ALBERT

Description: Background: Extramedullary disease (EMD), strictly defined as an infiltrate of clonal plasma cells at an anatomic site distant from the bone marrow or adjacent soft tissue in a patient with underlying multiple myeloma, is an uncommon manifestation of multiple myeloma. Comparatively little is known about this disease entity, with no large case series published in the last decade. Patients and Methods: 663 consecutive patients with multiple myeloma who underwent autologous or allogeneic stem cell transplantation at a single, large, academic medical center in the United States from January 2005 to December 2011 were assessed for the presence or absence of EMD, as well as baseline demographic and biochemical characteristics, treatment regimens, and response to therapy. Results: A cohort of 55 patients with biopsy-proven EMD was identified, comprising 8.3% of the total study population. Among the patients with EMD, 13 (23.6%) were found to have EMD at the time of initial presentation, while the remainder developed EMD during the course of their illness. Patients with EMD received a median of 5 different treatment regimens during the course of their illness, most commonly with combinations of dexamethasone, thalidomide, lenalidomide, and bortezomib, as well as autologous hemotopoietic stem cell transplantation. Patients had received a median of 3 lines of therapy prior to experiencing an extramedullary relapse. Patients with EMD had markedly elevated maximum serum LDH levels (median 613.5 units/L) and low minimum hemoglobin levels (median 7.8 g/dL). Common cytogenetic abnormalities included deletion 13q, deletion 11q, t(11;14), and deletion 17p. Available immunohistochemical data suggest that EMD specimens had frequent expression of CXCR4, CD44, and CD56. The median overall survival data of these patients was 3.2 years (range, 0.9-9.5) and the median time from diagnosis of EMD to death was 0.5 years (range, 0.002-3.2). Conclusions: This report describes a large series of multiple myeloma patients with EMD who were treated in the era of stem cell transplant at a single academic medical center. Further studies to examine the molecular characteristics of extramedullary multiple myeloma are necessary to better define this entity and characterize therapeutic options that can prolong survival in this otherwise very vulnerable population. Disclosures Ghobrial: Millennium/Takeda: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Laubach:Novartis: Research Funding; Onyx Pharmaceuticals: Research Funding. Schlossman:Millennium: Consultancy. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Research Funding; Johnson & Johnson: Research Funding; DFCI: patent submission on stromal co-culture technologies Patents & Royalties.

Description: Background Increased bone marrow (BM) microvessel density (MVD) has been associated with progression of multiple myeloma (MM). Endothelial progenitor cells (EPCs) are circulating precursors with the capacity to differentiate into endothelial lineage cells through a process known as “vasculogenesis” thus contributing to vessel formation. The role of these cells in MM pathogenesis remains largely unexplored. We studied EPC BM mobilization in several MM mouse models (5TGM1, MM1S and Vk*MYC) during disease progression and quantified these cells in peripheral blood (PB) from patients at different stages of MM disease. Methods EPCs were quantified using flow cytometry (circulating CD34+ VEGFR2+ cells) in PB from mice injected intravenously (i.v.) with either murine MM 5TGM1-turboRFP+ cells or human MM1S-GFP+/luc+ cells. Circulating EPCs were also enumerated in PB from mice previously transplanted with BM from SCID/GFP mice (GFP-BM SCID mice) after the i.v. injection of 5TGM1-turboRFP+ cells as circulating GFP+ CD34+ VEGFR2+ cells. Peripheral blood samples were obtained from transgenic Vk*MYC mice affected by smoldering (s) MM (M-spike less than 6% of total protein on SPEP); active (a) MM (M-spike higher than 6% of total protein on SPEP); or healthy syngeneic mice, and examined for EPC levels through flow-cytometry (circulating CD34+ VEGFR2+ cells). Finally, the level of EPCs was evaluated in PB from healthy controls, smoldering (s) MM patients, in remission (r) and active (a) MM patients by using flow-cytometry (CD34+VEGFR2+ cells) and in vitro by performing endothelial colony forming assays [endothelial cells colony forming units (EC-CFUs) and endothelial colony forming cells (ECFCs)]. Results An increase in EPCs was evident starting one week after i.v. injection of tumor cells in both murine 5TGM1 and human MM1S orthotopic models. Compared to control mice, this EPC increase became significant (P

Description: Key Points C1013G/CXCR4 acts as an activating mutation in WM leading to enhanced tumor growth, and as an inducer of drug resistance. BMS936564/MDX1338, a novel anti-CXCR4 moAb, successfully targets WM cells, either C1013G/CXCR4 mutated or wild-type.

