Peripheral Blood Of Multiple Myeloma Patients Research Articles

[Significance of Tim-3 and Its Ligand Galectin-9 in Th1/Th2 Imbalance in Patients with Multiple Myeloma].

To investigate the significance of Tim-3 and Galectin-9 in Th1/Th2 imbalance in patients with multiple myeloma (MM). 55 newly diagnosed MM patients and 20 healthy controls were included. Flow cytometry was used to detect the expression of Tim-3 on CD4+T cells, the proportion of Th1, Th2, Tim-3+Th1 and Tim-3+Th2 cells in peripheral blood. ELISA was used to detect the levels of cytokines IFN-γ and IL-4 in serum, and PCR was used to detect the level of Galectin-9 mRNA. Then the correlations between Galectin-9 mRNA expression and Th-cell subsets and related cytokine levels, as well as the relationship between Tim-3+Th1/Tim-3+Th2 ratio and corresponding clinical features were analyzed. Compared with the control group, the expression of Tim-3 on CD4+T cells in peripheral blood of MM patients was significantly increased (P<0.05), the proportions of Tim-3+Th1 cells, Tim-3+Th2 cells and Tim-3+Th1/Tim-3+Th2 ratio in MM patients were also increased (P<0.05), while the proportion of Th1 cells and Th1/Th2 ratio in MM patients were significantly decreased (P<0.05). The level of cytokine IFN-γ and IFN-γ/IL-4 ratio in MM patients were significantly decreased (P<0.05), while the level of cytokine IL-4 was increased (P<0.05). The mRNA levels of Galectin-9 in MM patients were significantly increased (P<0.05). The levels of Galectin-9 mRNA were positively correlated with Tim-3+CD4+T cells (r=0.663), Tim-3+Th2 cells (r=0.492) and IL-4 (r=0.470), while negatively correlated with IFN-γ (r=-0.593). The ratios of Tim-3+Th1/Tim-3+Th2 in MM patients were positively correlated with ISS stage (r=0.511), osteolytic damage (r=0.556) and chromosome abnormality (r=0.632). These results suggest that Tim-3 and Galectin-9 are involved in Th1/Th2 imbalance in MM patients, and the high ratio of Tim-3+Th1/Tim-3+Th2 is associated with poor clinical prognosis.

Heme Attenuates T Cell Exhaustion and Drives Effector Function: Implications for Immune and Adoptive T Cell Therapy

Introduction: Though frontline agents have been effective in multiple myeloma (MM) therapy, a significant proportion of MM patients relapse and succumb to refractory disease. B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T therapy represents an exciting novel therapeutic strategy for MM but still suffers from limited in vivo T cell expansion and CAR T cytotoxicity. Altering the CAR T manufacturing process to produce CAR T cells with less exhausted phenotypes and robust killing is an active area of research. Mitochondrial function and energetics play a central role in regulating T cell fate and function, and heme (iron-protoporphyrin IX) is critical for maintaining electron transport chain activity and oxidative phosphorylation. Heme can be sourced from the extracellular milieu, diet, or intrinsic heme biosynthesis and has a multitude of cellular functions throughserving as a cofactor for enzymatic and metabolic reactions. Additionally, heme is catabolized by heme oxygenase 1, producing the antioxidants biliverdin and bilirubin critical to preserving cellular redox homeostasis. In the present study, we investigate the effects of heme on CAR T cell efficacy. Our results suggest that heme supplementation during ex vivo CAR T manufacturing or supplementation during CAR T-tumor cell engagement can lead to decreased T cell exhaustion and increased tumor killing. Methods: Human healthy donor and MM patient T cells were activated and expanded with α-CD3/CD28 and IL-2 for 7 days +/- hemin, after which immunostaining and metabolic assays were conducted. For CAR T manufacture, T cells were transduced with CAR construct starting on day 3 and were co-cultured with antigen-expressing tumor cells in vitro on day 7-8. Cytotoxicity and cytokine production were assayed after 24 hours of co-culture. Results: In a dose-dependent manner, heme supplementation significantly decreases proportions of LAG3 + and TIM3 + and decreases proportions of CD62L + and TCF1 + cells in both CD4 + and CD8 + subsets isolated from healthy donor and MM patient peripheral blood. Supportive of these observations, inhibition of de novo heme biosynthesis leads to the opposite effect with an increase in proportions of PD1 +, LAG3 +, TIM3 +, and CD62-L + cells. CAR T cells manufactured with heme (heme-CAR T cells) have significantly reduced terminally exhausted phenotypes (PD1 +/LAG3 +/TIM3 +) and increased enrichment of effector memory cells (CD62L -CD45RA -) in both CD4 + and CD8 + subsets. Inquiry of cellular energetics of heme supplemented CAR T cells reveals reduced reactive oxygen species (ROS) and increased mitochondrial membrane potential, consistent with previous reports showing increased membrane potential in effector T cells for greater production of cytokines such as IL-17A, IL-17F, and IFNγ. In co-culture with tumor cells in vitro, heme-CAR T cells have higher tumor-specific lysis and production of TNFα and IL-2. CAR T-tumor cell engagement in the presence of heme resulted in significantly decreased proportions of exhausted CAR T cells and increased production of TNFα. Heme also increases BCMA surface expression in several MM cell lines, highlighting a viable strategy for enhancing BCMA CAR T engagement with MM. Lastly, MM patient bone marrow-localized T cells demonstrate significantly decreased terminally exhausted phenotypes and increased differentiation into effector subsets when expanded with heme. Conclusion: We show that heme increases effector T and CAR T cell function and decreases T cell exhaustion. Increased cytotoxicity of heme-supplemented CAR T cells was associated with reduced ROS and increased membrane potential. Ongoing in vivo studies aim to characterize the efficacy of heme-supplemented CAR T cells in MM xenograft models. Our studies have broader implications for investigating heme supplementation in ex vivo CAR T manufacturing for a wide range of hematological cancers and underscore the need for further investigation of heme uptake and metabolism for strategic enhancement of T-cell based immunotherapies.

[Effect of LINC00174 on the Malignant Proliferation of Multiple Myeloma Cells and Its Related Mechanism].

