Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program
Jean Gabert,Emmanuel Beillard,V H J van der Velden,Wanli Bi,David Grimwade,Niels Pallisgaard,Gisela Barbany,Giovanni Cazzaniga,Jean Michel Cayuela,Hélène Cavé,Fabrizio Pane,J. L. E. Aerts,D De Micheli,X Thirion,V Pradel,Marcos González,Susanne Viehmann,Maria Malec,Giuseppe Saglio,J. J. M. Van Dongen +19 more
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TLDR
The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels and is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.Abstract:
Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytc leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n = 278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.read more
Citations
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The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments
Stephen A. Bustin,Vladimir Benes,Jeremy A. Garson,Jan Hellemans,Jim F. Huggett,Mikael Kubista,Reinhold Mueller,Tania Nolan,Michael W. Pfaffl,Gregory L. Shipley,Jo Vandesompele,Carl T. Wittwer,Carl T. Wittwer +12 more
TL;DR: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency.
Journal ArticleDOI
Retinoic acid and arsenic trioxide for acute promyelocytic leukemia
Francesco Lo-Coco,Giuseppe Avvisati,Marco Vignetti,Christian Thiede,Simona Iacobelli,Felicetto Ferrara,Paola Fazi,Laura Cicconi,E. Di Bona,Giorgina Specchia,Simona Sica,Mariadomenica Divona,Alessandro Levis,Walter Fiedler,Elisa Cerqui,Massimo Breccia,Giuseppe Fioritoni,Mario Cazzola,L. Melillo,Enrica Morra,Bernd Hertenstein,Mohammed Wattad,Michael Lübbert,Mathias Hänel,Norbert Schmitz,Alessandro Rambaldi,G. La Nasa,Mario Luppi,Fabio Ciceri,Olimpia Finizio,Adriano Venditti,Francesco Fabbiano,Konstanze Döhner,M. Sauer,Arnold Ganser,Sergio Amadori,Franco Mandelli,Hartmut Döhner,Gerhard Ehninger +38 more
TL;DR: ATRA plus arsenic trioxide is at least not inferior and may be superior to ATRA plus chemotherapy in the treatment of patients with low-to-intermediate-risk APL.
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Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results
Timothy P. Hughes,Michael W. Deininger,Andreas Hochhaus,Susan Branford,Jerald P. Radich,Jaspal Kaeda,Michele Baccarani,Jorge E. Cortes,Nicholas C.P. Cross,Brian J. Druker,Jean Gabert,David Grimwade,Rüdiger Hehlmann,Suzanne Kamel-Reid,Jeffrey H. Lipton,Janina A. Longtine,Giovanni Martinelli,Giuseppe Saglio,Simona Soverini,Wendy Stock,John M. Goldman +20 more
TL;DR: Suggestions are made for harmonizing the differing methodologies for measuring BCR-ABL transcripts in patients with CML undergoing treatment and using a conversion factor whereby individual laboratories can express BCR -ABL transcript levels on an internationally agreed scale.
Journal Article
Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction.
Stephen A. Bustin,Tania Nolan +1 more
TL;DR: Real-time RT-PCR remains a research tool, and it is important to recognize the considerable pitfalls associated with transcriptome analysis, with the successful application of RTPCR depending on careful experimental design, application, and validation.
Journal ArticleDOI
Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program
Emmanuel Beillard,Niels Pallisgaard,V H J van der Velden,Wanli Bi,R Dee,E van der Schoot,Eric Delabesse,E Macintyre,Enrico Gottardi,Giuseppe Saglio,F Watzinger,Thomas Lion,J. J. M. Van Dongen,Peter Hokland,Jean Gabert +14 more
TL;DR: The ABL gene is proposed to be used as CG for RQ-PCR-based diagnosis and MRD detection in leukemic patients and these data are not only eligible for quantifying of fusion gene transcripts, but also for the quantification of aberrantly expressed genes.
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