ALBERT

Description: The bone marrow (BM) repopulating potential of hematopoietic stem cells (HSCs) is directly related to the cell cycle status of these cells. In general, only mitotically quiescent HSCs retain the ability to engraft and sustain long-term multilineage reconstitution in conditioned recipients. In a series of studies, our laboratory previously examined the effect of cell cycle status on the engraftment potential of human HSC from three different hematopoietic tissues. Only CD34+ cells in G0 phase of cell cycle (G0CD34+) from adult human BM or mobilized peripheral blood (MPB) engrafted successfully in conditioned NOD/SCID mice whereas those in G1 phase of cell cycle (G1CD34+) failed to do so. In contrast, both G0CD34+ and G1CD34+ cells from cord blood (CB) engrafted effectively. In the present study, we used the distinct in vivo behavior of these groups of adult and neonatal cells as the basis for genotypic and proteomic analyses in which it was possible to align multiple profiles of functional and non-functional HSC and therefore derive a genetic and protein fingerprint that may be associated with Engraftment potential of human stem cells. Human CD34+ cells from BM, MPB, and CB were sorted into G0 and G1 phases of cell cycle and the cell cycle status of each isolated fraction was further confirmed by the expression or lack thereof of Ki67 by qRT-PCR. Agilent Whole Human Genome Oligo Microarrays were used for genotyping (three independent samples from each tissue for a total of 18 groups) and a Linear Mixed Effect Model was used to identify differentially expressed genes, with at least a two-fold increase in expression and false discovery rate

Description: Abstract 2890 Background: Multiple myeloma is an incurable and fatal hematologic malignancy. Recent gene microarray studies showed distinct gene expression profiles defining MM subgroups and their association with cytogenetic abnormalities and treatment outcome. However, aside from transcriptional control, a variety of post-transcriptional/post-translational modifications likely play an important role in regulating protein expression and function, and ultimately may prove informative for predicting tumor behavior. Objectives: We hypothesize that the protein profile in MM cells is different than normal plasma cells. Methodology: Normal plasma cells and myeloma cells were isolated using CD138 immune magnetic beads from bone marrow aspirates from healthy volunteers or patients with newly diagnosed MM, respectively. CD138+ cells were frozen and subsequently analyzed in one batch. Proteins were digested by trypsin. Tryptic peptides were injected onto an HPLC system and analyzed on a Thermo-Fisher LTQ mass spectrometer. Peptide identification and quantification were carried out using proprietary algorithms. Identified proteins were categorized into priority groups based on the quality of the peptide identification by tandem mass spectrometry. Proteins with significant changes in expression level were further analyzed by bioinformatics tools for the determination of the biological significance. Results: In the discovery phase of this study, 433 proteins were identified and their expression levels were quantitatively compared. 169 of these proteins demonstrated a significant difference between normal plasma cells and MM cells. Among the significantly changed proteins, 18 were identified and quantified with high confidence, and were therefore chosen for further validation. The identified proteins are known to be involved in the glycolysis/gluconeogenesis pathway, the oxidative phosphorylation pathway, cysteine metabolism and the pentose phosphate pathway. None of these proteins are known to be of prognostic value or being currently targeted for therapy in MM. A high-throughput LC/MS-based multiple-reaction-monitoring (MRM) assay for quantitative validation of these candidates with clinical samples is ongoing. To date, using the MRM assay, we were able to detect MRM peptides for 13 of the 18 targeted proteins in clinical samples. The quantification of these peptides will be further confirmed using a separate set of clinical samples. Conclusion: Significant differences in protein expression were observed between MM and normal plasma cells. The study presents an important step toward using proteomics as a tool to develop diagnostic and/or prognostic biomarkers in the clinical setting. However, both follow-up analytical and clinical validations are required before they can serve as disease-specific biomarkers. Disclosures: Abonour: Celgene: Membership on an entity's Board of Directors or advisory committees.