discriminate patients achieving a sustainable response and could therefore be considered for treatment cessation studies from those patients in whom BCR-ABL1 transcripts are likely to re emerge.