Reference reagents for monitoring minimal residual disease

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Reference reagents for monitoring minimal
residual disease

• Jean Gabert, MD, PhD
• ERT MEIDIA APHM
• Marseille, France
• Gabert.j_at_jean-roche.univ-mrs.fr
• http//meidia.nord.univ-mrs.fr
• SoGAT XVII, Paris, May 27th

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Acute leukemia and CML Concept of minimal
residual disease (MRD)
Cell number
Morphological detection limit
1012
1010
PCR detection limit
106
I
Time
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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(921) or
  Ph Chr.
• - CCR status (18 months)
• Interferon ARA C 14.4
• Gleevec 74 plt0,0001
• 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

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• Europe Against Cancer Network
• Fusion Transcripts (n9) RQ PCR
• For leukemias
• Standardization
• Quality Controls

• Partners
• SANCO European Commission
• Applied Biosystems
• ABI 7 700
• IPSOGEN
• plasmid calibrators

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E. Beillard et al Gabert J et al Leukemia, dec
2003
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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 n03/03199

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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
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Freeze-dried cell lines International standards

Ratio BCR ABL/ABL dosage by RQ PCR
Neat 1/10 1/100
gt reproducible
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Accelerated ageing experiments
NCN normalized copy number
gt stable for 3 to 5 years
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Gene profilingreproducibility (2 K562 freeze
dried cell vials)on nylon membrane, cDNA (10 000
spots)
54 clones
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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)

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reference materials

• Required for innovative 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
• gt WHO international standards for K562 freeze
  dried cells
• gt the SAQUITO project national cancéropôle
  network

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Acknowledgments

• 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)