Detection and Quantification of BCR-ABL1 Fusion Transcripts by Droplet Digital PCR

Jennings, Lawrence J.; George, David; Czech, Jörg; Yu, Min; Joseph, Loren

doi:10.1016/j.jmoldx.2013.10.007

Cited by 112 publications

(89 citation statements)

References 42 publications

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“…The concept behind dPCR has been long established (1 ), but it has been made practical in recent years by the development of microfluidics and/or emulsion chemistries to simplify and automate the process. The frequently cited benefits of improved precision affording finer fold-change measurements and deeper rare variant detection have been highlighted and increasingly applied to clinical challenges, including oncology (2)(3)(4)(5) and infectious diseases (6 -10 ) as well as fetal genetic screening (11,12 ), and in predicting transplant rejection (13 ).…”

confidence: 99%

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

2015

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BACKGROUND Digital PCR (dPCR) is an increasingly popular manifestation of PCR that offers a number of unique advantages when applied to preclinical research, particularly when used to detect rare mutations and in the precise quantification of nucleic acids. As is common with many new research methods, the application of dPCR to potential clinical scenarios is also being increasingly described. CONTENT This review addresses some of the factors that need to be considered in the application of dPCR. Compared to real-time quantitative PCR (qPCR), dPCR clearly has the potential to offer more sensitive and considerably more reproducible clinical methods that could lend themselves to diagnostic, prognostic, and predictive tests. But for this to be realized the technology will need to be further developed to reduce cost and simplify application. Concomitantly the preclinical research will need be reported with a comprehensive understanding of the associated errors. dPCR benefits from a far more predictable variance than qPCR but is as susceptible to upstream errors associated with factors like sampling and extraction. dPCR can also suffer systematic bias, particularly leading to underestimation, and internal positive controls are likely to be as important for dPCR as they are for qPCR, especially when reporting the absence of a sequence. SUMMARY In this review we highlight some of the considerations that may be needed when applying dPCR and discuss sources of error. The factors discussed here aim to assist in the translation of dPCR to diagnostic, predictive, or prognostic applications.

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confidence: 99%

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

2015

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“…60 Moreover, there are preliminary data indicating that it may improve MRD detection compared with established standardized RT-qPCR assays in chronic myeloid leukemia. 61 …”

Section: Newer Molecular Technologies For Mrd Detectionmentioning

confidence: 99%

Defining minimal residual disease in acute myeloid leukemia: which platforms are ready for “prime time”?

Grimwade

Freeman

2014

Blood

231

212

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The past 40 years have witnessed major advances in defining the cytogenetic aberrations, mutational landscape, epigenetic profiles, and expression changes underlying hematological malignancies. Although it has become apparent that acute myeloid leukemia (AML) is highly heterogeneous at the molecular level, the standard framework for risk stratification guiding transplant practice in this disease remains largely based on pretreatment assessment of cytogenetics and a limited panel of molecular genetic markers, coupled with morphological assessment of bone marrow (BM) blast percentage after induction. However, application of more objective methodology such as multiparameter flow cytometry (MFC) has highlighted the limitations of morphology for reliable determination of remission status. Moreover, there is a growing body of evidence that detection of subclinical levels of leukemia (ie, minimal residual disease, MRD) using MFC or molecular-based approaches provides powerful independent prognostic information. Consequently, there is increasing interest in the use of MRD detection to provide early end points in clinical trials and to inform patient management. However, implementation of MRD assessment into clinical practice remains a major challenge, hampered by differences in the assays and preferred analytical methods employed between routine laboratories. Although this should be addressed through adoption of standardized assays with external quality control, it is clear that the molecular heterogeneity of AML coupled with increasing understanding of its clonal architecture dictates that a “one size fits all” approach to MRD detection in this disease is not feasible. However, with the range of platforms now available, there is considerable scope to realistically track treatment response in every patient.

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“…21 It measures the copy number of BCR-ABL transcripts in each amount of RNA obtained from blood. 22,23 In our samples, BCR-ABL was detected in both diagnostic and follow up samples, ABL being the control gene. The RT-qPCR must detect an endogeneous control transcript to assess the quality and quantity of RNA and to normalize for potential differences between tests.…”

Section: Discussionmentioning

confidence: 68%

Prognostic significance of micro RNA 150 marker in BCR-ABL positive chronic myeloid leukaemia patients on imatinib mesylate

Ezeanosike¹,

Afonne

2017

Int J Contemp Pediatr

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Background: Micro-RNAs control gene expression by destabilizing targeted transcripts and inhibiting their translation. In chronic myeloid leukaemia (CML), abnormal expressions of miRNAs have been described. The current treatment for newly diagnosed cases of CML is imatinib mesylate which produces rapid haematological responses. It is currently impossible to predict whether a patient will develop resistance to imatinib mesylate. This makes identification of predictors of resistance to imatinib an important goal in management of patients with CML. MicroRNA expression patterns can be used to predict outcome which can be remission or relapse. This study therefore, was set to assess the possible use of microRNA 150 for prognostication.Methods: Fifty peripheral blood samples previously collected from CML patients who were being treated with imatinib mesylate and stored in the refrigerator at +4°C were analyzed for the expression of microRNAs 150. Total RNA was extracted from guanidium isothiocynate (GITC) lysate of the blood samples using RNeasy mini spin column. The total RNA was converted to complimentary DNA by random hexamer priming using Murine Moloney Leukaemia Virus Reverse Transcriptase. Real time Multiplex PCR was used for detecting Breakpoint Cluster Region-Abelson Murine Leukaemia (BCR-ABL) transcript type.Results: The patients’ samples showed an expression of miRNA-150. Correlation of BCR-ABL ratio with miRNA-150 was done and the Spearman correlation coefficient (Rho) between BCR-ABL1 and miRNA-150 was 0.442 (p = 0.001; CI = 0.18-0.65) showing that there was a positive correlation between BCR-ABL1 and miRNA-150. The coefficient of determination was 20% (CI = 3-42%), which implies that about 20% of BCR-ABL1 ratio could be accounted for by the miRNA-150 values.Conclusions: Therefore, once patients who are on imatinib achieve molecular remission of the CML, the miRNA-150 can be useful in predicting outcome which could be relapse or complete molecular remission but is weak at diagnosis in predicting such outcome.

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Detection and Quantification of BCR-ABL1 Fusion Transcripts by Droplet Digital PCR

Cited by 112 publications

References 42 publications

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

Defining minimal residual disease in acute myeloid leukemia: which platforms are ready for “prime time”?

Prognostic significance of micro RNA 150 marker in BCR-ABL positive chronic myeloid leukaemia patients on imatinib mesylate

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