Description: Key Points LAPTM5 c403t and HCLS1g496a are potentially novel contributors for the genetic predisposition to familial WM. LAPTM5 c403t and HCLS1g496a represent possible candidates for screening in familial WM.

Description: Background Multiple Myeloma (MM) is the second most prevalent hematological malignancy and remains incurable, with a median survival of 3-7 years. However, despite the success of the new treatments, most patients still succumb to their disease. In about 20-25% of high-risk patients, MM progresses rapidly and does not respond to conventional therapies leading to rapid extramedullary disease and demise of these patients. One such regulator of dissemination and drug resistance is the dynamic process of oxygen deprivation or hypoxia. A number of studies show that hypoxia promotes neo-angiogenesis, cancer progression, epithelial-mesenchymal transition (EMT), acquisition of metastasis potential and stem-cell features, as well as resistance to therapy by activating adaptive transcriptional programs. Targeting hypoxia, and the metabolic pathways regulated by hypoxia in the tumor cells, could lead to novel opportunities for cancer therapy. Rapidly proliferating hypoxic cancer cells undergo a “metabolic switch” to anaerobic glycolysis. This altered energy metabolism has been shown to be associated with activated oncogenes and mutant tumor suppressors, which are more prevalent in patients with high-risk MM. We aimed 1) to examine the role of HIF-1a and HIF-2ain regulating drug resistance in vitro and in vivo and 2) to identify specific hypoxia-regulated genes and regulators of energy metabolism leading drug resistance in MM. Methods The effect of hypoxia was analyzed in different MM cell lines (MM1S, RPMI8226, U266 and H929) in basal conditions and after the treatment with bortezomib, dexamethasone or melphalan. The cytotoxicity was analyzed by means of MTT assay. Apoptosis studies were performed by flow cytometry. Gene expression profile of MM1S cells treated with bortezomib was compared in normoxia vs hypoxia using D-chip and GSEA softwares. Genes with expression changes greater or lower than 2 fold in either direction were selected. HIF1A and HIF2A knockdowns were performed in MM1S using lentiviral vectors. For metabolite collection, samples were re-suspended using HPLC grade water for mass spectrometry and analyzed using a 5500 QTRAP hybrid triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu). A total of 254 endogenous water soluble metabolites were analyzed. Results We observed that MM cell lines were resistant to bortezomib and melphalan in hypoxic conditions (12 hours at 0.5% of oxygen levels) compared to normoxic conditions. At transcriptional and protein level. cells treated with bortezomib in hypoxic conditions affected a large number of genes/proteins involved in cell cycle such us p21, p53 and p57, cell death and glucose metabolism. However, cell cycle arrest was not responsible for the resistance of MM cells to bortezomib that was observed in hypoxic conditions. Therefore, we investigated mechanisms that are mediated by hypoxia and can regulate drug resistance. HIF1A knockdown restored the effect of bortezomib in MM1S and increased the percentage of apoptosis in cells treated with bortezomib under hypoxic conditions. To further explore the role of hypoxia in the regulation of tumor metabolism downstream of HIF1A, metabolomic studies were performed to characterize metabolic alterations following bortezomib treatment in hypoxic and normoxic conditions. This analysis revealed that hypoxic tumor cells treated with or without bortezomib show significant metabolic changes involving multiple pathways, the most significant of which are intermediates in glucose metabolism such us glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate and phosphoenolpyruvate. We also observed a decrease in measured tricarboxylic acid cycle (TCA) cycle intermediates (citrate, fumarate and malate) after hypoxia exposure and a significant increase of LDHA levels. We assessed the metabolic response to several drugs and shRNAs targeting different glycolytic enzymes (HK2, PFKBP3, PFKBP4 and LDHA). Of these, the most significant changes were observed with LDHA knockdown where these overcame resistance to bortezomib in hypoxic conditions. Conclusion Hypoxic conditions are essential for drug resistance and glucose utilization. These data provide new therapeutic targets and associated biomarkers for the treatment of Multiple Myeloma. Disclosures: Ghobrial: Onyx: Membership on an entity’s Board of Directors or advisory committees; BMS: Membership on an entity’s Board of Directors or advisory committees; BMS: Research Funding; Sanofi: Research Funding; Novartis: Membership on an entity’s Board of Directors or advisory committees.