To explore the biological function of LINC00174 in multiple myeloma (MM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of LINC00174 and miR-150 in peripheral blood of MM patients and MM cell lines. EdU staining and flow cytometry were used to detect the effects of LINC00174 and miR-150 on the proliferation and apoptosis of MM cells. Western blot was used to detect the expressions of proliferation marker nuclear-related antigen Ki67, apoptosis-related protein cleaved caspase-3 and transcription factor forkhead box protein P1 (FOXP1). Bioinformatics and dual-luciferase reporter assay were used to verify the targeting relationship between LINC00174 and miR-150 and the targeting relationship between miR-150 and FOXP1. The level of LINC00174 was significantly increased in peripheral blood of MM patients and MM cell lines (P <0.05). Compared with NC-siRNA group, the expression of LINC00174 was significantly reduced in LINC00174-siRNA group, the proliferation of U266 cells was reduced, the apoptosis rate was significantly increased, the level of Ki67 protein was reduced, and the level of cleaved caspase-3 protein was increased (all P <0.05). LINC00174 targeted regulation of the expression of miR-150. Compared with LINC00174-siRNA+NC inhibitor group, the expression of miR-150 in U266 cells in LINC00174-siRNA+miR-150 inhibitor group was significantly reduced, the cell proliferation was enhanced, the apoptosis rate was reduced, the level of Ki67 protein was increased, and the level of cleaved caspase-3 was decreased (all P <0.05). FOXP1 is the target gene of miR-150. Compared with NC mimic group, the expression of FOXP1 protein in miR-150 mimic group was significantly reduced, the cell proliferation was reduced, the apoptosis rate was significantly increased, Ki67 protein level was decreased, and the level of cleaved caspase-3 was increased. Compared with miR-150 mimic + vector group, the expression of FOXP1 protein in miR-150 mimic + pcDNA-FOXP1 group was significantly increased, the cell proliferation was enhanced, the apoptosis rate was reduced, the level of Ki67 protein was increased, and the level of cleaved caspase-3 was decreased (all P <0.05). LINC00174 promotes the proliferation of MM cells and inhibits cell apoptosis by regulating the miR-150/ FOXP1 axis.

Characterization of monoclonal gammopathy of undetermined significance progression to multiple myeloma through meta-analysis of GEO data

The etiology of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) is still obscure as are the processes that enable the progression of MGUS to MM. Understanding the unique vs. shared transcriptomes can potentially elucidate why individuals develop one or the other. Furthermore, highlighting key pathways and genes involved in the pathogenesis of MM or the development of MGUS to MM may allow the discovery of novel drug targets and therapies.We employed STARGEO platform to perform three separate meta-analysis to compare MGUS and MM transcriptomes. For these analyses we tagged (1) 101 MGUS patient plasma cells from bone marrow samples and 64 plasma cells from healthy controls (2) 383 MM patient CD138+ cells from bone marrow and the 101 MGUS samples in the first analysis as controls (3) 517 MM patient peripheral blood samples and 97 peripheral blood samples from healthy controls. We then utilized Ingenuity Pathway Analysis (IPA) to analyze the unique genomic signatures within and across these samples.Our study identified genes that may have unique roles in MGUS (GADD45RA and COMMD3), but also newly identified signaling pathways (EIF2, JAK/STAT, and MYC) and gene activity (NRG3, RBFOX2, and PARP15) in MGUS that have previously been shown to be involved in MM suggesting a spectrum of molecular overlap. On the other hand, genes such as DUSP4, RN14, LAMP5, differentially upregulated in MM, may be seen as tipping the scales from benignity to malignancy and could serve as drug targets or novel biomarkers for risk of progression. Furthermore, our analysis of MM identified newly associated gene/pathway activity such as inhibition of Wnt-signaling and defective B cell development. Finally, IPA analysis, suggests the multifactorial, oncogenic qualities of IFNγ signaling in MM may be a unifying pathway for these diverse mechanisms and prompts the need for further studies.

[IL-6 Regulates the Chemosensitivity of Drug-Resistant Multiple Myeloma Cell Lines to Bortezomib through STAT3/Notch Signaling Pathway].

To investigate the effect of interleukin-6 (IL-6) on the chemosensitivity of drug-resistant multiple myeloma (MM) cell lines to bortezomib (BTZ) and its mechanism. Peripheral blood samples were collected from patients with BTZ-resistant MM before and after treatment. Human MM cell lines KM3 and KM3/BTZ were cultured in vitro. ELISA was used to detect the content of IL-6 in peripheral blood of MM patients, KM3 and KM3/BTZ cells. CCK-8 assay was used to detect the drug sensitivity of KM3 and KM3 / BTZ cells to BTZ. KM3 / BTZ cells were divided into KM3/BTZ control group (normal culture for 48 h), IL-6 neutralizing antibody Anti-IL-6 group (500 ng/ml Anti-IL-6 treated for 48 h), BTZ group (300 ng/ml BTZ treated for 48 h), BTZ + Anti-IL-6 group (300 ng/ml BTZ and 500 ng/ml Anti-IL-6 treated for 48 h). The proliferation activity of KM3 / BTZ cells was detected by CCK-8 assay. The cell cycle distribution of KM3/BTZ cells was detected by flow cytometry. The apoptosis of KM3/BTZ cells was detected by Annexin V-FITC/PI double staining. The mRNA expression levels of IL-6, Notch1, signal transducer and activator of transcription 3 (STAT3) in KM3/BTZ cells were detected by real-time fluorescent quantitative PCR (qRT-PCR), and the protein expression levels of IL-6, Notch1, STAT3 in KM3/BTZ cells were detected by Western blot. The level of IL-6 in peripheral blood of patients with BTZ-resistant MM after treatment was significantly higher than that before treatment (P<0.05). The level of IL-6 in KM3/BTZ cells was significantly higher than that in KM3 cells (P<0.05). The sensitivity of KM3/BTZ cells to BTZ was significantly lower than that of KM3 cells (P<0.05), and the resistance index (RI) was 19.62. Anti-IL-6 and BTZ could inhibit the proliferation of KM3 / BTZ cells, block cell cycle, and induce apoptosis (P<0.05). Compared with single drug treatment, the combined effect of Anti-IL-6 and BTZ was more obvious on KM3/BTZ cells (P<0.05), and significantly down regulated the mRNA and protein expression of IL-6, Notch1 and STAT3 in KM3/BTZ cells (P<0.05). Antagonizing IL-6 can increase the chemosensitivity of MM cells to BTZ, and IL-6 may reduce the sensitivity of MM cells to BTZ through STAT3/Notch signaling pathway.

