Multiple Myeloma Plasma Cells Research Articles

Multiple myeloma from the pathologist's perspective

Multiple myeloma (MM) is one of the most common hematological neoplasms and accounts for approximately 1% of human cancers. Description of current diagnostics and classification of MM and related plasma cell neoplasms from the pathology viewpoint. Current knowledge regarding pathology and genetics of MM is summarized and tissue-based diagnostics following international consensus classifications and the current S3 guideline are described. MM and related neoplasms are composed of malignant plasma cells that secrete amonoclonal immunoglobulin, which is an important parameter of disease activity. MM shows amultistage development. Almost all cases are preceded by aclinically inapparent precursor lesion, monoclonal gammopathy of undetermined significance (MGUS), which can progress to smoldering myeloma with ahigher tumor burden, but absence of organ damage. Systemic MM needs to be discerned from the localized forms, solitary osseous and primary extramedullary plasmacytoma. MM is genetically very heterogeneous and can be broadly subdivided into two cytogenetic groups, cases with primary IGH translocations and cases with hyperdiploidy. Intratumoral genetic heterogeneity is frequently pronounced and correlates with the size of focal lesions in imaging. Diagnosis of plasma cell neoplasms is done according to the criteria of the International Myeloma Working Group (IWMG) and requires interdisciplinary evaluation of clinical, serological, pathological and radiological features. In addition to clinical parameters, molecular markers, especially cytogenetic aberrations, are of great prognostic relevance.

OAB-041: Epithelial-mesenchymal-transition regulated by Junctional Adhesion Molecule-A (JAM-A) associates with aggressive extramedullary multiple myeloma disease

<h3>Background</h3> Interactions between multiple myeloma plasma cells (MMPC) and the multiple myeloma (MM) niche control cell proliferation, drug resistance, and disease spreading through various adhesion molecules and chemokine receptors. Among these, junctional adhesion molecule-A (JAM-A) mediates MMPC-endothelial interactions and propagates neovascularization. Here we addressed whether JAM-A signaling mediates MMPC invasive behavior orchestrating intra- and extramedullary MM dissemination. <h3>Methods</h3> We investigated JAM-A expression with flow cytometry and immunohistochemistry in 60 MM patient bone marrow (BM) aspirates and biopsies at different disease stages with and without extramedullary disease (EMD) manifestation. We compared these results with RNA-Seq data from 647 newly diagnosed MM (NDMM) patients collected in the MMRF CoMMpass study. Using bioinformatics, we investigated JAM-A-related pathways with hierarchical cluster analysis. Subsequently, we functionally validated these JAM-A-related pathways regarding epithelial-mesenchymal transition (EMT), invasion, and MM dissemination in vitro. <h3>Results</h3> The median OS differed significantly in subjects with elevated membrane JAM-A expression levels, dividing the patients in the EMD JAM-Ahigh group with a median OS of 84.1 months, whereas median OS in JAM-Alow patients was not reached, irrespective of the EMD status (log-rank=4.19, P=.04). Immunohistochemistry analysis of BM biopsies confirmed these findings. RNA-Seq analysis corroborated the prognostic impact of JAM-A (log-rank=3.8, P=.051). High-risk patients with EMD and JAM-A expression displayed a unique gene-expression signature. Additionally, we constructed a novel expression cluster subgroup model, revealing complex molecular disease patterns and associations between clinical traits and expression markers. Strikingly, MM patients clustered by JAM-A expression levels and EMD manifestation revealed properties of epithelial-mesenchymal-transition and induced focal adhesion pathways. Notably, functional knockdown of JAM-A in MMPC in vitro not only reduced cell viability but also diminished MMPC adhesion molecules and CD138 surface expression. Furthermore, mTOR/PI3K in vitro inhibition with BEZ235 significantly downregulated surface expression in JAM-Ahigh MM cells. Interestingly, when we inhibited mTOR/PI3K in JAM-Alow MM cells, we observed JAM-A induction. Finally, ensuing functional shRNA knockdown of JAM-A halted MM invasion (P<.002), angiogenesis (P<.0001), migration (P<.002), cell survival (P<.001), and expression of cellular-adhesion system molecules such as integrin-beta-1, fibronectin, RAC1 and RHOA (P<.001). Conversely, overexpressing JAM-A in MM.1S cells changed to an EMT-like phenotype compared to empty vector-infected MM.1S cell controls. <h3>Conclusions</h3> Collectively, these data reveal JAM-A-mediated signaling critical for EMT transition and invasive behavior. Based on these findings we propose JAM-A as a promising biomarker and novel theragnostic target in MM patients.

OAB-049: Poor post-vaccination anti-SARS-CoV-2-antibody response in patients with Multiple Myeloma correlates with low CD19+ B-lymphocyte count and anti-CD38 treatment

