The assessment of minimal residual disease versus that of ...
Reference reagents for monitoring minimal residual disease
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Transcript of Reference reagents for monitoring minimal residual disease
Reference reagents for Reference reagents for monitoring minimal residual monitoring minimal residual
diseasedisease
Jean Gabert, MD, PhDJean Gabert, MD, PhDERT MEIDIA & APHMERT MEIDIA & APHM
Marseille, FranceMarseille, France
[email protected]@jean-roche.univ-mrs.frhttp://meidia.nord.univ-mrs.fr
SoGAT XVII, Paris, May 27thSoGAT XVII, Paris, May 27th
Incidence of main cancers in France - (1995)
Sein
Colo rectum
Prostate
Poumon
ORL
Vessie
Estomac
Onco hématologie
LMNH
Hodgkin
Myelome
LeucemiesOnco hematology
= model in Oncology
3,6%
Acute leukemia and CMLAcute leukemia and CML
Concept of minimal residual disease (MRD)Concept of minimal residual disease (MRD)
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Cell number
Time
Morphological detection limit
PCR detection limit
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New molecular-based New molecular-based therapiestherapies
1.1. Therapeutic breakthroughTherapeutic breakthrough O Brien et al NEJM, 2003; 348; 994-1004O Brien et al NEJM, 2003; 348; 994-1004 GleevecGleevec a tyrosine kinase inhibitor and CML ( a tyrosine kinase inhibitor and CML (BCR ABLBCR ABL))
– Therapeutic follow up on cytogenetic: t(9;21) or Ph Chr.Therapeutic follow up on cytogenetic: t(9;21) or Ph Chr.– -- CCR status (18 months): CCR status (18 months):
• Interferon + ARA C: 14.4%Interferon + ARA C: 14.4%• Gleevec 74% p<0,0001Gleevec 74% p<0,0001
2.2. Biological surveyBiological survey::• Hughes TP et al NEJM 2003; 349: 1399-401Hughes TP et al NEJM 2003; 349: 1399-401
• 3 laboratories3 laboratories• reference value: pool of 30 CML at diagnosisreference value: pool of 30 CML at diagnosis• Responder: reduction of at least 3 logs by 12 months.Responder: reduction of at least 3 logs by 12 months.
Sudden need of reference biological material and Sudden need of reference biological material and Quality ControlsQuality Controls
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•Partners:Partners:• SANCO European CommissionSANCO European Commission
•Applied BiosystemsApplied Biosystems•ABI 7 700
•IPSOGENIPSOGEN• plasmid calibratorsplasmid calibrators
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‘ ‘Europe Against Cancer’ NetworkEurope Against Cancer’ Network
Fusion Transcripts (n=9) & RQ PCRFusion Transcripts (n=9) & RQ PCRFor leukemiasFor leukemias• StandardizationStandardization• Quality ControlsQuality Controls
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RQ PCR - EAC network- CONCLUSIONS -1. The PCR step was highly reproducible,2. Variations mainly due to the pre -PCR steps (RNA stability, RT),3. Normalisation by a control gene is mandatory,4. The EAC protocol allowed data comparison,5. Proposal: use of the EAC protocol as International reference
E. Beillard et al & Gabert J et al Leukemia, dec 2003
developpment of a biological reference developpment of a biological reference materialmaterial
• RationalRational– Allows data comparison between and within plat-forms / Allows data comparison between and within plat-forms /
laboratorieslaboratories • Needed for research, Needed for research, • Strategic for network programs,Strategic for network programs,• Indispensable for medical useIndispensable for medical use (ex. therapeutic decision (ex. therapeutic decision
made on gene profiling signatures)made on gene profiling signatures)
• Collaboration ERT MEIDIA & NIBSC (UK)Collaboration ERT MEIDIA & NIBSC (UK)– NAT Standards in virology (Dr. J. Saldanha)NAT Standards in virology (Dr. J. Saldanha)– Freeze dried cells?Freeze dried cells?
• can be sent at room temperature worldwidecan be sent at room temperature worldwide– French patent n°French patent n°03/0319903/03199
Marseille
Paris
Bordeaux
SalamancaAthens
Bologna
Naples
ViennaTorino, Monza
Frankfurt, MannheimRotterdam
Turku
Stockholm
Uppsala
London
DublinArhus
Seattle
Los Angeles
Houston
Adelaide
Sendai
Phase 3International study
Freeze-dried cell lines
International standards
Ratio BCR-ABL to ABL (%)
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Ratio BCR-ABL to ABL (%)
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22
18
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05
09
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11
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01
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19
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14
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05
09
10
02
05
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12
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0707
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Neat 1:10 1:100Neat 1/10 1/100=> reproducible
Ratio BCR ABL/ABL dosage by RQ PCR
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NCN
sensibility
t0 7 days 21 days1 month
2
months
45°C
0,0001
0,001
0,01
0,1
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NCN
sensibility
t0 7 days1 month2 months
37°C
6 months
=> stable for 3 to 5 years
NCN = normalized copy number
Accelerated ageing experiments
Gene profilingGene profiling
reproducibilityreproducibility (2 K562 freeze dried cell vials)(2 K562 freeze dried cell vials)
on nylon membraneon nylon membrane, , cDNA cDNA (10 000 spots)(10 000 spots)
54 clones repro lyophilisation K562y = 0,9504x + 0,0098
R2 = 0,8896
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-1,5 -1 -0,5 0 0,5 1 1,5 2 2,5
L1
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Control population
97 clonesIPSOGEN
MRD by gene expression analysis MRD by gene expression analysis (RQ PCR)(RQ PCR)
and Hematological malignanciesand Hematological malignancies
• Towards the definition of new biological Towards the definition of new biological makersmakers
• Potentially available wapon within the Potentially available wapon within the medical arsenal against hematological medical arsenal against hematological malignanciesmalignancies– at diagnosis (Lossos et al NEJM, 2004)at diagnosis (Lossos et al NEJM, 2004)– For therapeutic efficiency monitoring (MRD)For therapeutic efficiency monitoring (MRD)
reference materialsreference materials
• Required forRequired for innovative technologies innovative technologies development :development :– gene expression profiling, CGH array, SNP …gene expression profiling, CGH array, SNP …
– Proteomics, epigenetic event analysis…Proteomics, epigenetic event analysis…
• Pool of freeze-dried cell linesPool of freeze-dried cell lines• Stable material (RT)Stable material (RT)
• Reproducible Reproducible
• Standardization tool +++Standardization tool +++
• Controls each step of the process from the extractionControls each step of the process from the extraction
=> WHO international standards for K562 freeze dried => WHO international standards for K562 freeze dried cellscells
=> the SAQUITO project & national cancéropôle network => the SAQUITO project & national cancéropôle network
AcknowledgmentsAcknowledgments
• MEIDIA laboratoryMEIDIA laboratory– E. BeillardE. Beillard– C. PicardC. Picard– M SilvyM Silvy– N. ChaigneauN. Chaigneau
• U387 INSERMU387 INSERM – C. GenreC. Genre
• IPSOGENIPSOGEN– V. FertV. Fert
French networksFrench networks– E. MacintyreE. Macintyre
(GBMHM)(GBMHM)– JM Cayuela (Costly JM Cayuela (Costly
Innovation)Innovation)
• EAC networkEAC network– JJM van DongenJJM van Dongen
• International studyInternational study– J. Saldanha (NIBSC)J. Saldanha (NIBSC)