Measurable Residual Disease in Extracellular Vesicles from Bone Marrow and Peripheral Blood of Patients with Multiple Myeloma: A Proof-of-Concept Study

BACKGROUND: Multiple myeloma (MM) treatment improved substantially in the last years, with unprecedented survival outcomes. However, even when achieving complete remission, patients ultimately relapse. Therefore, monitoring measurable residual disease (MRD) is crucial to assess treatment response and define the depth of patients' remission status. However, this currently still requires invasive bone marrow (BM) aspirates, which severely hinders real-time monitoring of the disease. Therefore, the identification of biomarkers of MRD in the peripheral blood (PB) of patients would allow a more frequent and minimally invasive monitoring of MRD. Extracellular Vesicles (EVs) are small particles (30-1000nm) shed by all cells, which are found in all biofluids including the BM and PB. These particles carry a specific cargo from their cell of origin, including proteins, enclosed by a lipidic layer. Therefore, they have been described as a possible source of cancer biomarkers, with potential to monitor MRD. AIMS: This study aimed to implement a protocol for the isolation of EVs from the BM and PB of MM patients at distinct stages of the disease (diagnosis and remission), in order to detect and compare the levels of known MRD biomarkers in their cargo. METHODS: The study was previously approved by the Ethical Committee of CHSJ and patient's consent was obtained. EVs from BM and PB Platelet-Poor Plasma (PPP) were isolated by size-exclusion chromatography (SEC), and further concentrated by ultrafiltration (UF). Then, the EVs were characterized according to their size and concentration (by Nanoparticle Tracking Analysis), morphology (by Transmission Electron Microscopy), protein concentration (Lowry protein assay) and presence of EV-associated protein markers (Western Blot - WB). In addition, 16 known MRD and MM biomarkers were analyzed by WB in the isolated EVs from PB and BM of seven patients, at two main stages of the disease - diagnosis versus response after autologous stem cell transplant (ASCT). Clinical features regarding cytogenetics and immunophenotypic markers using multi-parameter flow cytometry (MFC) were analyzed and compared. RESULTS: The two-step protocol described allowed the isolation of size-resolved EVs from both PB and BM of MM patients. The EVs isolated (both from PB and BM) presented a size-range from 50 to 500nm and presented EV-associated protein markers, such as CD81 and CD63. Moreover, several MM MRD biomarkers (e.g. CD56, CD45, CD38 and light chain) were detected in the cargo of the EVs from BM and PB at diagnosis and complete remission. The biomarkers of MM and MRD detected in the cargo of PB EVs were mainly the same as the ones detected in the cargo of BM EVs. The complete remission after ASCT was mostly associated with a decrease in the expression of EV-associated MM markers in both the BM and the PB; however, in some patients a few of the markers persisted at this stage when compared to diagnosis. In fact, the expression of CD45 and HLA-DR persisted at the remission stage in 3 and 2, respectively, out of 5 patients presenting these markers at diagnosis. Moreover, an increased expression of CD56 was also detected at remission in 3 out of 7 patients. By correlating these data with patient's routine work-up it was found that patients with persistent CD45 didn't reach 10^-5 MRD negative by flow cytometry. CONCLUSIONS: Taken together, this work suggests that it is possible to detect MM markers in EVs from either BM or PB of MM patients and compare their expression at different stages of the disease (diagnosis and remission after ASCT). Importantly, our results demonstrate the importance and potential of analyzing EVs cargo from PB, suggesting the possibility of using them for minimally invasive monitoring of MRD in MM patients.ACKNOWLEDGEMENTS: The authors acknowledge Celgene/BMS for providing funding to this work (Project Looker - Grant_138800). The authors acknowledge Cytogenetics Laboratory, Department of Clinical Hematology, Centro Hospitalar e Universitário São João and Flow Cytometry Laboratory, Department of Clinical Pathology, Centro Hospitalar e Universitário São João. DisclosuresBergantim: Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Barbosa: BMS: Research Funding. Silva: BMS: Research Funding. Polónia: BMS: Research Funding. Caires: BMS: Research Funding. Guimarães: BMS: Research Funding; Amgen: Research Funding. Vasconcelos: BMS: Research Funding; Amgen: Research Funding.

Significance of Tim-3 in The Imbalance of Th17/Treg in Patients with Multiple Myeloma

To investigate the singnificance of Tim-3 in Th17/Treg balance in patients with multiple myeloma (MM). Fifty-six newly diagnosed MM patients and 30 healthy people were enrolled. Flow cytometry was used to detect the expression of Tim-3 on CD4+T cells, the proportion of Th17 and Treg cell, the Th17/Treg ratio, the expression of Tim-3 in Th17 and Treg cells, and the ratio of Tim-3+Th17/Tim-3+Treg cells in two groups. ELISA was used to detect the level of cytokines IL-17 and IL-10. Moreover, to analyze the relationship between Tim-3+Th17/Tim-3+Treg balance and clinical indicator. Compared with the control group, the expression of Tim-3 on CD4+T cells in peripheral blood of MM patients was increased (P<0.05), the ratio of Th17 cells and Th17/Treg in MM patients were higher than those in control group (P<0.05), and the ratio of Treg cells in MM patients was lower than those in control group, which the difference showed no statistically significant (P>0.05), and the level of cytokines IL-17, IL-10 and the ratio of IL-17/IL-10 in MM patients were significant higher than those in the control group (P<0.05). The expression level of Tim-3+Th17 cells and the ratio of Tim-3+Th17/Tim-3+Treg in MM patients were higher than those in control group (P<0.05), while the expression level of Tim-3+Treg cells in MM patients were lower than that in the control group (P<0.05). The ratio of Tim-3+Th17/Tim-3+Treg in MM patients were positive correlated with ISS staging, DS staging, chromosome abnormality and sFLCR (r=0.635, r=0.501, r=0.449, r=0.587). The ratios of Th17/Treg, IL-17/IL-10 and Tim-3+Th17/Tim-3+Treg increase in MM patients, among which Tim-3+Th17/Tim-3+Treg is correlated with ISS staging, DS staging, chromosomal abnormalities and sFLCR, which suggesting that Tim-3 is involved in the imbalance of Th17/Treg in MM patients.

Correlation of the Expression Alteration of Tim-3 with the T Cell and B Cell Dysfunction in Peripheral Blood of Multiple Myeloma Patients

To study the correlation of the expression alteration of Tim-3 with the T cell and B cell dysfunction in peripheral blood of multiple myeloma (MM) patients. 30 patients diagnosed as MM from October 2016 to October 2018 were selected and enrolled in MM group, and 30 healthy persons whose sex and age was matched with the MM patients were selected and enrolled in healthy control group (HC). The blood samples from MM patients and HC were collected, and the peripheral blood mononuclear cells (PBMNC) were separated by density gradient centrifugation, then the serum was kept for further study. The ratios of CD3+CD4+Tim-3+T cells, CD3+CD8+Tim-3+T cells and the CD19+CD20-CD38+B cells were analysed by flow cytometry (FCM)，and the concentration of T cell-related cytokines IFN-γ, TNF-αand B cell-related antibodies IgA, IgM and IgG were measured by ELISA. At the same time, the differences of the ratios of CCD3+CD4+Tim-3+T, CD3+CD8+Tim-3+T cells and plasmablast and the concentration of IFN-γ, TNF-α, IgA, IgM and IgG between the MM patient and HC were estimated, and the correlation of the ratio of CD3+CD4+Tim-3+T, CD3+CD8+Tim-3+T cells with the ratio of plasmablast and the concentration of IFN-γ, TNF-α, IgA, IgM and IgG in MM patients were analyzed. The ratio of CD3+CD4+Tim-3+T, CD3+CD8+Tim-3+T cells increased in MM patients, while the ratio of CD19+CD20- CD38+B cells and the concentration of IFN-γ, TNF-α, IgA, IgM and IgG decreased in MM patients. And there was a negative correlation of the ratio of CD3+CD4+Tim-3+T cells with CD19+CD20-CD38+B cells and the concentration of IFN-γ, IgA, IgM and IgG in MM patients, while the ratio of CD3+CD8+Tim-3+T cells just negatively correlated with the concentration of TNF-α. Expression of Tim-3 on CD4 and CD8 cells elevates in the peripheral blood of MM patients, which also correlates with the function suppression of T and B cells.

