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Reference reagents for monitoring minimal residual disease. Jean Gabert, MD, PhD ERT MEIDIA & APHM Marseille, France Gabert.j@jean-roche.univ-mrs.fr http://meidia.nord.univ-mrs.fr SoGAT XVII, Paris, May 27th. Acute leukemia and CML Concept of minimal residual disease (MRD). Cell number.
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Reference reagents for monitoring minimal residual disease Jean Gabert, MD, PhD ERT MEIDIA & APHM Marseille, France Gabert.j@jean-roche.univ-mrs.fr http://meidia.nord.univ-mrs.fr SoGAT XVII, Paris, May 27th
Acute leukemia and CMLConcept of minimal residual disease (MRD) Cell number Morphological detection limit 1012 1010 PCR detection limit 106 I Time
New molecular-based therapies • Therapeutic breakthrough O Brien et al NEJM, 2003; 348; 994-1004 Gleevec a tyrosine kinase inhibitor and CML (BCR ABL) • Therapeutic follow up on cytogenetic: t(9;21) or Ph Chr. • - CCR status (18 months): • Interferon + ARA C: 14.4% • Gleevec 74% p<0,0001 • Biological survey: • Hughes TP et al NEJM 2003; 349: 1399-401 • 3 laboratories • reference value: pool of 30 CML at diagnosis • Responder: reduction of at least 3 logs by 12 months. • Sudden need of reference biological material and Quality Controls
• • • • • • • • • • • • • • • • • ‘Europe Against Cancer’ Network • Fusion Transcripts (n=9) & RQ PCR • For leukemias • Standardization • Quality Controls • • • • • • • • • • • • • • • • • • • • • • • Partners: • SANCO European Commission • Applied Biosystems • ABI 7 700 • IPSOGEN • plasmid calibrators • • • • • • • • • • • •
developpment of a biological reference material • Rational • Allows data comparison between and within plat-forms / laboratories • Needed for research, • Strategic for network programs, • Indispensable for medical use (ex. therapeutic decision made on gene profiling signatures) • Collaboration ERT MEIDIA & NIBSC (UK) • NAT Standards in virology (Dr. J. Saldanha) • Freeze dried cells? • can be sent at room temperature worldwide • French patent n°03/03199
Phase 3 International study Seattle Los Angeles Sendai Houston Uppsala Turku Adelaide Stockholm Arhus Dublin Rotterdam London Frankfurt, Mannheim Paris Vienna Torino, Monza Bordeaux Bologna Marseille Salamanca Naples Athens
Freeze-dried cell lines International standards Ratio BCR ABL/ABL dosage by RQ PCR Neat 1/10 1/100 => reproducible
Accelerated ageing experiments NCN = normalized copy number => stable for 3 to 5 years
Control population 97 clones IPSOGEN Gene profilingreproducibility(2 K562 freeze dried cell vials)on nylon membrane, cDNA (10 000 spots) 54 clones
MRD by gene expression analysis (RQ PCR)and Hematological malignancies • Towards the definition of new biological makers • Potentially available wapon within the medical arsenal against hematological malignancies • at diagnosis (Lossos et al NEJM, 2004) • For therapeutic efficiency monitoring (MRD)
reference materials • Required forinnovative technologies development : • gene expression profiling, CGH array, SNP … • Proteomics, epigenetic event analysis… • Pool of freeze-dried cell lines • Stable material (RT) • Reproducible • Standardization tool +++ • Controls each step of the process from the extraction => WHO international standards for K562 freeze dried cells => the SAQUITO project & national cancéropôle network
MEIDIA laboratory E. Beillard C. Picard M Silvy N. Chaigneau U387 INSERM C. Genre IPSOGEN V. Fert French networks E. Macintyre (GBMHM) JM Cayuela (Costly Innovation) EAC network JJM van Dongen International study J. Saldanha (NIBSC) Acknowledgments