Assessment of droplet digital polymerase chain reaction for measuring BCR‐ABL1 in chronic myeloid leukaemia in an international interlaboratory study

Abstract

Summary Measurement of BCR activator of RhoGEF and GTPase ‐ABL proto‐oncogene 1, non‐receptor tyrosine kinase (BCR‐ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large‐scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR‐ABL1‐positive samples with levels ranging from molecular response (MR)1·0–MR5·0 were tested by 23 laboratories using RTddPCR with the QXDX BCR‐ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR‐ABL1 in all samples to MR4·0. Detection rates for deep‐response samples were 95·7% at MR4·5, 78·3% at MR4·7 and 87·0% at MR5·0. Interlaboratory coefficient of variation was indirectly proportional to BCR‐ABL1 level ranging from 29·3% to 69·0%. Linearity ranged from 0·9330 to 1·000 (average 0·9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment‐free remission.

Highly sensitive droplet digital polymerase chain reaction for BCR::ABL1 messenger RNA identifies patients with chronic myeloid leukaemia with a low probability of achieving treatment‐free remission

“…We note a recent study demonstrated similar sensitivity between optimised RT-qPCR as compared with ddPCR under standardised conditions. 13 In contrast, we demonstrated an increased assay sensitivity with ddPCR, using increased complementary DNA input and number of reactions performed. Our results have similar clinical implications to previous reports, 7,8 in particular Nicolini et al, 14 who showed a BCR::ABL1 of <0.0023% correlated with TFR success.…”

Performance characteristics of the first Food and Drug Administration (FDA)-cleared digital droplet PCR (ddPCR) assay for BCR::ABL1 monitoring in chronic myelogenous leukemia

“…In this study performance between sites and operators showed low variability in ddPCR, a challenge for many qPCR-based assays, yet essential for long term monitoring. A study used 10 BCR :: ABL1 positive samples across 11 labs, showed higher inter-lab CV% in RTq-PCR when compared head-to-head with RTddPCR [ 35 ]. The sum of these performance characteristics led to FDA regulatory clearance.…”

“…These include, 1) measuring actual molecules (positive droplets) rather than estimating starting template amount by amplification slope and standard curve samples; 2) insensitivity to sample inhibition due to the use of endpoint counting and droplet chemistry rather than amplification slope, the latter easily influenced by poor quality template and/or endogenous and exogenous interferents that might inhibit amplification, and 3) sampling a large number of droplets, which increases the precision of the assay and can increase the sensitivity of the assay. These characteristics also add robustness to the assay performance and allow high levels of reproducible results lab to lab, operator to operator, lot to lot, and even assay to assay [ 22 , 35 , 39 – 43 ].…”

Impact of BCR::ABL1 transcript type on RT-qPCR amplification performance and molecular response to therapy

“…An alternative approach is to use ddPCR, a technique which is relatively insensitive to differences in amplification efficiency as well as having other advantages such as producing results that are less variable that those produced by RT-qPCR and the lack of requirement for a standard curve [ 34 – 37 ]. Our initial data using control materials indicated that ddPCR results do not show the transcript-related differences that were seen using RT-qPCR.…”