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

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

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

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

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

Patient characteristics

Methods Affymetrix Protocole oligonucleotides transcripts human genes blood collectionPBMC gradient isolation Purification with anti-CD19 magnetic beads % purity RNA extraction (1,5µg)

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 _a_atZAP70 2< 1e _atZAP70 Gene symbol ZAP70high mean RatioProbe set Parametric p-value FDR ZAP70low mean qPCR validation

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

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

Gene set enrichment analysis GO categoriesDescriptionP-value GO actin cytoskeleton GO actin cytoskeleton organization and biogenesis GO actin filament-based process GO actine polymerization and/or depolimerization GO cell adhesion GO cell-cell adhesion GO cell-matrix adhesion GO chemotaxis GO cytoskeleton GO cytoskeleton organisation and biogenesis GO locomotion GO microtubule GO microtubule based movement Kegg PathwayDescriptionP-value hsa04514Cell adhesion molecules (CAMs) hsa04530tight junction hsa04520adherens junction hsa04540gap junction hsa04670transendothelial leukocyte migration <10 -7 hsa04810regulation of actin cytoskeleton <10 -7 hsa04510Focal adhesion <10 -7 Broad Pathway 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 lymphocytes h_integrinPathwayIntegrin Signaling Pathway h_lymphocytePathwayAdhesion Molecules on Lymphocyte h_cxcr4PathwayCXCR4 Signaling Pathway Lagneaux L. et al, Blood, 1998 Ghia P., Semin Cancer Biol, 2002

ZAP70 and CXCR4 modulation by ME CXCR4 on fresh samples

Adhesion and migration

MicroRNA expression TFS OS

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

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.