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

Gabert.j@jean-roche.univ-mrs.frGabert.j@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)

1012

1010

106

Cell number

Time

Morphological detection limit

PCR detection limit

I

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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1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Ratio BCR-ABL to ABL (%)

25 50 100 200 400 800 1600 3200 6400

14

19

21

21

1203

12

19

10

21

10

22

03

22

18

22

05

09

02

08

11

18

01

03

04

08

19

02

08

14

18

05

09

10

02

05

09

12

20

11

13

20

20

13

11

16

06

06

14

16

06

13

0707

16

07

15b

15b

15a 15a

17

17 17

Neat 1:10 1:100Neat 1/10 1/100=> reproducible

Ratio BCR ABL/ABL dosage by RQ PCR

0,0001

0,001

0,01

0,1

1

10

100

1000

NCN

sensibility

t0 7 days 21 days1 month

2

months

45°C

0,0001

0,001

0,01

0,1

1

10

100

1000

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

-1,5

-1

-0,5

0

0,5

1

1,5

2

2,5

-1,5 -1 -0,5 0 0,5 1 1,5 2 2,5

L1

L2

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)