Mechanical segregation and capturing of clonal circulating plasma cells in multiple myeloma using micropillar-integrated microfluidic device.

Multiple myeloma (MM), the disorder of plasma cells, is the second most common type of hematological cancer and is responsible for approximately 20% of deaths from hematological malignancies. The current gold standard for MM diagnosis includes invasive bone marrow aspiration. However, it lacks the sensitivity to detect minimal residual disease, and the nonuniform distribution of clonal plasma cells (CPCs) within bone marrow also often results in inaccurate reporting. Serum and urine assessment of monoclonal proteins, such as Kappa light chains, is another commonly used approach for MM diagnosis. Although it is noninvasive, the level of paraprotein elevation is still too low for detecting minimal residual disease and nonsecretive MM. Circulating CPCs (cCPCs) have been reported to be present in the peripheral blood of MM patients, and high levels of cCPCs were shown to correlate with poor survival. This suggests a potential noninvasive approach for MM disease progress monitoring and prognosis. In this study, we developed a mechanical property-based microfluidic platform to capture cCPCs. Using human myeloma cancer cell lines spiked in healthy donor blood, the microfluidic platform demonstrates high enrichment ratio (>500) and sufficient capture efficiency (40%-55%). Patient samples were also assessed to investigate the diagnostic potential of cCPCs for MM by correlating with the levels of Kappa light chains in patients.

PiRNA-823 delivered by multiple myeloma-derived extracellular vesicles promoted tumorigenesis through re-educating endothelial cells in the tumor environment.

Extracellular vesicles (EVs) can carry a wide array of RNAs in the tumor microenvironment, and are crucial for communication between tumor and surrounding stromal cells, including endothelial cells. Piwi-interacting RNAs (piRNAs) are important regulators implicated in the pathogenesis of multiple myeloma (MM). However, little is understood about the role of piRNA-823 in intercellular communication between MM and endothelial cells. In this study, we found that piRNA-823 mainly accumulated in EVs from peripheral blood of MM patients and EVs derived from MM cells (MM-derived-EVs). Increased piRNA-823 expression was associated with late stages and poor prognosis of MM. The MM-derived-EVs effectively transferred piRNA-823 to EA.hy926 endothelial cells. The piRNA-823 mimic and inhibitor were designed to upregulate or to suppress the endogenous function of piRNA-823. Transfection with piRNA-823 mimic or treatment with MM-derived-EVs significantly promoted the proliferation, tube formation, and invasion of EA.hy926 cells by enhancing the expression of VEGF, IL-6, and ICAM-1 and attenuating apoptosis. EA.hy926 cells transfected with piRNA-823 mimic or pre-treated with MM-derived-EVs promoted the growth of xenograft MM in mice. In contrast, the transfection with piRNA-823 inhibitor or treatment with EVs from piRNA-823 inhibitor-transfected-MM cells had diametrically opposite effects. Our findings demonstrated that piRNA-823 carried by MM-derived-EVs is essential for the re-education of ECs toward a unique environment amenable to the growth of MM cells by altering its biological characteristics. Our findings may pave the way for the development of new piRNA-mediated prognostic stratification and therapeutic strategies for MM.

Abstract 1559: Detection and retrieval of multiple myeloma cells with single-cell molecular confirmation from peripheral blood by multiparameter immunofluorescence, automated scanning, and image analysis

Abstract Background: The detection of multiple myeloma (MM) cells is currently performed by analysis of bone marrow aspirates. Detection of MM cells from the peripheral blood would allow for less invasive and more frequent assessments to monitor residual disease. Using the RareCyte platform, we developed a 5-parameter assay to visualize and retrieve MM cells from peripheral blood and applied it to MM patient samples under an IRB-approved protocol. We also used this to identify plasma cells from the peripheral blood of healthy volunteer donors. Materials and Methods: Healthy volunteer or MM patient peripheral blood was collected into RareCyte blood collection tubes. RPMI8226 cells spiked into normal donor blood samples were used as a model for MM cell assay development. Buffy coats isolated from 7.5mL of blood by AccuCyte® separation were spread onto slides. Slides were stained with a 5-marker panel that included antibodies to CD138, CD38, CD56, CD45, and a nuclear dye on the Leica Bond Rx auto-stainer. Slides were scanned with CyteFinder® and MM cells identified by positive CD138 and CD56 staining and negative CD45 staining. Identified MM cells and WBC control cells were mechanically retrieved with the integrated CytePicker module, whole genome amplified, and Sanger sequencing was performed for specific mutations in four genes (IDH1, NRAS, PHLDA1, RPH3AL) identified by genomic analysis of a bone marrow sample. Plasma cells from normal donors were identified by co-expression of CD138 and CD38. Results: In a pretreatment MM sample, the 5-parameter assay identified 55 MM cells per mL of whole blood. In isolated single cells with at least one successful sequencing reaction, 0 of 13 WBCs contained mutations in at least one of the genes. Mutations were found in 9 of 15 MM cells (defined as CD138+, CD56+, CD38-, and CD45-). In healthy volunteer samples, 5 plasma cells (defined as CD138+, CD38+, and CD45 dim/negative) were identified per mL of whole blood. Conclusions: The 5-parameter assay successfully detected rare cells with an MM phenotype from the peripheral blood of an MM patient. Individually retrieved MM cells were demonstrated to contain mutations specific to the patient's tumor, confirming that the cells identified were MM cells. This approach may allow for less invasive assessment of MM and monitoring of residual disease. Citation Format: Lance U'Ren, Nolan Ericson, Daniel Campton, Arturo Ramirez, C. Anthony Blau, Eric Kaldljian. Detection and retrieval of multiple myeloma cells with single-cell molecular confirmation from peripheral blood by multiparameter immunofluorescence, automated scanning, and image analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1559.

High levels of FLT3-ligand in bone marrow and peripheral blood of patients with advanced multiple myeloma.