BackgroundUp to now, only few data are available regarding the efficacy of anti-SARS-CoV-2 vaccines in patients with plasma cell neoplasia. This ongoing observational study aimed to describe the level of post-vaccination anti-SARS-CoV-2-antibodies depending on B lymphocyte count, current therapy, and remission status of patients with multiple myeloma (MM) and related plasma cell dyscrasias, after the first dose of anti-SARS-CoV-2 vaccination.MethodsIn this single-center study, patients aged 18 years and older with a confirmed diagnosis of MM, monoclonal gammopathies of clinical significance (MGCS) or systemic light-chain amyloidosis (AL) who were eligible for Anti-SARS-CoV-2 vaccination according to the International Myeloma Society recommendations were included. Data were collected between January 1st and May 21th, 2021, at the department of oncology and hematology at the University Medical Center Hamburg-Eppendorf, Germany. The primary aim of this study was to evaluate a possible correlation between antibody titers and CD19+ B lymphocyte count and secondary to identify other possible factors influencing immune response. This study is part of the COVIDOUT trial (NCT04779346). Written informed consent was provided by each patient according to local requirements.ResultsA total of 82 patients were included in this study. The median age was 67.5 years (range 40-85 years). 78 patients had MM, 2 MGCS, and 2 AL. Overall, 63 patients were vaccinated with mRNA-based and 19 with vector-based vaccines, respectively. At the time of vaccination, 69 (84.1%) patients were under current anti-myeloma treatment. In total, 34 (41%) patients received anti-CD38-targeting therapies and 52 (63%) patients received IMiD-based therapies. 57 (69.5%) patients were in deep remissions (very good partial remission or better) at the time of vaccination. Assessment of anti-SARS-CoV-2 spike protein antibody titer (SP-AbT) took place on a median of 25 days after the first vaccination (SD ± 11.8). A positive SP-AbT was detected in 23% (17/74) of assessable patients. A cut-off value of ≥ 30 CD19+ B cells/µl was significantly positively correlating with higher SARS-CoV-2 SP-AbT. Individuals with current anti-CD38-antibody treatment showed lower SP-AbT and the likelihood for positive titer results was significantly lower in patients with current anti-CD38 treatment compared to those without. Treatment with immunomodulatory drugs did not harm the development of antibody titers. Furthermore, in multivariable linear regression, higher age and insufficiently controlled disease (≤ partial remission) were significantly negatively correlated with SARS-CoV-2 SP-AbT.ConclusionBased on our results, the majority of myeloma patients respond poorly after receiving the first dose of any anti-SARS-CoV-2 vaccination. Since both low CD19+ B lymphocyte count and CD38-directed therapy negatively impact vaccination response, booster vaccination seems therefore of utter importance.

Pathogenesis and treatment of multiple myeloma bone disease.

Multiple myeloma (Plasma cell myeloma), a malignancy of the plasma cells, exhibits tumor expansion preferentially in the bone marrow and the development of bone-destructive lesions. Multiple myeloma is still an incurable disease with changes in the bone marrow microenvironment in favor of the survival and proliferation of multiple myeloma cells and bone destruction. In this review, we described the recent findings on the regulators involved in the development of myeloma bone diseases, and succinctly summarize currently available therapeutic options and the development of novel bone modifying agents for myeloma treatment.

Multiple Myeloma Relapse Is Associated with Increased NFκB Pathway Activity and Upregulation of the Pro-Survival BCL-2 Protein BFL-1

Simple SummaryMultiple myeloma (MM) is a blood cancer that is still incurable because patients become insensitive to available treatments. The cancerous cells in MM misuse a signaling route, called the NFκB pathway, to make MM more difficult to treat. In our study, we used a new method to measure NFκB pathway activity in cancer cells of MM patients at different stages of disease. We found that NFκB pathway activity remains unchanged during disease development, is independent of the life expectancy of MM patients, and does not predict how well the cancer cells will respond to treatment. However, the cancer cells that survive after treatment of MM patients do show higher NFκB pathway activity. In a subgroup of these patients, the cancer cells also showed higher BFL-1 gene expression. BFL-1 can enhance cancer cell survival after treatment and may therefore be a potential new candidate to target in these patients.Multiple myeloma (MM) is a hematological malignancy that is still considered incurable due to the development of therapy resistance and subsequent relapse of disease. MM plasma cells (PC) use NFκB signaling to stimulate cell growth and disease progression, and for protection against therapy-induced apoptosis. Amongst its diverse array of target genes, NFκB regulates the expression of pro-survival BCL-2 proteins BCL-XL, BFL-1, and BCL-2. A possible role for BFL-1 in MM is controversial, since BFL-1, encoded by BCL2A1, is downregulated when mature B cells differentiate into antibody-secreting PC. NFκB signaling can be activated by many factors in the bone marrow microenvironment and/or induced by genetic lesions in MM PC. We used the novel signal transduction pathway activity (STA) computational model to quantify the functional NFκB pathway output in primary MM PC from diverse patient subsets at multiple stages of disease. We found that NFκB pathway activity is not altered during disease development, is irrespective of patient prognosis, and does not predict therapy outcome. However, disease relapse after treatment resulted in increased NFκB pathway activity in surviving MM PC, which correlated with increased BCL2A1 expression in a subset of patients. This suggests that BFL-1 upregulation, in addition to BCL-XL and BCL-2, may render MM PC resistant to therapy-induced apoptosis, and that BFL-1 targeting could provide a new approach to reduce therapy resistance in a subset of relapsed/refractory MM patients.

Development of antibody response to SARS‐CoV‐2 following asymptomatic infection in patients with plasma cell disorders on immunomodulatory therapy

Development of antibody response to SARS‐CoV‐2 following asymptomatic infection in patients with plasma cell disorders on immunomodulatory therapy

Post-Vaccination Anti-SARS-CoV-2-Antibody Response in Patients with Multiple Myeloma Correlates with Low CD19+ B-Lymphocyte Count and Anti-CD38 Treatment

