Abstract 4034


Multiple myeloma (MM) is a monoclonal gammopathy characterized by the uncontrolled proliferation of plasma cells (PCs). The lack of knowledge about MM cell biology compared to normal PCs is hindering the discovery of myeloma specific targeted therapeutics. Current therapeutics target broad cellular functions such as suppression of the bone marrow environment, myeloma cell proliferation and induction of apoptosis. The objective of our study was to determine biomarkers of the disease and identify new potential targets for future therapeutics, and therefore increase treatment options for MM. We utilized quantitative proteomics using an iTRAQ-based approach to identify biomarkers that can distinguish between MM and normal PCs.


Tonsil tissues, removed from patients suffering from sleep apnea syndrome who consented for tissue repository, were the source of normal PCs. First, the tonsil cells were depleted of T-cells, granulocytes and macrophages using RosetteSep® antibody cocktail and, subsequently, CD138+ PCs were isolated by EasySep® magnetic bead selection. Bone marrow aspirates from MM patients who consented for IRB-approved MM repository protocol, were enriched for PCs with RosetteSep® antibody cocktail. PC percentage for purity assessment was performed by Wright-Giemsa staining of cytospin preparations. PCs (250,000) were lysed and proteomic profiles were generated by iTRAQ 4-plex methods where 2 tonsil PCs (TPC) and 2 MM plasma cells (MMPC) were in each 4-plex. After labeling with iTRAQ tags, the proteins were fractionated by cation exchange chromatography followed by LC-MS/MS analysis on a MALDI-TOF/TOF analyzer. The data were analyzed and quantification performed using ProteinPilot software. Real time PCR of cDNA from TPC and two independent MMPC samples was performed to validate the results.


We consistently obtained 100–250,000 normal PCs from each tonsil sample, at a purity of >80%. To obtain reliable data from proteomics we required >200,000 cells and therefore tonsil pools were utilized wherever necessary. Three types of MM patient samples were studied: newly diagnosed MM, relapsed MM and plasma cell leukemia. We detected and quantified 848 proteins with high confidence from three 4-plex iTRAQ experiments (FDR <5%). Proteins were determined as differentially expressed (MMPCs vs TPCs) if 5 of 6 MMPCs showed difference in expression in 3 independent iTRAQ experiments, or all 4 of 4 MMPCS in two experiments. We identified 11 proteins that qualified as differentially expressed in MMPCs vs TPCs, irrespective of MM subset. Of the 11 proteins, 3 were downregulated and 8 were upregulated in MMPCs. The differential expression of 7 proteins, considered possibly relevant in PC biology, was validated at the mRNA level by real time PCR assay. Two proteins, Clu1 (clusterin) and Basp1 (brain acid soluble protein 1, Nap-22) were expressed at lower levels in MMPCs. Their down-regulation was validated in two independent MM samples by real time PCR. These two proteins also enriched a network (P=1.24E-6, z score=46.43) identified in GeneGo Metacore platform for pathway analysis. This network showed that clusterin and Basp1 might play a role in pathways that regulate pro-apoptotic proteins Bax and Bak, which are in turn regulated by c-Myc, a key transcription factor that controls growth of myeloma cells. Validation by immunoblotting for the biomarkers identified is in progress.


We have successfully isolated a sufficient number of PCs from tonsils to compare proteomic profiles of tonsilar PCs, from subjects without malignancy, with PCs from bone marrows of MM patients. Our analysis has identified clusterin and Basp1 as proteins that are differentially expressed between TPCs and MMPCs, and therefore might play a role in disease physiology. Regulatory pathways identified in this study might be candidates for myeloma-specific therapeutic intervention.

This study was partly supported by a grant from the Multiple Myeloma Research Foundation.


Jakubowiak:Millennium, Celgene, Bristol-Myers Squibb, Johnson & Johnson Ortho-Centocor: Honoraria; Millennium, Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millennium, Celgene, Centocor-Ortho Biotech: Speakers Bureau.

Author notes


Asterisk with author names denotes non-ASH members.