RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia
| Autor: | Jane F. Apperley, Simon Cowen, Susanna Akiki, Michael J. Griffiths, Letizia Foroni, Mary Alikian, Alexandra S. Whale, Celia Torrado, Alistair Reid, Jim F. Huggett, Helen E. White, Kim Piechocki, Thet Myint |
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| Přispěvatelé: | Imperial College Healthcare NHS Trust- BRC Funding, National Institute for Health Research, CANCER RESEARCH UK, Imperial College Healthcare NHS Trust |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Neoplasm Residual Clinical Biochemistry Fusion Proteins bcr-abl Real-Time Polymerase Chain Reaction Bioinformatics 03 medical and health sciences 0302 clinical medicine Leukemia Myelogenous Chronic BCR-ABL Positive hemic and lymphatic diseases 1101 Medical Biochemistry And Metabolomics Humans Medicine Digital polymerase chain reaction Proto-Oncogene Proteins c-abl General Clinical Medicine ABL Reverse Transcriptase Polymerase Chain Reaction business.industry 1004 Medical Biotechnology Biochemistry (medical) Myeloid leukemia Cancer 1103 Clinical Sciences Gold standard (test) medicine.disease Leukemia 030104 developmental biology Real-time polymerase chain reaction Fusion transcript 030220 oncology & carcinogenesis business |
| Zdroj: | Clinical Chemistry. 63:525-531 |
| ISSN: | 1530-8561 0009-9147 |
| Popis: | 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 fusion transcript BCR-ABL1 (breakpoint cluster region–c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1IS (%BCR-ABL1IS) 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-ABL1IS 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. |
| Databáze: | OpenAIRE |
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