RT-qPCR and RT-Digital PCR: a comparison of different platforms for the evaluation of residual disease in chronic myeloid leukemia

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Title: RT-qPCR and RT-Digital PCR: a comparison of different platforms for the evaluation of residual disease in chronic myeloid leukemia
Authors: Alikian, M
Whale, AS
Akiki, S
Piechocki, K
Torrado, C
Myint, T
Cowen, S
Griffiths, M
Reid, AG
Apperley, J
White, H
Huggett, JF
Foroni, L
Item Type: Journal Article
Abstract: BACKGROUND: 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 BCR, RhoGEF, and GTPase activating protein-ABL proto-oncogene 1, non-receptor tyrosine kinase fusion transcript BCR-ABL1(IS) (%BCR-ABL1(IS)) 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. METHODS: 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. RESULTS: Measurements on all instruments correlated well when the %BCR-ABL1(IS) 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. CONCLUSIONS: 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.
Issue Date: 27-Jan-2017
Date of Acceptance: 9-Nov-2016
URI: http://hdl.handle.net/10044/1/43780
DOI: https://dx.doi.org/10.1373/clinchem.2016.262824
ISSN: 1530-8561
Publisher: American Association for Clinical Chemistry
Start Page: 525
End Page: 531
Journal / Book Title: Clinical Chemistry
Volume: 63
Issue: 2
Copyright Statement: © 2016 American Association for Clinical Chemistry
Sponsor/Funder: Imperial College Healthcare NHS Trust- BRC Funding
National Institute for Health Research
Imperial College Healthcare NHS Trust
Funder's Grant Number: RDB05 79560
Keywords: General Clinical Medicine
1004 Medical Biotechnology
1101 Medical Biochemistry And Metabolomics
1103 Clinical Sciences
Publication Status: Published
Conference Place: United States
Appears in Collections:Department of Medicine
Faculty of Medicine

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