Simple SummaryThe global impact of the current COVID-19 pandemic has led to the impressively rapid development of multiple anti-SARS-CoV-2 vaccines. However, only few data are available regarding the efficacy of anti-SARS-CoV-2 vaccines in patients with hematological malignancies, and, in particular, plasma cell neoplasia. This ongoing observational study aimed to describe the level of post-vaccination anti-SARS-CoV-2-antibodies depending on multiple clinical factors including B lymphocyte count and current therapy of 82 patients with multiple myeloma and related plasma cell neoplasia, after the first dose of anti-SARS-CoV-2 vaccination. A positive SARS-CoV-2 spike protein antibody titer (SP-AbT) was detected in 23% of assessable patients. SARS-CoV-2 SP-AbT was significantly higher in patients with higher CD19+ B lymphocyte counts and current treatment with anti-CD38-antibodies has led to significantly reduced SP-AbT titers. Based on our results, the majority of myeloma patients respond poorly after receiving the first dose of any anti-SARS-CoV-2 vaccination and need booster vaccination.Few data are available regarding the efficacy of anti-SARS-CoV-2 vaccines in patients with hematological malignancies, and particular, plasma cell neoplasia. This ongoing single-center study aimed to describe the level of post-vaccination anti-SARS-CoV-2-antibodies depending on B lymphocyte count, current therapy, and remission status of patients with multiple myeloma and related plasma cell dyscrasia, after the first dose of anti-SARS-CoV-2 vaccination. The 82 patients included in this study received SARS-CoV-2 vaccines (including mRNA- and vector-based vaccines) as a routine measure. After the first vaccination, a positive SARS-CoV-2 spike protein antibody titer (SP-AbT) was detected in 23% of assessable patients. SARS-CoV-2 SP-AbT was significantly higher in patients with higher CD19+ B lymphocyte counts. A cut-off value of ≥30 CD19+ B cells/µL was significantly positive correlating with higher SARS-CoV-2 SP-AbT. In contrast, current treatment with anti-CD38-antibodies has led to significantly reduced SP-AbT titers. Furthermore, in multivariable linear regression, higher age and insufficiently controlled disease significantly correlated negatively with SARS-CoV-2 SP-AbT. Conversely, treatment with immunomodulatory drugs did not harm the development of antibody titers. Based on our results, the majority of myeloma patients respond poorly after receiving the first dose of any anti-SARS-CoV-2 vaccination and need booster vaccination.

Desmoglein-2 expression is an independent predictor of poor prognosis patients with multiple myeloma.

Multiple myeloma (MM) is the second most common haematological malignancy and is an incurable disease of neoplastic plasma cells (PC). Newly diagnosed MM patients currently undergo lengthy genetic testing to match chromosomal mutations with the most potent drug/s to decelerate disease progression. With only 17% of MM patients surviving 10‐years postdiagnosis, faster detection and earlier intervention would unequivocally improve outcomes. Here, we show that the cell surface protein desmoglein‐2 (DSG2) is overexpressed in ~ 20% of bone marrow biopsies from newly diagnosed MM patients. Importantly, DSG2 expression was strongly predictive of poor clinical outcome, with patients expressing DSG2 above the 70th percentile exhibiting an almost 3‐fold increased risk of death. As a prognostic factor, DSG2 is independent of genetic subtype as well as the routinely measured biomarkers of MM activity (e.g. paraprotein). Functional studies revealed a nonredundant role for DSG2 in adhesion of MM PC to endothelial cells. Together, our studies suggest DSG2 to be a potential cell surface biomarker that can be readily detected by flow cytometry to rapidly predict disease trajectory at the time of diagnosis.

Belantamab Mafodotin (GSK2857916) Drives Immunogenic Cell Death and Immune-mediated Antitumor Responses In Vivo.

B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug conjugate approved for the treatment of relapsed/refractory multiple myeloma. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation in vitro and in vivo GSK2857916 treatment enhances intratumor immune cell infiltration and activation, delays tumor growth, and promotes durable complete regressions in immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4-hBCMA). Responding mice are immune to rechallenge with EL4 parental and EL4-hBCMA cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor antigen spreading, which are abrogated upon depletion of endogenous CD8+ T cells. Combinations with OX40/OX86, an immune agonist antibody, significantly enhance antitumor activity and increase durable complete responses, providing a strong rationale for clinical evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune responses, including T-cell-directed checkpoint modulators.

CD56+ Clonal Plasma Cells In Multiple Myeloma Are Associated With Unique Disease Characteristics and Have a Counterpart Of CD56+ Normal Plasma Cells With Increased Maturity