Description: Introduction. Waldenström Macroglobulinemia (WM) is a low- grade B-cell lymphoma with a heterogeneous clinical presentation. In many patients, the diagnosis is preceded by an asymptomatic precursor state of IgM monoclonal gammopathy of undetermined significance (MGUS) and smoldering WM. However, little is known about the mechanisms involved in the progression from asymptomatic WM to symptomatic WM. Exosomes are small vesicles secreted by cells that enable the transfer of nucleic acids, proteins and lipids between distant cells in the organism. Because of the active role of exosomes in promoting tumor growth and metastasis, we investigated the microRNA content of circulating exosomes in patients at different stages of WM in order to identify possible markers of progression. Methods.We isolated exosomes by ultracentrifugation from the peripheral blood plasma of 101 patients with WM and 10 normal donors. The WM cases included 7 patients with IgM MGUS, 42 patients with smoldering WM and 52 patients with symptomatic WM. We first profiled microRNAs from the exosomes of 14 patients and 5 normal controls using the TaqMan Array Human MicroRNA A Card (Thermo Fisher Scientific) which enables the quantitation of 377 human microRNAs. Exosomes from an additional group of 14 individuals (2 normal donors, 8 patients with asymptomatic WM and 4 patients with symptomatic WM) were profiled with the Oncology Panel of the Firefly Multiplex Circulating miRNA Assay (Abcam), which measures the expression level of 68 different human microRNAs. Following these analyses, a group of 33 microRNAs deregulated between patients' groups was selected and a custom microRNA panel was built to allow quantitation of these microRNAs with the Firefly Multiplex Circulating miRNA Assay (Abcam). The level of expression of these 33 microRNAs was measured in a group of 80 individuals comprised of healthy controls (n=8), patients with asymptomatic WM including IgM MGUS and smoldering WM (n=34) and patients with previously untreated symptomatic WM (n=11), relapsed WM (n=21) and refractory WM (n=6). Additionally, exosomes were imaged using electron microscopy with immunogold labeling for the detection of the exosome-specific receptors CD63 and CD81. Results. Imaging with electron microscopy showed vesicles with a size ranging from 50 to 150 nm and expressing CD63 and CD81, thus validating our ultracentrifugation method for exosome isolation. Among the 33 microRNAs analyzed, 12 were significantly deregulated between WM patients and healthy controls (P

Description: Introduction: Massive parallel sequencing of tumor cells obtained from the bone marrow (BM) of patients with multiple myeloma (MM) has demonstrated significant clonal heterogeneity with a median of five clones present in each sample. However, it could be envisioned that such clonal diversity may be even higher since single BM samples only represent a small fraction of the whole BM compartment, and the pattern of BM infiltration in MM is typically patchy. Accordingly, it remains unknown whether using liquid biopsies (i.e.: patients' genetic characterization performed in peripheral blood -PB- samples) can provide a more complete profile of MM clonal diversity. Moreover BM biopsies and cannot be repeated multiple times during the course of therapy, indicating a need for less invasive methods to genomically characterize MM patients. We aimed to determine the overall applicability of performing genomic characterization of MM patients non-invasively, and define if the mutation profile of circulating tumor cells (CTCs) reflected that of patient-paired BM clonal PCs. Methods: We performed CTC enumeration using multiparameter flow cytometry (MFC) in 50 newly-diagnosed patients with symptomatic MM who were prospectively enrolled on the Spanish clinical trial PETHEMA/GEM2010MAS65 as well as 64 patients with MM with relapsed disease or in remission/on