IntroductionMultiple myeloma (MM) is still incurable due to resistance against various therapies. Thus, the identification of biomarkers predicting progression is urgently needed. Here, we evaluated four biomarkers in bone marrow and peripheral blood of MM patients for their prognostic significance.Materials & methodsBone marrow- and peripheral blood plasma levels of FLT3-L, soluble TIE2, endostatin, and osteoactivin were determined in patients with monoclonal gammopathy of undetermined significance (MGUS, n = 14/n = 4), patients with newly diagnosed MM (NDMM, n = 42/n = 31) and patients with relapsed/refractory MM (RRMM, n = 27/n = 16) by sandwich ELISA.ResultsMedian FLT3-L expression increased from MGUS (58.77 pg/ml in bone marrow; 80.40 pg/ml in peripheral blood) to NDMM (63.15 pg/ml in bone marrow; 85.05 pg/ml in peripheral blood) and was maximal in RRMM (122 pg/ml in bone marrow; 160.47 pg/ml in peripheral blood; NDMM vs. RRMM p<0.001). A cut-off value of FLT3-L >92 pg/ml in bone marrow and >121 pg/ml in peripheral blood was associated with relapse or refractoriness in MM patients. FLT3-L was found to be a high predictive marker for discrimination between NDMM and RRMM as well in bone marrow as in peripheral blood (AUC 0.75 in bone marrow; vs 0.84 in peripheral blood).ConclusionHigh levels of FLT3-L in bone marrow and peripheral blood of MM patients identify patients with progressive disease and are associated with relapse or refractoriness in MM patients. FLT3-L could be useful as a marker to identify RRMM patients and should be evaluated as target for future therapies.

Lenalidomide Increases Human Dendritic Cell Maturation in Multiple Myeloma Modulating Both Monocyte Differentiation and Mesenchymal Stromal Cell Inhibitory Properties through Ikaros and Casein Kinase 1 Degradation, Respectively

The use of the immunomodulatory drugs (IMiDs®), including lenalidomide (LEN), to reverse tumor-mediated immune suppression and amplify multiple myeloma (MM)-specific immunity is currently being explored. Particularly LEN effects on antigen presenting cells are still unknown. In this study we investigated LEN potential effect on Dendritic Cell (DC) differentiation and activity and on the immunosuppressive properties of human mesenchymal stromal cells (hMSCs) on DCs.DCs were obtained either from THP1 (THP1-DC), a human monocytic cell line, or from CD14+ cells, purified by an immunomagnetic method both from peripheral blood (PB) and bone marrow (BM) samples of a total cohort of 19 patients with monoclonal gammopathies including 13 with active MM.Monocyte-derived DC (mo-DC) differentiation was induced by the treatment with GM-CSF and IL-4 for 8 days and TNF-α for the last 24 hours. LEN treatment was performed at concentration ranging from 0.1 to 2 μM. Then, non-adherent cells were analyzed for DC maturation markers (CD83, HLA-DR, CD80, CD86 and CD209) by flow cytometry. The levels of soluble factors involved in the immune response (CCL2, CCL5, CXCL10, IL-6, IL-8, IL-10, IL-12, IFN-γ, TNF-α) were measured in the conditioned media (CM) of mo-DCs by a Bio-Plex® Multiplex System. Moreover, the effect of LEN treatment on DC ability to stimulate T-cell proliferation was investigated through an MTT assay on CD3+ cells, purified from PB of MM patients, co-cultured for 6 days with LEN pretreated autologous mo-DCs, in RPMI medium at 15% AB human serum. Ex vivo studies were also performed in order to evaluate mo-DC differentiation of CD14+ cells purified from PB of 4 MM patients before and after 7 days of LEN treatment at 25 mg/day without Dexamethasone. Moreover, mo-DC differentiation was performed in the presence of CM of human TERT transfected (hTERT) -hMSCs treated for 5 days with LEN (0.1-2 μM). The expression levels of factors with an inhibitory effect on DC development (CCL5, IDO1, IL6, PTGS2, TGFB) were tested in LEN treated hTERT-hMSCs by real time PCR. Finally, the protein levels of Cereblon and its transcription factor targets Aiolos, Ikaros, IRF-4, p62 and Casein kinase (CK) 1 and 2 were evaluated either in THP1-DCs or in hTERT-hMSCs LEN treated cells by Western Blotting.Despite a reduction of both number and % of mature mo-DCs, LEN at the concentration range reached in vivo in MM patients significantly increased the median intensity expression of HLA-DR (LEN vs vehicle, p<0.0001) and CD86 (LEN vs vehicle, p<0.0001) by mo-DCs derived from BM. Similarly, increased HLA-DR (LEN vs vehicle, p=0.047) and CD86 (LEN vs vehicle, p<0.0001) expressions have been seen in mo-DCs derived from PB of both active MM and smoldering MM patients. LEN treatment also enhanced the production of IL-8 in a dose dependent manner (LEN 2 μM vs vehicle, p<0.0001), CCL2 (LEN 2 μM vs vehicle, p=0.0207) and TNF-α levels (LEN 0.1 μM vs vehicle, p=0.0054). Moreover, LEN pretreated mo-DCs showed an increased ability to stimulate CD3+ cell proliferation. Interestingly, even in vivo LEN treatment of MM patients enhances mo-DC differentiation of CD14+ cells in ex-vivo culture (HLA-DR, LEN day7 vs LEN day0 p=0.047; CD209, LEN day7 vs LEN day0 p=0.017). LEN effect on DC differentiation was associated to Ikaros degradation as it was observed a significant down-regulation in THP1-DCs after LEN treatment, with a dose dependent effect. LEN also blunted hTERT-hMSC inhibitory effect on mo-DC differentiation and significantly down-regulated PTGS2 gene expression levels at all concentrations tested (p<0.05). These effects were likely to be not mediated by Aiolos, Ikaros, and IRF-4 because they were not expressed by hMSCs; meanwhile p62, CK1, CK2 were expressed but only CK1 expression was down-regulated after LEN treatment with a dose dependent effect.In conclusion, LEN increased the expression of mature DC markers both in vitro and in ex vivo cultures, enhancingDC ability to stimulate T cell proliferation and to release chemokines involved in the immune response. LEN treatment also reduces the immunosuppressive properties of hMSCs, suggesting new possible effects of IMiDs® on the alloreactivity against MM cells. DisclosuresGiuliani:Celgene: Research Funding; Janssen: Research Funding.