Neural cell adhesion molecule (NCAM)/CD56 plays a crucial role in supporting normal hematopoiesis by mesenchymal stem cells, albeit it is currently suggested that its expression is largely restricted to natural killer cells and a subset of T cells among normal hematopoietic cells. Expression of this molecule by clonal plasma cells (PC) is found in around 50-80% of multiple myeloma (MM) patients and might be associated with a favorable prognosis. The biological function of CD56 expression by clonal - and potentially also normal - PC is still unknown.We studied the immunophenotype of normal (i.e. polyclonal) PC originating from tonsil (n=11), peripheral blood (PB, n=24) and bone marrow (BM, n=24) with 8-color multiparameter flow cytometry. Hereby, a large panel of 41 different monoclonal antibodies directed against antigens involved in differentiation (e.g. CD45, CD138), survival (e.g. CD95, bcl-2) and adhesion/homing (e.g. CXCR4, CD31, CD49d, CD56) was used. This also included analysis of CD56- and CD56+ normal PC populations - the latter was detected in a small PC fraction of all analyzed normal BM samples -. Clinical and biological characteristics of MM with CD56-vs. CD56+ clonal PC were also studied in a cohort of 401 patients enrolled in two prospective trials of the Spanish Multiple Myeloma group.Normal PC originating from tonsil, PB and BM showed a gradual increase in CD44, CD49d and CXCR4 expression, whereas the expression intensities of other markers (e.g. CD45, CD81, CD95, HLADR, CXCR5) were gradually decreased. Normal PC from tonsil and PB were systematically CD56-. In turn, a small fraction of CD56+ PC (median 9% of all PC) was detected in all investigated normal BM samples. CD56+vs. CD56- normal BM PC further showed a more mature phenotype with decreased CD19, CD45 and CD95 expression, but increased CD31, CD44, CD49d, CD138 and bcl-2 expression intensities. These results suggest a more mature immunophenotypic profile, less prone to undergo apoptosis of CD56+vs. CD56- normal BM PC.In MM, CD56 positivity of clonal PC was significantly associated with absence of CD19, CD27 and CD28 expression. Hierarchical cluster analyses using median fluorescence intensities further showed that CD56 expression had the most important impact on clustering of clonal PC by their overall immunophenotypic profile. In addition, we found that cases carrying t(4;14) were virtually always CD56+ (98%) (P < .001). Conversely, presence of t(14;16) and t(11;14) were significantly (P < .001) associated with infrequent CD56 expression (13% and 42%, respectively). CD56 positivity of clonal PC was also associated with other important disease characteristics such as DNA hyperdiploidy by flow cytometry (65% vs. 36%, P < .001) and a higher frequency of major bone lesions (39% vs. 24%, P < .05). Cytogenetic abnormalities and the expression status of CD27 and CD81 - but not of CD56 - were of prognostic value.In summary, we found that there systematically exists a small subset of CD56+ PC in normal BM; such cells displaying a mature immunophenotype vs. CD56- BM PC. These results suggest that CD56-vs. CD56+ MM could mainly represent accumulation of clonal PC at different maturation stages, but also with slightly different cytogenetic patterns. Disclosures:No relevant conflicts of interest to declare.

HIF-2α Upregulates CCR1 to Promote Dissemination of Plasma Cells in Multiple Myeloma

Introduction: Multiple myeloma (MM) disease progression and relapse is dependent on the ability of the MM plasma cells (PC) to leave the bone marrow, re-enter the circulation and disseminate to other sites throughout the skeleton. Recent data suggests that increased bone marrow hypoxia is associated with increased recirculation of MM PC (Azab et. al. Blood 2012), suggesting that hypoxia may act as a stimulus for dissemination of MM PC. However, the mechanisms responsible for this phenomenon remain unclear. In this study, we identified a potential driver of hypoxia-mediated dissemination of MM PC.Methods and results: To identify which genes were regulated by hypoxia in MM PC, we utilised microarrays to assess the effects of HIF-2α overexpression on gene expression in the human MM cell line, LP-1. The most highly upregulated gene was the C-C chemokine receptor 1 (CCR1) (6.5-fold upregulated), the receptor for C-C chemokine ligand 3 (CCL3; also known as macrophage inflammatory protein-1α [MIP-1α]), a potent inducer of MM PC migration in vitro . In addition, overexpression of HIF-2α in the human MM cell line RPMI-8226 resulted in an almost three-fold increase in migration towards CCL3, indicating that upregulation of CCR1 in response to HIF-2α confers an increase in the migratory response to CCL3.Previous studies have indicated that over two-thirds of newly diagnosed MM patients have circulating MM PC, with increased PC numbers associated with a poorer prognosis (Nowakowski et. al. Blood 2005). CCR1 expression on peripheral blood MM PC, as assessed by flow cytometry, positively correlated with the number of circulating MM PC. Moreover, in silico analysis of CCR1 expression in publically-available microarray data from newly diagnosed MM patients found that above median CCR1 expression was associated with poor prognosis in these patients in an independent dataset (p=0.025; HR=2.3 [95% CI: 1.1-4.9]; n=142 patients; E-TABM-1138).To identify the potential role of CCR1 in driving MM PC dissemination, the migration and proliferation of MM PC expressing CCR1 was assessed in vitro and in vivo . The role of CCR1 in tumour growth and dissemination of MM PC was investigated using the C57BL/KalwRijHsd mouse model of MM. In this model, luciferase-expressing mouse 5TGM1 MM PC cells were injected into the tibia, where they establish in the bone marrow and initiate systemic MM disease. Using flow cytometry, our studies show that C57BL/KalwRijHsd mice bearing 5TGM1 CCR1 overexpressing cells (5TGM1-CCR1) have increased numbers of circulating tumour cells, when normalized to tumour burden, compared with mice bearing control 5TGM1 cells. These data suggest that expression of CCR1 drives the egress of MM PC from the bone marrow into the circulation in vivo .Conclusion: This study identified CCR1 as a novel driver for hypoxia-driven MM PC migration both in vitro and in vivo . Furthermore, this study highlights an association between increased CCR1 expression, poorer prognosis and an increase in circulating tumour cells in newly diagnosed MM patients. Importantly, these findings suggest that therapies targeting CCR1 may inhibit MM PC egress from the bone marrow and limit disease progression and relapse. DisclosuresNo relevant conflicts of interest to declare.