maintenance therapy seen at the Dana-Farber Cancer Institute. For whole exome sequencing studies, we obtained 8 samples of newly-diagnosed untreated patients whose bone marrow, CTC and germline T lymphocytes were available and selected for exome sequencing. We sequenced the whole exome of BM clonal PCs and CTCs up to 200x, and germline cells up to 50x. Whole genome amplification (WGA) was performed for CTCs, and two independent libraries were constructed from the sample, followed by sequencing up to 100x for each duplicate. For samples with WGA, only single nucleotide variants (SNVs) shared in both parallel libraries were used. Results: Before investigating if CTCs could represent a reliable non-invasive alternative to perform genomic characterization of MM patients, we first aimed to define its true applicability at different disease stages. Using sensitive MFC, we showed that CTCs were detectable in 40/50 (80%) newly-diagnosed MM patients, and in 71/130 (55%) of multiple sequential samples from patients with relapsed disease or in remission/on maintenance. As for the prognostic value of CTC enumeration, 19 of the 40 newly-diagnosed cases displaying PB CTCs had relapsed (median time-to-progression of 31 months); by contrast, only 1 of the 10 patients with undetectable CTCs has relapsed (median time-to progression not reached; P=.08). Afterward, we investigated whether dynamic changes in the kinetics of CTCs in sequential PB samples from patients with relapsed disease or in remission/on maintenance therapy was also predictive of outcome. Accordingly, increasing CTC counts were associated with poor overall survival (P= .01), indicating that both the absolute numbers of CTCs and trend of CTC are predictive of outcome in MM. After demonstrating that CTCs can be readily detected in the majority of MM patients, we then determined the mutational profile of CTCs and compared it to that of patient-paired BM clonal PCs. We identified a median of 223 and 118 SNVs in patient-paired BM clonal PCs and CTCs, respectively. The concordance of somatic variants found in matched BM clonal PCs and CTCs was of 79%. Noteworthy, upon investigating specific mutations implicated in MM (eg. KRAS, NRAS, BRAF) a total of 18 nonsynonymous SNVs (NS-SNVs) in 13 genes were identified in our cohort, and most of these NS-SNVs were simultaneously detected in matched BM clonal PCs and CTCs from the same patients. That notwithstanding, we also identified several unique mutations present in CTC or BM clonal PCs; of those, up to 39 NS-SNV were identified as CTC specific, and 6 NS-SNVs in 4 genes (CR1, DPY19L2, TMPRSS13, HBG1) were detected in CTC from multiple patient samples. A significant concordance for the pattern of copy number variations (CNVs) between matched BM and PB tumor cells was also observed. Conclusion: This study defines a new role for CTCs in the prognostic and molecular profiling of MM patients, and provides the rational for an integrated flow-molecular algorithm to detect CTCs in PB and identify candidate patients for noninvasive genomic characterization to predict outcomes. Disclosures Paiva: Sanofi: Consultancy; Millennium: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Binding Site: Consultancy; BD Bioscience: Consultancy; EngMAb AG: Consultancy. Richardson:Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Laubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Schlossman:Millennium: Consultancy. San Miguel:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction Disease assessment in Waldenstrom’s Macroglobulinemia (WM) is dependent on percent involvement of B-cell neoplasm in bone marrow and immunoglobulin M (IgM) paraprotein in the serum. A subset of patients also demonstrate extramedullary involvement and is infrequently examined. Its role in diagnosis and prognosis of WM is poorly understood. In this study, we sought to understand the role of extramedullary disease (EMD) in patients with WM. Methods Database for patients seen at Dana-Farber Cancer Institute was searched and 985 documented WM cases were identified between June 1994 and April 2013. Medical charts were reviewed for patients who had biopsy of one or more extramedullary sites during the course of disease. Involvement of lymph nodes, spleen and amyloid deposits were excluded. Results Among the 985 WM patients screened, 50 (5%) had evidence of extramedullary involvement during the course of disease. 