1. ¹Department of Hematology Biology, Institut Paoli Calmettes, France
2. ²Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
3. ³Applied Biosystems, Foster City, CA, USA
4. ⁴Department of Haematology, Division of Medical and Molecular Genetics, Guy's, King's & St Thomas' School of Medicine, University College London Hospitals, London, UK
5. ⁵Department of Immuno-haematology, Aarhus University Hospital, Aarhus, Denmark
6. ⁶Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden
7. ⁷Pediatric Clinic, University of Milan Bicocca, San Gerardo Hospital, Monza, Italy
8. ⁸Central Laboratory of Hematology and INSERM U462, Hôpital Saint Louis, Paris, France
9. ⁹Department of Genetic Biochemistry, Hôpital Robert Debré, Paris, France
10. ¹⁰Department of CEINGE & Department of Biochemistry & Medical Biotechnology, University Hospital 'Federico II', Naples, Italy
11. ¹¹Department of Haematology, University Hospital, Leuven, Belgium
12. ¹²Clinical and Biological Science, University of Turin, Ospedale San Luigi Gonzaga, Orbassano-Torino, Italy
13. ¹³Department of Medical Information, University Hôpital Sainte Marguerite/Biostatistics Laboratory Timone, Université de la Méditerranée, Marseille, France
14. ¹⁴Department of Hematology, Hospital Clínico Universitario, Salamanca, Spain
15. ¹⁵Biogenetics Laboratory, Children's University Hospital, Giessen, Germany
16. ¹⁶Department of Hematology, Karolinska, Stockholm, Sweden

Correspondence: Professor J Gabert, Biochemistry and Molecular Biology Laboratory, IFR Jean Roche, Faculte de Medecine NORD, Bd Pierre dramard, 13916 Marseille Cedex 20, France. Fax: +33 491 69 87 51; E-mail: gabert.j@jean-roche.univ-mrs.fr

¹⁷First co-authors

¹⁸Present affiliation: Department of Biochemistry & Molecular Biology, Hôpital Universitaire Nord, ERT MEIDIA, IFR Jean Roche, Université de la Méditerranée, Marseille, France

¹⁹Consultant for Ipsogen

²⁰Present affiliation: Department of Genetics and Pathology, Uppsala University Hospital, Uppsala, Sweden

Received 27 August 2002; Accepted 31 May 2003; Published online 9 October 2003.

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.