MiR- 16 Regulates Crosstalk in NF-κB Inflammatory Signaling between Myeloma Cells and Bone Marrow Macrophages

Introduction: Multiple myeloma plasma cells (MM-PCs) home to and dynamically interact with the bone marrow (BM) microenvironment (BM-ME). Of the different factors secreted by the BM-ME, NF-κB-induced cytokines including IL-6 play a central role in MM-PC survival and resistance to treatments. Previously published data have shown that in the BM of MM patients the presence of CD163+ tumor associated macrophages (TAM) correlates with IL-6 production. The canonical NF-κB pathway is induced by most physiological stimuli to produce low levels of inflammatory cytokines, through the activation of the IκB kinase complex (IKKβ, IKKα, and IKKγ). This pathway seems constitutively activated in TAM, where instead the activation of the non-canonical pathway seems to play a critical role in the anti-cancer phenotype of BM macrophages (BM-Mϕ).Extracellular vesicles (EV) are membrane-covered cell fragments released by all cell types, and recently they have been shown to play a pivotal role in creating a tumor supportive BM-ME. Our group was the first to show that MM cells produce EV (Harshman et.al. 2013). Previous studies have shown that miR- 16 encapsulated inEV(EV-miR- 16) down-modulates IL-6 production in the BM-ME, and we showed that higher levels of circulating miR- 16 is a prognostic factor that correlates with better survival in newly diagnosed MM patients treated with proteasome inhibitor (PI) based therapy (Rocci et al. 2013). Here, we investigated whether MM derived EV can play a pivotal role in supporting monocyte to TAM differentiation and defined the molecular mechanisms of IL-6 modulation by miR- 16 .Methods: Monocyte to TAM differentiation was evaluated by flow cytometry analysis, light microscopy, q-RT-PCR and phagocytosis assays. EV were isolated via a serial centrifugation method. EV RNA content was analyzed by NanoString. EV isolated from producing cells (0.5x106cells/ml) were used to differentiate 1 monocyte (4:1 effector:target ratio). Cytokine arrays in the supernatant of BM-Mϕ and in the BM-Mϕ depleted fractions were performed. Cloning, Western blot and RT-PCR were used to confirm IKK complex targeting by miR- 16 . Single cell flow and NF-kB activation assays were conducted.Results: Our in vitro data show that EV isolated from the supernatant of MM cell lines (MM-EV) can promotemonocyte differentiation to CD163+ TAM. Mϕ differentiation was also observed when peripheral blood monocytes (PB-M) obtained from both MM and healthy donors were treated with EV isolated from the BM acellular fraction of MM patients (n=4). Limited differentiation was instead observed when the acellular fraction was EV-depleted (Fig. 1). Our data show that PB-M differentiation was partially reversed by the addition of an EV-miR- 16 mimic. We found that miR- 16 is dramatically down-regulated when PB-M is induced by macrophage colony stimulating factor to differentiate to TAM, and it is also down-regulated in primary BM-Mϕ as compared to PB-M of the same patients and to the BM-Mϕ of 3 age matched healthy donors. We learned that BM-Mϕ isolated from MM patients are the main source of NF - κB-induced cytokines including IL-6 and IL-10 under unstimulated conditions as well as IL-6, IL-8, IL-10, TNF-α and VEGF under Toll-like receptor stimulated conditions, as the Mϕ-depleted BM cellular fraction had significantly low levels. We also found that miR -16 ectopic expression in MM cells and in a human monocytic cell line downregulates both IKKβ/IKKα protein and mRNA levels, with subsequent downregulation of NF-κB activation signaling and decreased expression of inflammatory cytokines. Further, cell growth and apoptotic assays show that the attenuation of PI-based anti-MM activity by TAM is partially reversed by ectopic miR- 16 in both TAM and MM-PCs.Conclusions: MM-EV support monocyte differentiation to TAM, causing miR- 16 downregulation and an increase in NF-κB inflammatory signaling. We also show that this effect can be impaired by an ectopic miR- 16 mimic. Since constitutive activation of NF-κB decreases MM cells dependency on extrinsic growth factors, at the onset of the disease miR- 16 downregulation in MM-PCs can support NF-kB intrinsic activation and increased fitness of MM cells. Because NF-kB activation in MM-PCs counterbalances the anti-MM activity of PI, it is not surprising that miR- 16 expression can potentiate the killing effect of PI. We are exploring these hypotheses through use of miR -16 knock out mouse models. [Display omitted] DisclosuresHofmeister:Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Karyopharm: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Roche: Research Funding; Thrassos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding. Krishnan:Takeda: Speakers Bureau; Onyx: Speakers Bureau; Sutro: Consultancy; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Equity Ownership, Speakers Bureau.

Analysis of the Metabolic State of Residual Multiple Myeloma Plasma Cells Reveals a Proliferative Subset with Glycolytic Gene Expression