28 (56%) were male and 22 (44%) were female, with median age of 57 years (min 37.8; max 71.1) at the time of diagnosis. At diagnosis, 11 (22%) patients were asymptomatic with monoclonal gammopathy of unknown significance (MGUS) or smoldering WM, 21 (42%) were low risk, 15 (30%) were intermediate risk, and 3 (6%) were high risk based on the Morel ISS-WM study (ISS-WM). Laboratory data included median beta-2 microglobulin of 3.7 g/dL (min 1.26; max 10.9), IgM of 3.3 g/dL (min 0.4; max 10.1), and percent bone marrow involvement of 37% (min 5%; max 90%). At the time of data collection, one patient was previously untreated, 14 (28%) had 1 line of therapy, 9 (18%) had 2 lines, 9 (18%) had 3 lines, 8 (16%) had 4 lines, and 9 (18%) had 5 or more lines of therapy. Among the 50 patients identified with EMD, 12 (24%) patients presented with involvement at diagnosis, while 38 (76%) developed EMD after receiving therapy. One patient had EMD at diagnosis, which continued post-diagnosis. 7 patients were identified with multiple sites of involvement during the course of disease. Overall, 60 extramedullary samples were noted with 47 (78%) occurring post-therapy. Extramedullary sites involved included: Pulmonary (n=14; 23%), Soft tissue (n=12; 20%), Cerebrospinal fluid (n=10; 17%), Renal (n=5; 8%), Bone (n=4; 7%), Peripheral blood (n=3; 5%), Neck mass (n=3; 5%), Skin (n=2; 3%), Breast (n=1; 2%), Conjunctiva (n=1; 2%), Liver (n=1; 2%), Gallbladder (n=1; 2%), Small Bowel (n=1; 2%), Prostate (n=1; 2%), and Colon (n=1; 2%). Immunophenotypic data was obtained for 58 of the 60 extramedullary samples and was analyzed by flow cytometry (n=24), immunohistochemistry (n=17), or both (n=17). All samples were of B-cell lineage (CD19+, CD20+). Monotypic surface immunoglobulin kappa or lambda light chain was analyzed in 49 samples, of which 43 were kappa (88%) and 6 were lambda (12%). In the group of 38 patients that presented with EMD post-therapy median time to EMD presentation from diagnosis was 75.7 months (min 1.5; max 213.7 or approximately 6 years) with a median of 2 lines of treatment (min 1; max 6). Therapies prior to EMD presentation in this group included rituximab based regimen in 31 of 38 cases (82%), cyclophosphamide based regimen in 15 cases (39%), fludarabine/cladribine based regimen in 12 cases (32%), and bortezomib based regimen in 11 cases (29%). Treatments for EMD in both groups included bendamustine based regimen resulting in minimal response (MR) or better in 8 of 9 cases (89%), rituximab based regimen resulting in MR or better in 24 of 30 cases (80%), cyclophosphamide based regimen resulting in MR or better in 11 of 14 cases (79%), and bortezomib based regimen resulting in MR or better in 6 of 8 cases (75%). At the time of data collection, 32 of 50 patients were alive (64%), 10 patients were lost to follow-up (20%), and 8 patients died due to progressive disease (16%). Conclusion This is the first description and analysis of the EMD in WM as a clinical entity and further studies in understanding the molecular mechanisms that govern EMD in WM should be examined. Disclosures: Treon: Millennium: Consultancy. Ghobrial:Onyx: Membership on an entity’s Board of Directors or advisory committees; BMS: Membership on an entity’s Board of Directors or advisory committees; BMS: Research Funding; Sanofi: Research Funding; Novartis: Membership on an entity’s Board of Directors or advisory committees.

