Gene expression profiling

Workpackage 5

Objectives

We will work on the hypothesis that the MSC is a P-MSC, included in the minor fraction of proliferating multiple myeloma cells (MMC) in vivo or in the minor fraction of peripheral blood MM. The objective is to compare the gene expression profiling (GEP) of the fraction of MMC that proliferate in vivo (CD45+CD11a+ MMC), with that of CD45-CD11a- MMC, and with that of circulating MMC, and with those of normal plasmablasts and mature bone marrow plasma cells. We will also determine the GEP of bone cells that may form the MSC niche, osteoclasts and mesenchymal stem cells.

Description of work

Purification of CD45+CD11a+ MMC and of CD45-CD11a- MMC and of circulating MMC from 20 newly diagnosed patients and collection of samples of bone cells using a FacsAria. For each patient all the clinical, biological and cytogenetics characteristics will be collected in the shared data base. Bone marrow plasma cells, normal plasmablasts and memory B cells, osteoclasts and bone marrow mesenchymal stem cells (10 samples each) will be purified.

Identification of the gene signatures of plasmablastic and mature MMC. We will use the same approach we recently described for characterizing TACIhigh and TACIlow MMC (Moreaux, Blood, 2005). The availability of GEP of normal plasmablasts and mature bone marrow plasma cells will make it possible to look for whether, for a same patient, the CD45+CD11a+ MMC or circulating MMC have a plasmablastic signature and the CD45-CD11a- MMC a mature plasma cell one, in particular regarding cell cycle genes. We will determine the differentially expressed genes between CD45+ and CD45- MMC coding for the main cell-to-cell communication factors proteins that may be associated with interactions with osteoclasts and mesenchymal stem cells: chemokines/chemokines receptors, adhesion molecules, metalloproteinases, receptors with protease activity, MMC growth factors and other factors that can alter the bone marrow microenvironment. We will also look to the genes coding for intracellular signalling pathways. This analysis will make it possible to determine whether, in a same patient, the CD45+CD11a+ MMC or circulating MMC have the characteristic of a P-MSC, defined by an increased proliferation rate, and a specific homing close to bone marrow mesenchymal stem cells and osteoclasts. We will point out the genes coding for membrane proteins or signalling pathways that may suggest novel possibilities to target the P-MSC.

Prognostic value of remarkable genes. Using a large series of GEP of purofied MMC from 300 newly-diagnosed patients treated with autologous stem cell transplantation, we will investigate the clinical impact of the remarkable genes picked up in this research project. Thses genes may encode for putative new therapeutic targets.

Deliverables

• Determination of a gene signatures discriminating plasmablastic and mature myeloma cells and linked with the bone cell niche. 24 months.
• Determination of genes linked with patient's response to high dose chemotherapy and stem cell transplantation. 12 months at the end of the project.
• Will serve as a core facility to perform gene expression profiling with Affymetrix platform. And bioinformatic analyses using web available tools developed by partner 7: Rage, rage.montp.inserm.fr, and Amazonia, amazonia.montp.inserm.fr.

Milestones

• Analysis for stemcell genes in present databases.
• Purification of malignant plasmablasts and mature plasma cells and circulating MMC of 20 newly-diagnosed patients.
• Determination and analysis of the gene expression profiling. Identification of novel genes linked with treatment response and encoding for putative novel therapeutic targets.