BackgroundTumor cells are characterized by high reactive oxygen species (ROS) levels, low mitochondrial content and increased glycolytic capacity. Whereas cancer stem cells are reported to utilize oxidative phosphorylation (OxPhos) to produce ATP, a metabolic phenotype associated with stemness that constitutes a platform to understand the biology of residual and drug resistant cancer cells.We have shown that residual multiple myeloma plasma cells (MMPCs) contain more mitochondria than normal PCs and have lower ROS levels than MMPCs from active disease. To recapitulate the metabolic characteristics of residual MMPCs, myeloma cell lines (MMCLs) were cultured in medium with galactose substituted for glucose to force cells to use oxidative phosphorylation. These cells exhibited reduced ROS levels, increased mitochondrial content, and were more sensitive to drugs that target OxPhos and more resistant to those that target glycolysis than cells grown in glucose. To detect the transition from dormancy to proliferative disease at the earliest stage, we defined the metabolic state of residual MMPCs.MethodsWe used FACS to analyze ROS (H2DCFDA), superoxide (DHE) and mitochondrial content (Mitotracker) in residual MMPCs (0.28-8.9%) in whole bone marrow from 98 patients receiving therapy. Serum LDH was used as a marker of glycolytic activity and proliferation. Ki67, a proliferation marker, was detected by FACS. We compared glucose uptake (NBDG) by FACS in MMCLs cultured in either glucose or galactose media. Expression of genes associated with glycolysis (HK2, MYC) or OxPhos (PPARGC1a) were detected by qRT-PCR in MMCLs and by gene expression profiling (GEP) in clinical samples.ResultsA subset of residual MMPCs have higher ROS (p=0.0165) and superoxide (p<0.0001) than normal PCs; in most cases this was mutually exclusive. Superoxide levels were lower in residual MMPCs from cases in remission (n=16) than those not in remission (n=82, p=0.0261). Of the cases with archived GEP data; HK2 (p=0.0004) and MYC (p=0.001) expression were weakly correlated with ROS level and PPARGC1a was negatively correlated with mitochondrial content (p=0.003, suggesting low mitochondrial activity). Similarly, MMCLs cultured in galactose had lower expression of MYC and HK2, but unlike residual MMPCs,higher expression of PPARGC1a . MMCLS cultured in galactose had lower glucose uptake than cells cultured in glucose.In an effort, to understand the subset of residual cases with higher ROS levels we developed a metabolic score (MS) by defining quartiles for mitochondrial content, superoxide, ROS and LDH level from measurements of residual MMPCs. High superoxide and mitochondrial content (lower score) were considered to indicate dormancy and high ROS and LDH (higher score) indicate glycolytic and proliferative activity. Of the cases analyzed, the MS of 29% indicated dormancy (≤ -2), 48% had an intermediate MS, and 23% had an MS ≥ 2 indicating glycolytic activity and possible transition to the proliferative state. Residual MMPCs from 10 cases were examined for Ki67 production; 0.1- 12.2% of cells were positive for Ki67, with 3 samples > 4%. The MS for these samples ranged from -3 to +4 including one outlier (low MS, high Ki67), however there is clear potential to phenotype residual cases. Repeated measurements after 3 months in 3 cases showed that the MS for Pt 1 improved from 2 to -3, Pt 2 remained at 2, and Pt 3 had a slight change from -1 to 0, suggesting that in the absence of external stimuli the metabolic state of MMPCs is dynamic and most likely responds during dormancy to cues from the microenvironment.ConclusionsWe examined metabolic and proliferative parameters in residual MMPCs. Our data suggest that in a dormant state, most residual MMPCs have lower ROS and higher superoxide and mitochondrial content. However, some residual cases had higher ROS levels, possibly indicating loss of dormancy and transition to active disease. We developed a metabolic score to define the metabolic and proliferative activity for each case to allow precision targeting of residual MMPCs and will test this in an additional cohort to determine its sensitivity and specificity. Additionally, these parameters will be used to predict risk of progression prior to clinical relapse, and to test drugs targeting dormant residual cells prior to progression. DisclosuresDavies:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Morgan:Bristol Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Transcriptome Alterations of Prospectively Isolated Bone Marrow Stromal Cells Provide Potential Cues to MGUS and Multiple Myeloma Pathobiology

Multiple myeloma (MM) is the second most common hematopoietic malignancy characterized by the clonal population of plasma cells that invade and destroy adjacent bone tissue. Monoclonal gammopathy of undetermined significance (MGUS) is the premalignant precursor of MM with 1% of patients progressing to MM every year. Although both MGUS and MM plasma cells depend on interactions with bone marrow stromal cells for their survival and growth, little is known about the exact mechanisms of this crosstalk and if specific genetic events in the stromal compartment are correlated with disease state.Primary bone marrow mesenchymal stromal cells (BMSC) are essential components of the hematopoietic microenvironment and they give rise to the hematopoietic stroma in vivo. We have previously reported the phenotype of the bona-fide stroma stem cells in human bone marrow (Li et al., Stem Cell Reports, 3(6):965-74, 2014), and surface marker expression profiling studies on primary lin-CD45-CD71-CD235a-CD31-CD271+ BMSC from patients with MM (n=5), MGUS (n=8) and healthy age-matched controls (n=7) demonstrated clear differences in the expression of signature MSC surface markers, such as CD146, CD105, CD106, CD230, CD51, CD73, and W5C5.Motivated by these striking findings we then went on to transcriptionally interrogate prospectively isolated primary BMSC from MGUS and MM patients using RNAseq. Primary lin-CD45-CD71-CD235a-CD31-CD271+ stromal stem cells from patients undergoing diagnostic bone marrow examination (n=43) and age-matched healthy donors (n=7) were isolated from aspirated bone marrow samples by FACS. Fifteen and five of these patients were diagnosed with MGUS and MM, respectively, and RNA was extracted from bulk sorted populations of primary MM-BMSC, MGUS-BMSC and control-BMSC. The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing was used for cDNA synthesis. Sequencing libraries were prepared using Nextera XT DNA Library Prep kit (Illumina) and run on a NextSeq500 system (Illumina) using the NextSeq 500/550 High Output Kit v2 (300 cycles).Unsupervised clustering of the data showed that MGUS-BMSC samples clustered partly with healthy controls, but partly also with MM-BMSC or as a distinct and completely separate cluster. Clearly, some MGUS-BMSC showed features of healthy donor BMSC and some exhibited already a transcriptome profile that was more similar to MM-BMSC. In the next step, we therefore restricted the analysis to those BMSC samples that clustered separately and thus represented the MGUS-, MM- and normal BMSC specific transcriptional profiles. Comparison of the transcription patterns of these three distinct groups (MGUS-BMSC, n=3; MM-BMSC, n=5; control-BMSC, n=5) showed that a total of 3,758 genes were differentially expressed between the three groups. Further clustering of the data into 16 non-overlapping subsets of differentially expressed gene groups showed intriguing expression patterns. In one cluster, 78 genes were downregulated only in the MGUS-BMSC group, whereas three clusters contained genes that were downregulated specifically in MM-BMSC, and two clusters showed genes that were upregulated in both, MGUS-BMSC and MM-BMSC compared to control-BMSC. GSEA analysis revealed enrichment of genes reported to be downregulated in MM plasma cells, genes involved in cell adhesion and angiogenesis as well as genes involved in osteoclast differentiation in MM-BMSC versus MGUS-BMSC. Furthermore, MM-BMSCs were enriched for genes involved in integrin-mediated signaling pathway, cell-adhesion, and growth factors and chemokines associated with MM support compared to both control-BMSC and MGUS-BMSC.Taken together, surface marker profiling of prospectively isolated BMSC identified marked differences between these important stromal elements in MGUS, MM, and normal bone marrow. RNAseq of the stroma stem cells identified genes that were specifically upregulated/suppressed in MM-BMSC compared to MGUS-BMSC and control-BMSC. These genes thus represent potential candidates that might be implicated in the disruption of normal stroma function, thus leading to disease development and progression. Accordingly, studies are underway to investigate the effects of selected genes on stroma-myeloma crosstalk (dys)function. DisclosuresNo relevant conflicts of interest to declare.