Description: Background Clonal evolution involves simultaneous evolution of multiple co-existant subclones. Recent studies have suggested that clonal heterogeneity is critical during the progression of Multiple Myeloma (MM). Circulating tumor cells (CTCs) have been identified in many patients with solid tumors and hematological malignancies. Recent studies have suggested that CTCs can be identified in patients with Multiple Myeloma. The aims of this study were to identify the phenotypic characteristics of CTCs in patients with Mutliple Myeloma at different stages of the disease, to determine whether somatic mutations present in the bone marrow clones are also identified in CTCs or whether specific subclones are more prone to enter the systemic circulation. These subclones may have a higher likelihood of inducing dissemination into extramedullary sites and potential for drug resistance. Methods We analyzed the peripheral blood samples of 466 patients diagnosed with Multiple Myeloma at different stages of progression. Two plasma cell leukemia patients were included in the study. Freshly collected peripheral blood was processed to obtain white blood cell fractions. The cells were stained with eight antibodies including CD19, CD38, CD138, CD45, CD56, CD28, CD44, and CD183 and CTCs were purified by gating on CD19-/CD38+/CD138+ cells. Among them, ten of the CTC samples were selected to analyze somatic mutation using whole exome sequencing. Briefly, 1µg of genomic DNA was extracted form sorted cells followed by shearing, end repair, and ligation to barcoded adaptors. The DNA was size-selected, subjected to exonic hybrid capture and sequenced on Illumina HiSeq flow cells with an average depth of coverage of 100x. Results Of the 466 samples analyzed, the number of CTCs identified ranged from 0.01% to 61% of total WBC count. CTCs were detected in 61.4% of all samples analyzed. CTCs were detected in 64.5% of relapsed MM, 63.4% of newly diagnosed MM, 24.0% of smoldering MM, and 25.0% of MGUS patients. Significant differences of the surface markers including CD45, CD28, CD56, and CD44 were not observed in the different stages of MM disease progression. For further characterization of CTCs, we performed whole exome sequencing of CTCs in 10 MM samples, of which 5 had sequencing of their matched tumor cells collected from BM as well as matched normal germline cells to examine whether circulating tumor cells possess any distinctive somatic mutations. The sequence analysis revealed that both CTCS and marrow restricted tumor cells have substantial numbers of protein-coding mutations. CTCs and bone marrow cells shared 5-38% similar mutations, while interestingly the rest of the mutations were exclusively present in either the CTCs or bone marrow samples. We identified a total of 347 somatic mutations, which included 199 CTC specific mutations. Several known driver mutations were observed, i.e. BRaf V600E mutation present in the CTC samples but not in the matching bone marrow samples in one patient. Twelve of these CTC mutations were shared at least in two patients including ZNF721, NBPH10, F5, and PRDM15. Conclusion These data suggest subclonal out growth of CTCs from one of the parent clones with acquisition of additional mutation over time outside of the bone marrow microenvironment. Further validation of the unique mutations in CTCs may provide mechanistic insight into myeloma cell dissemination, and so potentially inform treatment strategies. Disclosures: Tai: Onyx: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership. Munshi:Celgene Corporation: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Millennium: The Takeda Oncology Company: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Onyx Pharmaceuticals Inc: Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Noxxon: Research Funding; BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisoryboard Other; Sanofi: Research Funding.

Description: Background. Endothelial progenitor cells (EPCs) are circulating precursors with the capacity to differentiate into endothelial lineage cells through a process known as “vasculogenesis” thus contributing to vessel formation. We studied the role of endothelial progenitor cells in multiple myeloma (MM) pathogenesis. Methods. EPC levels were evaluated in peripheral blood (PB) of patients with smoldering MM (SMM), and active myeloma (MM) and in PB of healthy controls, by using flow-cytometry (CD34+VEGFR2+ cells), and by performing endothelial colony forming assays. EPC levels were studied in PB from transgenic Vk*MYC mice harboring either early MM (smoldering-like stage); or late (active MM-like stage); as well as in mice injected intravenously with either murine MM 5TGM1-turboRFP+ cells or human MM1S-GFP+/luc+ cells. Healthy syngeneic mice were used as controls. GFP-bone marrow (BM) transplantation and sub-cutaneous femur implantation were performed in recipient mice to study EPC BM mobilization (GFP+CD34+VEGFR2 cells) and EPC BM recruitment to the implanted femurs during 5TGM1 MM model progression. Transgenic ID1+/-ID3-/- mice (with a defect in EPC mobilization and differentiation ability) were injected with transplantable Vk*MYC cells and followed for survival. Wild type littermates were used as controls. Similar experiments were performed using transgenic ID1+/-ID3-/- mice transplanted with BM of wild type littermates. Therapeutic activity of DC101 anti-murine VEGFR2 Ab was evaluated in the MM1S human orthotopic xenograft model, using both early and late treatment approaches. Results. EPC levels were significantly increased in PB of MM patients, including SMM, compared to healthy controls (P