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Experimental Hematology, Institut Jules Bordet (ULB)

Comparison of Chronic Lymphocytic Leukemia patients expressing high or low level of ZAP70 mRNA: new prognostic factors, differences in microRNA expression and interaction with the microenvironment.

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Experimental Hematology, Institut Jules Bordet (ULB)

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  1. Comparison of Chronic Lymphocytic Leukemia patients expressing high or low level of ZAP70 mRNA: new prognostic factors, differences in microRNA expression and interaction with the microenvironment. B. Stamatopoulos, B. Haibe-Kains, C. Equeter, N. Meuleman, A. Sorée, C. De Bruyn, D. Hanosset, D. Bron, P. Martiat, L. Lagneaux Experimental Hematology, Institut Jules Bordet (ULB) 23rd annual general meeting of the BHS, January 25, 2008

  2. Introduction • CLL : 2 distinct evolutions • Prognostic factors : IgVH mutational status • … and its surrogate : ZAP70, LPL,… • ZAP70 measured by flow cytometry

  3. ZAP70 measured by quantitative real time PCR TFS in Univ. Univ. Multiv. 1 year 2 years Assoc. Strength of AUC Conc. Assoc. Assoc. case of Cox Cox Cox AUC AUC with ass. with predict. with with with discord. predict. predict. predict. predictor predictor MS MS of MS MS TFS OS with MS of TFS of OS of TFS of TFS of TFS Mut. status (MS) - - - - S S - S S NS 70% 77% Zap-70 (qPCR) S very strong 89% 86% S S S S S S 74% 83% Zap-70 (FC) S strong 85% 78% S S NS S S S 79% 84% LPL (qPCR) S strong 76% 75% S NS NS S NS NS 69% 69% CD38 (FC) S substantial 70% 67% S NS NS S NS NS 63% 66% S: significant; NS: non significant TFS OS (Stamatopoulos B. et al, Clin. Chem, 2007)

  4. Aims of the study • To determine gene expression profiles linked to ZAP70 expression using Affymetrix technology • To find genes associated with prognosis but also with biology • To confirm differentially expressed microRNA and link them to TFS and OS

  5. Patient selection ZAP70 mRNA expression among 108 patients by qPCR Low high

  6. Mut. Status PATIENTS Zap-70 by qPCR Zap-70 by FC. Treatment Death Num Group Sex Stage Status Status Status Status Age qPCR % cell. 1 high F 69 A UM 1507.7 + 66 + yes yes 2 high M 68 C UM 1475.8 + 41 + yes no 3 high F 57 B UM 1179.2 + 57 + yes yes 4 high F 67 A M 1137.0 + 39 + yes yes 5 high M 61 C UM 1074.7 + 22 + yes yes 6 high F 74 C UM 650.3 + 42 + yes yes 7 high M 73 B UM 643.9 + 77 + yes yes 8 low F 67 A M 61.8 - 2 - no no 9 low F 68 A M 50.4 - 2.5 - no no 10 low F 59 B M 18.9 - 14 - no no 11 low M 67 B M 14.2 - 1 - no no 12 low M 72 B M 9.5 - 3 - yes no 13 low M 77 A M 7.5 - 12 - yes no 14 low M 69 A M 2.7 - 10 - yes no Patient characteristics

  7. Methods Purification with anti-CD19 magnetic beads 98-100% purity blood collection PBMC gradient isolation (1,5µg) RNA extraction • - 50 000 oligonucleotides • 47 000 transcripts • 39 500 human genes Affymetrix Protocole

  8. Parametric ZAP70low ZAP70high Gene FDR Ratio Probe set p-value mean mean symbol 1 < 1e-07 < 1e-07 17.9 124.4 6.94 1555613_a_at ZAP70 2 < 1e-07 < 1e-07 31.9 168.9 5.29 214032_at ZAP70 Results • 937 probe sets with P<0.05 • 135 probe sets with P<0.001 • 43 probe sets with FDR<10% • Top of the list: qPCR validation

  9. Novel genes and clinical outcome TFS OS … but also TLR7, LPL, PCDH9, MYBL1, …

  10. 4.7% 36.5% 25.7% 56.2% PDE8A: a novel therapeuthic target ? Control (ethanol) Dipyridamol 15µM Propidium Ioide - PE Annexine V - FITC

  11. Kegg Pathway Description P-value hsa04514 Cell adhesion molecules (CAMs) 0.0020 GO categories Description P-value -7 hsa04530 tight junction 2.6.10 -6 hsa04520 adherens junction 2.5. 10 GO0015629 actin cytoskeleton 0.0021 -5 Biocarta pathways hsa04540 gap junction 3.9 . 10 GO0030036 actin cytoskeleton organization and biogenesis 0.0027 -7 GO0030029 actin filament-based process 0.0014 hsa04670 transendothelial leukocyte migration <10 h_lympathway adhesion and diapedis of lymphocytes 0.0012 -7 GO0008154 actine polymerization and/or depolimerization 0.0010 hsa04810 regulation of actin cytoskeleton <10 h_integrinPathway Integrin Signaling Pathway 0.0015 -7 -7 GO0007155 cell adhesion 8.10 hsa04510 Focal adhesion <10 h_lymphocytePathway Adhesion Molecules on Lymphocyte 0.0012 GO0016387 cell-cell adhesion 0.0053 Broad Pathway h_cxcr4Pathway CXCR4 Signaling Pathway 0.0006 GO0007160 cell-matrix adhesion 0.0003 GO0006935 chemotaxis 0.0023 0.0121 cell_adhesion_receptor_activity_h GO0005856 cytoskeleton 0.0002 GO0007010 cytoskeleton organisation and biogenesis 0.0009 0.0069 SIG_Regulation_of_the_actin_cytoskeleton_by_Rho_GTPases_h GO0040011 locomotion 0.0060 0.0011 cell_adhesion_molecule_activity_h GO0005874 microtubule 0.0003 GO0007018 microtubule based movement 0.0108 0.0005 cell_motility_h 0.0004 cell_adhesion_h -5 1.9 . 10 h_ST_Integrin_Signaling_Pathway Gene set enrichment analysis Lagneaux L. et al, Blood, 1998 Ghia P., Semin Cancer Biol, 2002

  12. CXCR4 on fresh samples ZAP70 and CXCR4 modulation by ME

  13. Adhesion and migration

  14. MicroRNA expression TFS OS

  15. Conclusions • ZAP70 + and – patients : distinc gene expression profiles • new prognostic factors : genes and microRNA • new potential therapeutic target • gene set implicated in migration and adhesion • ZAP70 + : better adhesion and migration capacities into their microenvironment • Better survival of ZAP70 + cells and the agressiveness of the disease

  16. Acknowledgments PhD director: Laurence Lagneaux and ALL MEMBERS of the Laboratory of Experimental Hematology of Pr. Philippe Martiat CLL sample collection: Nathalie Meuleman Dominique Bron Philippe Martiat Philippe Herman (St-Pierre Hospital) Alain Kentos (Erasme Hospital –ULB) (Bordet Institute – ULB) This work was financed by F.R.I.A. grant and the Télévie fund, both of which are affiliated with the F.R.S-F.N.R.S. Thank you for your attention.

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