Combined Infusion of CD19 and Bcma-Specific Chimeric Antigen Receptor T Cells for RRMM: Initial Safety and Efficacy Report from a Clinical Pilot Study

Background: Relapsed/refractory MM (RRMM) patients remain highly unmet need. Chimeric antigen receptor (CAR) T cells targeting B-Cell Maturation Antigen (BCMA) in pre-clinical trials have displayed encouraging anti-tumor activity. Though CD19 is expressed only rarely on MM plasma cells (PC), rare CD19+ B cells can be identified in MM patients that are clonally related to the MM PC. These clonotypic B cells may exhibit properties of cancer stem cell. Thus, CD19 is also a potential therapeutic target in conjunction with other therapies that target MM PC. This study was designed to observe the safety and efficacy of combined infusion of CD19 and BCMA-Specific CART Cells for RRMM (NCT 03196414).Methods: Human T cells were collected from auto/allo-logous peripheral blood mononuclear cells (PBSC) and cultured with an anti-CD3 monoclonal antibody to activate proliferation. The cells were transduced with recombinant lentiviral verctors which respectively contained the anti-BCMA or anti-CD19 single chain variable fragment (scFv), the cytoplasmic portion of the OX40 and CD28 costimulatory moiety, and the CD3z T-cell activation domain. This is the new third generation CAR technique applied in clinic. In this ongoing, RRMM patients (pts) receive 3 doses of 300mg/m2 of cyclophosphamide and 3 doses of 30mg/m2 of Fludarabine iv qd×3 days on day -5,-4 and -3 as lymphodepleting conditioning , then are infused into CART-19 (1×107/kg on day 0) and CART-BCMA cells as split-dose infusions (40% on day 1 and 60% on day 2). BCMA expression on MM cells and CART-related cytokine release syndrome (CRS) are analyzed by flow cytometry. Levels of CAR-transduced cells are also measured by qPCR using a transgene-specific primer/probe pair. Chimerism (short tandem repeats, STRs) was determined by donor/receptor microsatellite detection for patients with infusion of allogeneic CART cells. Responses are assessed by IMWG criteria. The median follow-up times was 5 (2 ∼; 20) weeks.Results: To date, 8 pts have completed the CART cells infusion (cohort 1). Because pts 04 and 05 are invalid for previous autologous CART-BCMA immunotherapy, CART cells in this trial for these two pts were prepared from relative haploidentical donor peripheral blood T cells. The most common adverse event of CART therapy is acute CRS, which occurred in 8 patients (100%). High-grade CRS was associated with elevated levels of IL-6, IFNγ and IL-10. The cytokine levels increased significantly in two patients (pts 01 and 08) ,especially IL-6 up thousands of times from the baseline. But according to the criterias of CTCAE_V4.02, pt 01 was evaluated grade 2 of CRS and pt 08 was grade 3 due to abnormal renal and blood coagulation function. These two corresponding patients fully recovered within a week after tocilizumab (4mg/kg) treatment. Other outstanding toxicities to date include fatigue (n=8), cytopenia (n=8), anemia (n=6), coagulopathy (n=5), elevated bilirubin and AST(n=4), pulmonary edema (n=3) and pleural effusions and ascites (n=1). All patients did not need to use the vascular active drug. No neurologic complications and treatment related mortality (TRM) occurred in this cohort. There did not happend reactions of Graft-versus-host (GVH) and rejection of CART by host-versus-graft (HVD). STRs is both 2%-3% in pts 04 and 05.Among the 8 infused patients, 5 patients were monitored for a period of more than 4 weeks. 100% (5/5) pts had prolonged cytopenias. 4 out of 5 cases acquired above effects of partial remission (PR), which may be eventually further improved. The three patients who were followed up for less than 4 weeks have an asymptomatic living. Pt 02 had progressive MM with extramedullary lesion persisted in his right psoas muscle mass, with gradually increasing. He received local injection of 0.5×107/kg CART-BCMA cells on day 2 into the tumor mass under ultrasound guidance. Miraculously, 4 weeks later, imaging revealed a complete disappearance of the tumor and free light chain levels in his peripheral blood decreased below the detection limit.Conclusions: Combined administration of auto/allo-logous CART-19 and CART-BCMA cells can be manufactured from heavily-pretreated MM pts, and demonstrate promising in vivo expansion and clinical activity. Toxicities to date can be controlled and reversed, without TRM. Expanded accrual in cohort 1 is ongoing, with updated data to be presented at the meeting. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

