title: RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia.
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 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-ABL1ISwas ≥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.
authors.affiliation: Imperial Molecular Pathology, Imperial Healthcare Trust, Hammersmith Hospital, London, UK; m.alikian@imperial.ac.uk.|Centre for Haematology, Faculty of Medicine, Imperial College London, London, UK.
authors.forename: Mary
authors.initials: M
authors.lastname: Alikian
authors.affiliation: Molecular and Cell Biology Team, LGC, Queens Road, Teddington, UK.
authors.forename: Alexandra S
authors.initials: AS
authors.lastname: Whale
authors.affiliation: West Midlands Regional Genetics Laboratories, Birmingham Women's NHS Foundation Trust, Birmingham, UK.
authors.forename: Susanna
authors.initials: S
authors.lastname: Akiki
authors.affiliation: West Midlands Regional Genetics Laboratories, Birmingham Women's NHS Foundation Trust, Birmingham, UK.
authors.forename: Kim
authors.initials: K
authors.lastname: Piechocki
authors.affiliation: Imperial Molecular Pathology, Imperial Healthcare Trust, Hammersmith Hospital, London, UK.
authors.forename: Celia
authors.initials: C
authors.lastname: Torrado
authors.affiliation: Imperial Molecular Pathology, Imperial Healthcare Trust, Hammersmith Hospital, London, UK.
authors.forename: Thet
authors.initials: T
authors.lastname: Myint
authors.affiliation: Statistics Team, LGC, Queens Road, Teddington, UK.
authors.forename: Simon
authors.initials: S
authors.lastname: Cowen
authors.affiliation: West Midlands Regional Genetics Laboratories, Birmingham Women's NHS Foundation Trust, Birmingham, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Griffiths
authors.affiliation: Imperial Molecular Pathology, Imperial Healthcare Trust, Hammersmith Hospital, London, UK.|Centre for Haematology, Faculty of Medicine, Imperial College London, London, UK.
authors.forename: Alistair G
authors.initials: AG
authors.lastname: Reid
authors.affiliation: Centre for Haematology, Faculty of Medicine, Imperial College London, London, UK.|Clinical Haematology, Imperial College Healthcare NHS Trust, London, UK.
authors.forename: Jane
authors.initials: J
authors.lastname: Apperley
authors.affiliation: National Genetics Reference Laboratory (Wessex), Salisbury District Hospital, Salisbury, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: White
authors.affiliation: Molecular and Cell Biology Team, LGC, Queens Road, Teddington, UK.|School of Biosciences & Medicine, Faculty of Health & Medical Science, University of Surrey, Guildford, UK.
authors.forename: Jim F
authors.initials: JF
authors.lastname: Huggett
authors.affiliation: Centre for Haematology, Faculty of Medicine, Imperial College London, London, UK.|Clinical Haematology, Imperial College Healthcare NHS Trust, London, UK.
authors.forename: Letizia
authors.initials: L
authors.lastname: Foroni
mesh_terms: D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis / Q000235:genetics*; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics*; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D016044:Fusion Proteins, bcr-abl; D016315:Proto-Oncogene Proteins c-abl