Abstract 4736: Absolute quantification of BCR-ABL RNA using multiplexed digital RT-PCR
Authors: Arcila Maria E. , Benayed Ryma , Kiecka Iwona , Milbury Coren , Mauro Michael , Samuels Michael L. 
Published: August 2015

Abstract
Reciprocal translocation and fusion of the genes encoding BCR (chromosome 22) and ABL1 (chromosome 9) are typical and causative for approximately 95% of chronic myeloid leukemias (CMLs). Specific and effective drugs targeting the protein products of these gene fusions have been developed and are used in current clinical practice. Molecular diagnostic methods are used for patient identification, for tracking drug effectiveness, and monitoring minimal residual disease (MRD). However, the use of quantitative PCR (qPCR) for this purpose has been problematic, particularly for determination of low limits of detection (LOD) and for comparing results between labs. International standards have been developed to ameliorate these issues, but there are significant ongoing challenges with these efforts. Here we address these challenges through the development of a multiplexed reverse transcription digital PCR (RT-dPCR) assay, and provide a direct comparison between samples run using the International Standardized qPCR and the new multiplexed RT-dPCR method.
Digital PCR isolates individual single target molecules into compartments for endpoint PCR, with absolute quantification provided by simply counting the number of ‘PCR-positive’ compartments. Using the RainDrop dPCR system, we partitioned each sample's target RNA molecules into a single-molecule droplet format, together with a ‘one-step’ reverse transcription master mix (SuperScript III Platinum Taq, LifeTech) and standard qPCR hydrolysis probes/primers (IDT), for endpoint PCR and absolute quantification. We used a ‘4-plex’ multiplexed assay that counts 1) the e13:a2 isoform of BCR:ABL; 2) the e14:a2 isoform of BCR-ABL; 3) GUSB RNA as an endogenous counting control, and; 4) a synthetic RNA as an exogenous control.
Initial characterization of the multiplexed assay's specificity and sensitivity were performed using RNA from cell lines (K562 for e14:a2; Invivoscribe clonal cell line for e13:a2 and e1:a2; U937 for a negative control). Samples derived from blood were split and quantified using either qPCR together with International Standard Protocols and reagents, or using the 4-plex RT-dPCR assay. Concordance and comparison of the two assay's LODs will be presented. Sensitivity assessment based on control generated data provides evidence of detection as low 1 in 200,000 without hitting a detection limit.
The use of dPCR provides significant advantages over standard qPCR methods including absolute quantification which eliminates the need for standard curves, the ability to use RNA directly, the use of an exogenous control (non-ABL) and easy isoform analysis.
Citation Format: Maria E. Arcila, Ryma Benayed, Iwona Kiecka, Coren Milbury, Michael Mauro, Michael L. Samuels. Absolute quantification of BCR-ABL RNA using multiplexed digital RT-PCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4736. doi:10.1158/1538-7445.AM2015-4736