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) 23 rd 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. (Stamatopoulos B. et al, Clin. Chem, 2007) TFS OS  ZAP70 measured by quantitative real time PCR

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 Low high ZAP70 mRNA expression among 108 patients by qPCR

6. Patient characteristics

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

8. 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: 1< 1e-07 17.9124.46.941555613_a_atZAP70 2< 1e-07 31.9168.95.29214032_atZAP70 Gene symbol ZAP70high mean RatioProbe set Parametric p-value FDR ZAP70low mean qPCR validation

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

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

11. Gene set enrichment analysis GO categoriesDescriptionP-value GO0015629actin cytoskeleton0.0021 GO0030036actin cytoskeleton organization and biogenesis0.0027 GO0030029actin filament-based process0.0014 GO0008154actine polymerization and/or depolimerization0.0010 GO0007155cell adhesion8.10 -7 GO0016387cell-cell adhesion0.0053 GO0007160cell-matrix adhesion0.0003 GO0006935chemotaxis0.0023 GO0005856cytoskeleton0.0002 GO0007010cytoskeleton organisation and biogenesis0.0009 GO0040011locomotion0.0060 GO0005874microtubule0.0003 GO0007018microtubule based movement0.0108 Kegg PathwayDescriptionP-value hsa04514Cell adhesion molecules (CAMs)0.0020 hsa04530tight junction 2.6.10 -7 hsa04520adherens junction 2.5. 10 -6 hsa04540gap junction 3.9. 10 -5 hsa04670transendothelial leukocyte migration <10 -7 hsa04810regulation of actin cytoskeleton <10 -7 hsa04510Focal adhesion <10 -7 Broad Pathway 0.0121 0.0069 0.0011 0.0005 0.0004 1.9. 10 -5 cell_adhesion_h h_ST_Integrin_Signaling_Pathway cell_adhesion_receptor_activity_h SIG_Regulation_of_the_actin_cytoskeleton_by_Rho_GTPases_h cell_adhesion_molecule_activity_h cell_motility_h Biocarta pathways h_lympathwayadhesion and diapedis of lymphocytes0.0012 h_integrinPathwayIntegrin Signaling Pathway0.0015 h_lymphocytePathwayAdhesion Molecules on Lymphocyte0.0012 h_cxcr4PathwayCXCR4 Signaling Pathway0.0006 Lagneaux L. et al, Blood, 1998 Ghia P., Semin Cancer Biol, 2002

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

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. CLL sample collection: Nathalie Meuleman Dominique Bron Philippe Martiat Philippe Herman (St-Pierre Hospital) Alain Kentos (Erasme Hospital –ULB) PhD director: Laurence Lagneaux and ALL MEMBERS of the Laboratory of Experimental Hematology of Pr. Philippe Martiat Thank you for your attention. (Bordet Institute – ULB) Acknowledgments 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.