Novel Flow Cytometry-Based Biomarkers Predict Recurrence in Myeloma Patients through Detection of MRD in the Bone Marrow and CTCs in Peripheral Blood

Introduction: The presence of a minimal residual disease (MRD) that remains after treatment is one of the main reasons for recurrence and relapse in multiple myeloma (MM) patients. Diagnosis of MRD is becoming vital to assess the effectiveness of the treatment as well as to predict survival among patients with complete remission. CD138 (syndecan-1) is the gold standard marker for detecting MM cells using multiparametric flow cytometry analysis or immunohistochemistry (IHC) staining of BM biopsies. However, the presence of highly clonogenic, drug resistant and stem-cell like CD138-negative MM cells have been previously demonstrated. Moreover, hypoxia which is known to drive MM progression, drug resistance and metastasis was shown to significantly decrease CD138 expression in MM cells. In this study, we utilized a novel set of biomarkers to detect MRD in the bone marrow as well as circulating tumor cells (CTCs) in the blood, in order to predict progression free survival (PFS) in MM patients defined as complete remission according to the detection of CD138+ cells in their bone marrow.Methods: To detect myeloma cells, we utilized a novel set of biomarkers, independent of CD138 expression and hypoxic state of MM cells (CD38+/CD3-/CD19-/CD14-/CD16-/CD123-) by two-color flow cytometry. We detected MM cells in the bone marrow of 25 patients with complete remission and very good partial response, whose bone marrow was defined as a CD138-negative (less than 0.5%). In order to retrospectively correlate the involvement of MM cells (%) with PFS, we used 24 months as a cut-off and compared the results acquired with the new method to traditional CD138-based flow or histology. In addition, based on the percentage of detected MM cells with the 2% cut-off, we analyzed the time to progression (months). Moreover, we detected MM cells in the peripheral blood from matching MM patients and performed analogous analysis. Furthermore, we also detected CTCs in 7 newly diagnosed patients and compared to 7 patients with progressive disease.Results: A study conducted on 25 patients with their bone marrow defined as a CD138-negative (less than 0.5%) showed that the novel strategy to detect MM cells was more precise than CD138-based flow or histology in predicting PFS. Patients who relapsed in less than 24 months had an average (±SEM) of 3.53±0.82%, while patients who relapsed later than 24 months had an average of 1.15±0.27% MM cells in the bone marrow when detected by the new method (p=0.004). In addition, the median PFS with <2% or ≥2% of MM cells detected by novel strategy was 31.6±2.8 and 18.5±2.2 months, respectively (p=0.004). Similarly, while CD138 detected very low amounts of CTCs and demonstrated no difference between fast and slow relapsing patients; we found that the new biomarkers detected significant differences in the number of circulating MM cells in patients who relapsed fast and patients who relapsed late or did not relapse. Patients who relapsed in less than 24 months had 1.74±0.42%, while patients who relapsed later than 24 months had 0.4±0.08% CTCs in the peripheral blood (p=0.01). In addition, we detected significantly more CTCs in patients with progressive disease (1.27±0.24%) compared to newly diagnosed MM patients (0.63±0.12%), respectively (p=0.03).Conclusions: The novel flow cytometry-based set of biomarkers provides an alternative strategy to detect MRD in the bone marrow and CTCs in the peripheral blood of MM patients, and both allowed prediction of PFS in MM patients. DisclosuresDe La Puente:Cellatrix LLC: Other: Co-founder. Vij:Amgen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy; Jazz: Consultancy; Shire: Consultancy; Karyopharma: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy. Azab:Targeted Therapeutics LLC: Other: Founder and owner; Cleave Bioscience: Research Funding; Verastem: Research Funding; Cell Works: Research Funding; Karyopharm: Research Funding; Vasculox: Research Funding; Glycomimetics: Research Funding; Cellatrix LLC: Other: Founder and owner; Selexys: Research Funding.

A CD138-independent strategy to detect minimal residual disease and circulating tumour cells in multiple myeloma.

CD138 (also termed SDC1) has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug-resistant residual and circulating MM cells were shown to have lower expression of this marker. In this study, we have shown that residual MM cells following bortezomib treatment are hypoxic. This combination of drug exposure and hypoxia down-regulates their CD138 expression, thereby making this marker unsuitable for detecting residual or other hypoxic MM cells, such as circulating tumour cells, in MM. Hence, we developed an alternative biomarker set which detects myeloma cells independent of their hypoxic and CD138 expression status in vitro, in vivo and in primary MM patients. The new markers were able to identify a clonal CD138-negative population as minimal residual disease in the bone marrow and peripheral blood of MM patients. Further investigation to characterize the role of this population as a prognostic marker in MM is warranted.

Isolation of Human CD138+ Microparticles from the Plasma of Patients with Multiple Myeloma

The confinement of multiple myeloma (MM) to the bone marrow microenvironment requires an invasive bone marrow biopsy to monitor the malignant compartment. The existing clinical tools used to determine treatment response and tumor relapse are limited in sensitivity mainly because they indirectly measure tumor burden inside the bone marrow and fail to capture the patchy, multisite tumor infiltrates associated with MM. Microparticles (MPs) are 0.1- to 1.0-μm membrane vesicles, which contain the cellular content of their originating cell. MPs are functional mediators and convey prothrombotic, promalignant, proresistance, and proinflammatory messages, establishing intercellular cross talk and bypassing the need for direct cell-cell contact in many pathologies. In this study, we analyzed plasma cell–derived MPs (CD138+) from deidentified MM patients (n = 64) and normal subjects (n = 18) using flow cytometry. The morphology and size of the MPs were further analyzed using scanning electron microscopy. Our study shows the proof of a systemic signature of MPs in MM patients. We observed that the levels of MPs were significantly elevated in MM corresponding to the tumor burden. We provide the first evidence for the presence of MPs in the peripheral blood of MM patients with potential applications in personalized MM clinical monitoring.

Targeting Mechanisms for Natural Killer Cell Dysfunction in Patients with Multiple Myeloma

