RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2017-02, Vol.63 (2), p.525-531
• Main Authors: Alikian, Mary, Whale, Alexandra S, Akiki, Susanna, Piechocki, Kim, Torrado, Celia, Myint, Thet, Cowen, Simon, Griffiths, Michael, Reid, Alistair G, Apperley, Jane, White, Helen, Huggett, Jim F, Foroni, Letizia
• Format: Article
• Language: English
• Subjects: Blood; Calibration; Experiments; Funding; Fusion Proteins, bcr-abl - genetics; Humans; Laboratories; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive - diagnosis; Leukemia, Myelogenous, Chronic, BCR-ABL Positive - genetics; Neoplasm, Residual - diagnosis; Neoplasm, Residual - genetics; Plasmids; Proto-Oncogene Proteins c-abl - genetics; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2016.262824

Tyrosine kinase inhibitors (TKIs) are the cornerstone of successful clinical management of patients with chronic myeloid leukemia (CML). Quantitative monitoring of the percentage of the fusion transcript BCR-ABL1 (breakpoint cluster region-c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1 (%BCR-ABL1 ) by reverse transcription-quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to TKIs and classification into prognostic subgroups. However, this approach can be challenging to perform in a reproducible manner. Reverse-transcription digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for truly standardized patient stratification. BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. 3 commercially available digital PCR platforms (QS3D, QX200 and Raindrop) were compared with the platform routinely used in the clinic for RT-qPCR using the EAC (Europe Against Cancer) assay. Measurements on all instruments correlated well when the %BCR-ABL1 was ≥0.1%. In patients with residual disease below this level, greater variations were measured both within and between instruments limiting comparable performance to a 4 log dynamic range. RT-dPCR was able to quantify low-level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without use of a calibration curve. Adaptions to the protocol to increase the amount of RNA measured are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing on a dPCR platform.