Old and new immunophenotypic markers in multiple myeloma for discrimination of responding and relapsing patients: The importance of "normal" residual plasma cell analysis

Background Multiple myeloma is an incurable disease characterized by proliferation of clonal malignant plasma cells (CPCs), which can be immunophenotypically distinguished from polyclonal plasma cells (PPCs) by multiparameter flow cytometry (MFC). The utility of PPCs analysis in detecting prognostic and predictive information is still a matter of debate. Methods: we tested the ability of 11 MFC markers in detecting differences in the immunophenotype of CPCs and PPCs among patients in various disease stages; we verified if these markers could be associated with disease stage/response to therapy despite the role of clinical parameters. Results: significant changes in the expression of markers occurred both in CPCs and PPCs. CD58 on PPCs of responding patients was downregulated compared with PPC of relapsing group. Fraction of CD200 expressing PCs was lower in control subjects than in PPCs from MGUS and myeloma groups. CD11a levels of expression on both CPCs and PPCs showed an upregulation in newly diagnosed and relapsing patients versus PCs of controls; CD20 was less expressed on control PCs than on MGUS CPCs and PPCs. CD49d revealed to be advantageous in discrimination of PPCs from CPCs. In our multiple regression model, CD19 and CD49d on CPCs, and CD45, CD58 and CD56 on PPCs maintained their association with groups of patients independently of other prognostic variables. Conclusions: we provide a feasible start point to put in order ranges of expression on PPCs in healthy and myeloma subjects; we propose a new approach based on PPC analysis to monitor the stages of the disease

Very Late Antigen-4 (α<inf>4</inf>β<inf>1</inf> Integrin) Targeted PET Imaging of Multiple Myeloma

Biomedical imaging techniques such as skeletal survey and 18F-fluorodeoxyglucose (FDG)/Positron Emission Tomography (PET) are frequently used to diagnose and stage multiple myeloma (MM) patients. However, skeletal survey has limited sensitivity as it can detect osteolytic lesions only after 30-50% cortical bone destruction, and FDG is a marker of cell metabolism that has limited sensitivity for intramedullary lesions in MM. Targeted, and non-invasive novel probes are needed to sensitively and selectively image the unique molecular signatures and cellular processes associated with MM. Very late antigen-4 (VLA-4; also called α4β1 integrin) is over-expressed on MM cells, and is one of the key mediators of myeloma cell adhesion to the bone marrow (BM) that promotes MM cell trafficking and drug resistance. Here we describe a proof-of-principle, novel molecular imaging strategy for MM tumors using a VLA-4 targeted PET radiopharmaceutical, 64Cu-CB-TE1A1P-LLP2A. Cell uptake studies in a VLA-4-positive murine MM cell line, 5TGM1, demonstrated receptor specific uptake (P<0.0001, block vs. non-block). Tissue biodistribution at 2 h of 64Cu-CB-TE1A1P-LLP2A in 5TGM1 tumor bearing syngeneic KaLwRij mice demonstrated high radiotracer uptake in the tumor (12±4.5%ID/g), and in the VLA-4 rich organs, spleen (8.8±1.0%ID/g) and marrow (11.6±2.0%ID/g). Small animal PET/CT imaging with 64Cu-CB-TE1A1P-LLP2A demonstrated high uptake in the 5TGM1 tumors (SUV 6.6±1.1). There was a 3-fold reduction in the in vivo tumor uptake in the presence of blocking agent (2.3±0.4). Additionally, 64Cu-CB-TE1A1P-LLP2A demonstrated high binding to the human MM cell line RPMI-8226 that was significantly reduced in the presence of the cold targeting agent. These results provide pre-clinical evidence that VLA-4-targeted imaging using 64Cu-CB-TE1A1P-LLP2A is a novel approach to imaging MM tumors. © 2013 Soodgupta et al

Rationally engineered nanoparticles target multiple myeloma cells, overcome cell-adhesion-mediated drug resistance, and show enhanced efficacy in vivo

In the continuing search for effective cancer treatments, we report the rational engineering of a multifunctional nanoparticle that combines traditional chemotherapy with cell targeting and anti-adhesion functionalities. Very late antigen-4 (VLA-4) mediated adhesion of multiple myeloma (MM) cells to bone marrow stroma confers MM cells with cell-adhesion-mediated drug resistance (CAM-DR). In our design, we used micellar nanoparticles as dynamic self-assembling scaffolds to present VLA-4-antagonist peptides and doxorubicin (Dox) conjugates, simultaneously, to selectively target MM cells and to overcome CAM-DR. Dox was conjugated to the nanoparticles through an acid-sensitive hydrazone bond. VLA-4-antagonist peptides were conjugated via a multifaceted synthetic procedure for generating precisely controlled number of targeting functionalities. The nanoparticles were efficiently internalized by MM cells and induced cytotoxicity. Mechanistic studies revealed that nanoparticles induced DNA double-strand breaks and apoptosis in MM cells. Importantly, multifunctional nanoparticles overcame CAM-DR, and were more efficacious than Dox when MM cells were cultured on fibronectin-coated plates. Finally, in a MM xenograft model, nanoparticles preferentially homed to MM tumors with ∼10 fold more drug accumulation and demonstrated dramatic tumor growth inhibition with a reduced overall systemic toxicity. Altogether, we demonstrate the disease driven engineering of a nanoparticle-based drug delivery system, enabling the model of an integrative approach in the treatment of MM

Characterization of Cyclin E Expression in Multiple Myeloma and Its Functional Role in Seliciclib-Induced Apoptotic Cell Death

Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator – cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator - cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion–mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy

Utility of CD54, CD229, and CD319 for the identification of plasma cells in patients with clonal plasma cell diseases

Background: Multiparameter flow cytometry (MFC) identification and characterization of plasma cells (PCs) is a useful tool to support diagnosis, prognostication, and monitoring of PC diseases (PCD). Currently, the number of MFC markers suited for the identification of PC remains limited. Moreover, antibody therapies against PC-associated markers further compromise the utility of the most widely used reagents (e.g., CD38). Despite markers other than CD38 and CD138 are recognized as potentially useful PC-identification markers, no study has comparatively evaluated their performance in combination with CD38 and CD138. Here we compared the utility of CD229, CD54, and CD319 for the identification of normal and aberrant PCs. Methods: Bone marrow (BM) samples from 5 healthy controls, two noninfiltrated nonHodgkin lymphoma cases and 46 PCD patients plus 3 extraosseous plasmocytomas, and normal peripheral blood (PB) specimens, were studied. Results: Our results showed adequate performance of all three markers once combined with CD38. In contrast, when combined with CD138 for the identification of PC, only CD229 provided a good discrimination between PCs and all other cells for all BM and PB samples analyzed; in contrast, CD54 and CD319 showed limited utility for the identification of PCs, mainly because of significant overlap of the staining for these two markers on PCs and other myeloid cells in the sample. Conclusions: From the three markers evaluated, CD229 may be considered as the most reliable marker to replace CD38 or CD138 for the identification of PCs in patients undergoing anti-CD38 or anti-CD138 therapy, until a better alternative is available. (C) 2015 International Clinical Cytometry Societ