Patients with multiple myeloma (MM) have a multi-faceted immune dysfunction with poor NK cell cytotoxic function. We show that NK cells in MM have a disturbed homeostasis, featured by an increased frequency of terminally differentiated CD57+ NK cells and almost complete absence of the less mature CD56hi NK cell subset. MM patient NK cells have reduced levels of killer activating receptors (KAR) [Nkp46, Nkp44 and NKG2D] and increased levels of killer inhibitory receptor (KIR) [KIR2DL1 and KIR2DL3]. Despite expressing normal levels of the NK effector transcription factors Tbet and eomesdermin, and the effector protein perforin, MM patient NK cells demonstrate poor cytotoxic function against myeloma cell lines and lower levels of the cytotoxic proteins granzyme B (gzmB).Elotuzumab (Elo, Bristol Myers Squibb) is a human IgG1 antibody which targets SLAM-F7, a protein highly expressed on myeloma plasma cells and on specific subsets of immune cells. Elo mediates dual activation of NK cells and has shown promising clinical responses when combined with lenalidomide; however the complete mechanism(s) whereby Elo mediates these effects have not been fully described. Thus, this project investigates the Elo mechanisms of action in myeloma. To explore whether MM patient NK cells were capable of ADCC, we examined NK cell subset degranulation (CD107a) and IFN-γ secretion when co-cultured with the SLAM-F7 expressing OPM-2 myeloma cells and Elo. Healthy donor NK cells degranulated in response to Elo-labeled OPM-2 cells, which correlated with loss of the CD56lo CD16+ NK cell subset. In contrast, newly diagnosed (NEW) and refractory/relapsed MM (RRMM) patient NK cells had a decreased response to Elo-labeled OPM-2 cells as the CD56lo CD16+ NKsubset was retained, and they degranulated at a significantly lower level than that observed in healthy donor NK cells. Therefore, all MM patient NK subsets (CD56lo CD16+, CD161+ and CD57+) assessed were low responders to Elo-labeled OPM-2 cells.We then visually examined NK cell killing kinetics of Elo-labeled myeloma target cells using time lapse live video microscopy (TLLVM). NK cells used ADCC to kill OPM-2 cells in the presence of Elo, the killing kinetics of was equivalent to direct killing induced in presence of the human IgG isotype control, the delivery of the lethal hit occurring in under three minutes. Elo also mediated serial killing of OPM-2 cells. Interestingly, Elo did delay the NK cell detachment from dying myeloma plasma cell targets, which results in prolonged signaling to the NK cell and consequently higher levels of secreted cytokines and pro-inflammatory chemokines. Taken together, this data indicates Elo-induces a profound change in the NK-mediated killing kinetics of myeloma cells and that likely recruits other immune effectors to the myeloma killing site.Given that NK cells can also express SLAM-F7, we assessed whether they were likely to be a target for other NK cells when bound to Elo, and induce fratricide. We used killing assays, flow cytometry and TLLVM to show that in the presence of Elo, healthy donor NK cells were capable of NK fratricide under the assay conditions tested. Interestingly, fratricide induced death saw the depletion of the CD56lo CD16+ NKsubset. This data may partially explain why there is a rapid but transient reduction of NK cells from the peripheral blood of MM patients who received single agent Elo.In summary, MM patient NK dysfunction is due to aberrant NK cell homeostasis featured by terminal differentiation of the NK cells to a subset with poor natural cytotoxic function and high KIR expression. In vitro, Elo is less effective at inducing ADCC responses by NK cells from MM patients compared to normal donors. Nonetheless, Elo induces efficient normal donor NK cell killing of myeloma target cells, the killing kinetics is featured by a protracted immune synapse time and secretion of high levels of pro-inflammatory cytokines/chemokines by 24 hours, plus serial killing of myeloma target cells. These data likely explain the single agent efficacy of Elo in RRMM patients. The data also underlines the exciting potential of Elo to induce NK mediated ADCC of myeloma plasma cells, and recruit multiple arms of the immune system to the killing site. To harness its full potential, combination of Elo with other drugs (eg. Lenalidomide and anti-KIR) maybe required to normalize MM patient NK cell function and initiate the immune attack. DisclosuresRobbins:Bristol-Myers Squibb: Employment, Other: shareholder. Neeson:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

CAR-Engineered T Cells Specific for the Elotuzumab Target SLAMF7 Eliminate Primary Myeloma Cells and Confer Selective Fratricide of SLAMF7+ Normal Lymphocyte Subsets

Background: SLAMF7 (CS1, CD319) is uniformly and highly expressed in multiple myeloma (MM) where it promotes adhesion and survival of malignant plasma cells (mPCs) in the bone marrow niche. It is absent on normal solid organ tissues but known to be expressed on lymphocyte subsets (T, B and NK cells). Clinical evaluation of the anti-SLAMF7 monoclonal antibody (mAb) Elotuzumab (huLuc63) has resulted in marked reversible lymphodepletion and conferred potent anti-MM efficacy in combination therapy. Here, we evaluated the potential to generate SLAMF7-directed chimeric antigen receptor (CAR) modified T cells from previously treated MM patients and analyzed their potency against autologous mPCs as well as fratricidal activity against normal lymphocyte subsets.Methods: Flow cytometric analyses for SLAMF7 expression on mPCs and normal lymphocyte subsets of MM patients (n=67) and healthy donors (n=20) was performed using specific mAbs and matched isotype controls. A SLAMF7-specific CAR was constructed using the VH/VL targeting domains of mAb huLuc63, fused to an Ig-Fc spacer and a signaling module of CD3ζ and CD28. Lentiviral gene transfer was performed into CD3/CD28-bead stimulated bulk CD4+ and CD8+ T cells of MM patients (n=7). CAR transgene positive T cells were enriched using an EGFRt transduction marker and expanded for functional analyses.Results: We confirmed high SLAMF7 expression levels on mPCs in all analyzed samples and detected SLAMF7 expression on a fraction of normal lymphocytes obtained from peripheral blood of MM patients, including naïve and memory CD4+ (95% CI: 33-59%) and CD8+ T cells (75-95%), B cells (25-35%) and NK cells (94-98%). Remarkably, the proportion of SLAMF7+ cells was significantly higher in MM patients compared to healthy donors in all corresponding lymphocyte subsets (p<0.05). Despite high level SLAMF7 expression on the input T cell population, functional CD4+ and CD8+ T cells expressing the SLAMF7-CAR could be readily generated in all 7 MM patients, and expanded to therapeutically relevant doses in a single expansion cycle following enrichment (>107 cells). We analyzed the kinetics of SLAMF7 expression on CD4+ and CD8+ CAR T cells during the manufacturing process and detected rapid disappearance of SLAMF7+ T cells in T cell lines modified with the SLAMF7-CAR. By contrast unmodified T cells and T cell lines expressing a CD19-CAR retained a significant proportion of SLAMF7+ T cells, suggesting that expression of the SLAMF7-CAR induced killing of SLAMF7+ T cells. In vitro functional testing of SLAMF7-CAR CD4+ and CD8+ T-cell lines confirmed potent specific lysis of SLAMF7+ MM cell lines including MM1.S and OPM-2 and stable SLAMF7-transfectants of K562, as well as antigen specific IFNγ secretion and productive proliferation. In a flow cytometry based cytotoxicity assay, co-incubation of mPCs with autologous (or allogeneic) SLAMF7-CAR T cells resulted in elimination of >90% of mPCs after a 4-hour incubation period, whereas CD19-CAR or unmodified T cells had no discernible effects. Moreover, in an in vivo xenograft MM model (NSG/MM1.S) a single administration of SLAMF7-CAR T cells resulted in complete MM clearance and long-term survival, whereas mice treated with CD19-CAR or unmodified T cells rapidly expired from progressive disease. Finally, we analyzed the fratricidal potential of SLAMF7-CAR T cells to predict hematologic toxicities that might occur in a clinical setting. Co-incubation of purified CD4+ and CD8+ primary T cells, B cells and NK cells with SLAMF7-CAR T cells resulted in rapid and specific elimination of only SLAMF7+ subsets, whereas SLAMF7- cells remained viable and functional as confirmed for CD4+ and CD8+ T cells by inducible IFNγ secretion.Conclusion: Our data demonstrate that SLAMF7-specific CAR T cells can be reproducibly generated from MM patients and exert remarkable anti-myeloma efficacy in pre-clinical models in vitro and in vivo. Lymphocytic fratricide does not preclude the manufacture of SLAMF7-CAR T cells but might be associated with acute (cytokine storm) or chronic (viral infections) side effects in a clinical setting. However, such toxicities may be prevented e.g. by preparative lymphodepletion and antiviral prophylaxis and enable the implementation of SLAMF7-CAR T cell therapy as a safe and effective modality in the treatment of MM. DisclosuresKnop:Celgene Corporation: Consultancy. Einsele:Novartis: Consultancy, Honoraria, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Early Trafficking of Bone Marrow Derived-Endothelial Progenitor Cells Promotes Multiple Myeloma Progression

