Fast‐mode duplex qPCR for <i>BCR‐ABL1</i> molecular monitoring: Innovation, automation, and harmonization

摘要

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is currently the most sensitive tool available for the routine monitoring of disease level in patients undergoing treatment for BCR-ABL1 associated malignancies. Considerable effort has been invested at both the local and international levels to standardise the methodology and reporting criteria used to assess this critical metric. In an effort to accommodate the demands of increasing sample throughput and greater standardization, we adapted the current best-practice guidelines to encompass automation platforms and improved multiplex RT-qPCR technology. Chronic myeloid leukemia (CML) and Philadelphia positive acute lymphoblastic leukemia (Ph+ALL) are the two hematological malignancies most commonly associated with the t(9;22)(q34;q11) translocation (Philadelphia Chromosome, Ph) and the resultant BCR-ABL1 fusion gene. This in turn encodes a dysregulated tyrosine kinase oncoprotein, affecting a multitude of downstream targets leading to aberrant mitogenic signalling, altered cell adhesion, suppression of cell death pathways and leukemogenesis [1]. The fusion-gene transcript, being unique to the leukemic clone offers both a diagnostic and therapeutic target, exploited via RT-qPCR and tyrosine kinase inhibitors (TKI), respectively. The transcript reduction kinetics in response to TKI therapy is a powerful prognostic tool, allowing the clinician to make informed choices about an individual's therapeutic course at defined time points [2, 3]. Currently, many centers in the United Kingdom and Europe use RT-qPCR protocols based on the Europe Against Cancer (EAC) collaboration, which produced standardized sample preparation, primer-probe sequences, and reaction conditions for use in the molecular monitoring of fusion-gene positive leukemias [4, 5]. The standardization process has since continued through national and international collaborative groups, resulting in the publication of a number of guidelines [6-8]. However, due to the dual pressures of increasing sample throughput and economic restriction, it has become necessary to employ new technological solutions whilst retaining compliance within the ongoing standardization frameworks. Consequently, we adapted the EAC BCR-ABL1 qPCR protocol to make use of current hardware and chemistry capabilities to reduce cost and increase throughput. We had previously introduced a duplex assay that was an incremental modification of our incumbent in-house qPCR methodology, but it suffered from an increasingly problematic high background and an unexplained sensitivity to interbatch variations in qPCR master mix [9]. In addition, because of the use of legacy standard controls, the assay was not EAC compatible, which was problematic when undertaking cooperative standardization exercises. We, therefore, decided to overhaul the qPCR assay and introduce automation at key points in the processing workflow and then perform validation to ensure performance. A detailed summary of the changes made can be found in Table I and the primer/probe sequences in Table II. To reduce the problem with assay noise levels, minor-groove binding (MGB) probes [Applied Biosciences (ABI), UK], using a non-fluorescent quencher, were chosen. The corresponding EAC probe sequences were then truncated to compensate for the inherent increase in Tm associated with the MGB moiety [10]. Additionally, a fast-mode qPCR master mix was used to take advantage of the fast-run capabilities of our ABI 7900HT real-time analyser, and in so doing reduced the run time from 1 hr 45 min to 55 min. The reaction volume was also decreased from 25 to 20 μl, ensuring optimal fast-mode reaction kinetics. The BCR-ABL1 and ABL1 probes were differentially labelled with 6-FAM and VIC, respectively, to allow duplexing of the qPCR reaction. To replace our EAC incompatible standard curve plasmid, we chose a four-target plasmid construct, developed and kindly provided by the Wessex Regional G