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<0.05). PB mononuclear cells (PBMCs) from SMM and aMM patients showed an increase ability to form both EC-CFUs (P<0.01) and ECFCs compared to those from healthy controls (P<0.05).EPC levels were significantly increased in PB of transgenic Vk*MYC mice at both early and advanced stage of MM development compared to healthy syngenic mice. In orthotopic MM models we found that levels of circulating EPCs increase early after i.v. injection of either human MM1S-GFP+/luc+ cells (1.5 and 2 times higher after 1 week and 2 weeks, respectively) or murine 5TGM1-turboRFP+ MM cells (2.5 and 3 times higher after 2 and 3 weeks, respectively) compared to naïve mice. By coupling GFP-BM transplantation and subcutaneous femur implantation, we generated a new model that allows the study of BM derived-cell trafficking from one site to another of the BM. The levels of BM-derived GFP+ EPC increased early after i.v. injection of 5TGM1-turboRFP+ cells (2 times increase after 2 weeks, P<0.01); also, the percentage of both total GFP+ cells (7.92% versus 2.635%, P<0.05) and GFP+ EPCs (0.22% versus 0.068%, P<0.05) increased in the BM of implanted femurs of tumor cell injected mice compared to controls. Survival of ID1+/-ID3-/- mice was significantly increased compared to that of wild type littermates after injection of MM Vk*MYC cells (62 versus 27 days median survival time, P=0.0005); this survival advantage was lost after wild type BM was transplanted in ID1+/-ID3-/- mice (34 versus 35 days median survival time). In human MM1S orthotopic xenograft model, treatment with anti-murine VEGFR2 DC101 Ab was able to inhibit MM progression when treatment was started early during smoldering like stages, but not when it was started after significant tumor burden (50.5, 41.5 and 40 days median survival time in DC101 early, late and untargeted IgG treated mice respectively)Conclusion. These results show that EPC mediated vasculogenesis is an important pathogenic process that occurs during the early/smoldering MM stage. Knockout mouse experiments confirm that EPCs are essential for MM tumor progression. These results indicate that anti-angiogenic agents could be effective as an early therapeutic intervention in SMM patients. DisclosuresGhobrial: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.

Enhancing the Cytotoxicity of NK Cells Toward Multiple Myeloma Using an Antibody Specific to SLAMF7

Multiple myeloma (MM) is a malignancy of plasma cells characterized by a monoclonal expansion of tumor cells in bone marrow and accumulation of a monoclonal protein in blood and urine, which ultimately leads to end-stage organ damage. Despite advances in therapy, MM remains an incurable disease. Recent investigations have revealed a new therapeutic target for MM, SLAMF7 (CS1), which is expressed at high levels on MM tumor cells. SLAMF7 is also found on NK, CD8+ T, CD4+ T and activated B cells. Elotuzumab (HuLuc63) is a humanized IgG1 anti-SLAMF7 antibody, which can facilitate killing of primary MM cells by NK cells via antibody-dependent cellular cytotoxicity (ADCC) and has shown promise in treating relapsed MM. A key question in understanding elotuzumab’s mechanism of action is whether it can directly activate NK cells via SLAMF7 binding, in addition to Fc-mediated activation via binding of CD16 (FcγRIIIA).We have examined the in vitro effect of elotuzumab on NK cells in fresh human peripheral blood from healthy donors and MM patients incubated with or without MM cell lines that have varied levels of SLAMF7 surface expression. Cytotoxic degranulation of NK cells was quantified by staining for CD107a (marker of degranulation) after 2 hours, and direct effects of elotuzumab on NK cells were assessed by measuring upregulation of the CD69 activation marker after 24 hours of antibody exposure.Degranulation of fresh primary NK cells toward SLAMF7-bearing MM target cell lines was stimulated by addition of elotuzumab. Intensity of the degranulation response was dependent upon the concentration of elotuzumab and the expression level of SLAMF7 on the MM target cells. While elotuzumab alone could stimulate low-level degranulation of NK cells in fresh PBL from healthy donors, the degranulation response was stronger if SLAMF7-expressing MM target cells were added in combination with elotuzumab. Addition of F(ab’)2 elotuzumab or an Fc mutant form of elotuzumab that cannot bind CD16 did not stimulate degranulation, implying that binding of the antibody to SLAMF7 on NK cells and MM cells, without concomitant engagement of NK cell Fc receptors, is insufficient to trigger degranulation.Soluble intact elotuzumab stimulated CD69 expression and degranulation by fresh primary NK cells in the absence of MM target cells, which peaked at 1 µg/ml and only modestly increased up to 100 µg/ml elotuzumab. In the same assays, we only observed modest stimulation of CD69 expression using soluble F(ab’)2 or Fc mutant elotuzumab. In contrast, plate-bound F(ab’)2 elotuzumab stimulated robust CD69 expression, indicating that the antibody can induce direct stimulation of NK cells through SLAMF7, but strong stimulation requires aggregation of the antibody. In soluble form, a non-fucosylated version of elotuzumab with greater affinity for CD16 stimulated significantly stronger degranulation and CD69 expression than intact elotuzumab, both in the presence or absence of MM target cells, and with both high and low SLAMF7 expression on target cells. Importantly, elotuzumab was found to induce similar degranulation by NK cells from freshly isolated peripheral blood of MM patients as compared to healthy donors.Taken together, our data support the hypothesis that binding of soluble elotuzumab to SLAMF7 can weakly activate NK cells, but strong activation and degranulation require antibody aggregation or engagement of the Fc domain with CD16. Elotuzumab variants with enhanced CD16 binding, such as a non-fucosylated version, can augment ADCC against myeloma cell lines, and warrant further study as a potential strategy to improve clinical efficacy in MM patients. DisclosuresCohen:Celgene: Member, Independent Response Adjudication Committee Other; Janssen: Advisory Board, Advisory Board Other; Bristol-Myers Squibb: Advisory Board, Advisory Board Other, Research Funding; Onyx: Advisory Board, Advisory Board Other. Campbell:Bristol-Myers Squibb: Advisory Board Other, Research Funding; Janssen: Advisory Board, Advisory Board Other; Conkwest: Consultancy, Patented NK-92 cell lines, Patented NK-92 cell lines Patents & Royalties, Research Funding.