T Cells Genetically Modified to Express an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor with a CD28 Costimulatory Moiety Cause Remissions of Poor-Prognosis Relapsed Multiple Myeloma

▪B-cell maturation antigen (BCMA) is expressed by normal plasma cells and the malignant plasma cells of multiple myeloma (MM). We developed the first anti-BCMA chimeric antigen receptor (CAR-BCMA). CAR-BCMA includes an anti-BCMA single-chain variable fragment, a CD28 costimulatory domain, and a CD3-zeta T-cell activation domain (Carpenter et al. Clinical Cancer Research 2013. Vol. 19. 2048-2060). We used a gamma-retroviral vector to genetically modify autologous T cells to express CAR-BCMA. We are conducting a phase I clinical trial of CAR-BCMA T cells. On this trial, patients receive a chemotherapy regimen of 300 mg/m2 of cyclophosphamide and 30 mg/m2 of fludarabine on days -5 to -3 followed by an infusion of CAR-BCMA T cells on day 0. Chemotherapy is administered to increase the activity of CAR T cells by depleting endogenous leukocytes. Twenty-three patients have received CAR-BCMA T cells on one of 4 dose levels, 0.3x106, 1x106, 3x106, and 9x106 CAR+ T cells/kg of bodyweight. All patients treated on the lower dose levels and 2 patients treated on the highest dose level have been reported previously (Ali et al. Blood 2016. 128(13). 1688-1700). This abstract exclusively covers the 13 patients treated to date on the highest dose level of 9x106CAR-BCMA T cells/kg.The median age of the 13 patients was 54 years (range 43-66). The patients had a median of 11 prior lines of therapy. Five patients (12, 19, 20, 23, and 25), had a history of the poor-prognosis chromosome 17p-deletion prior to protocol enrollment. The monoclonal protein produced by each patient's MM and the number of prior therapies of each patient are shown in the table.Toxicities were consistent with prior reports of cytokine-release syndrome (CRS) after infusions of CAR T cells. The most prominent, signs of CRS were fever, tachycardia, hypotension, and reduced cardiac function. CRS grades are in the table. Of thirteen patients, 4 received the interleukin (IL)-6-receptor antagonist tocilizumab to treat CRS; 2 of these 4 patients also received corticosteroids. While CAR-BCMA T-cell toxicity was severe in some cases, the toxicities were mainly limited to the first 2 weeks after CAR-BCMA T-cell infusion. Because of Grade 3 and 4 CRS experienced by some patients with high bone marrow myeloma burdens, we modified the clinical protocol to only allow enrollment of patients with lower myeloma burdens defined as MM making up 30% or less of bone marrow cells. Two patients (16 and 18) experienced delayed neutropenia and thrombocytopenia that started approximately 1 month after CAR T-cell infusion when blood counts had recovered from the chemotherapy administered before CAR-BCMA T-cell infusions. These patients were treated with filgrastim, eltrombopag, and prednisone based on the hypothesis that the cytopenias were caused by CAR T cells in the patient's bone marrow. At the time of delayed cytopenias, 15 percent of Patient 16's bone marrow T cells were CAR+, and 16.5 percent of Patient 18's bone marrow T cells were CAR+. In both cases, cytopenias resolved after approximately 1 month.CAR-BCMA T cells exhibited clear anti-myeloma activity. Nine of 11 evaluable patients obtained objective anti-myeloma responses with 2 stringent complete responses, 5 very good partial responses, and 2 partial responses; the duration of responses varied. The longest response to date is 66 weeks (Table). Eight of 10 evaluable patients obtained minimal residual disease negative status by bone marrow flow cytometry. Consistent with BCMA-specific T-cell activity, plasma cells were reduced on bone marrow core biopsies in all 8 evaluated patients and absent in 4 of these patients 2-3 months after CAR-BCMA T-cell infusion.CAR+ cell levels have been quantified in the blood of patients. CAR T cell levels peaked in the first 2 weeks after infusion and persisted at lower levels for many months in some cases. Cytokines were measured in the serum of all patients. In patients with CRS, multiple cytokines including interferon-gamma, IL-6, IL-8, and IL-17A were consistently elevated in the serum.These results demonstrate that CAR-BCMA T cells can induce responses in poor-prognosis MM. Accrual to this trial continues. Toxicity was significant but limited in duration and controllable. A compelling atribute of CAR T-cells is that they have a mechanism of action different from other MM therapies. The results reported here should encourage research to improve CAR T-cell therapies for MM. [Display omitted] DisclosuresWang:Acerta Pharma: Consultancy, Research Funding; Asana Biosciences: Research Funding; BeiGene: Research Funding; Celgene: Honoraria, Research Funding; Dava Oncology: Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; June Therapeutics: Research Funding; Kite Pharma: Research Funding; Onyx: Research Funding; Pharmacyclics: Research Funding; Proteolix: Honoraria, Research Funding. Kochenderfer:Bluebird bio: Research Funding; Kite Pharma: Research Funding; N/A: Patents & Royalties: I have multiple patents in the CAR field.

Protein Kinase CSNK1A Regulates the Multiple Myeloma Plasma Cell-Microenvironment Interplay

Multiple myeloma (MM) is a plasma cell malignancy characterized by a strong dependency on the bone marrow microenvironment, which makes plasma cells resistant to pro-apoptotic stimuli and favours the myeloma-associated bone disease (MMABD). Alteration of Wnt/β-catenin, Hedgehog (Hh) and NF-κB signaling by plasma cell-intrinsic or bone marrow stroma-delivered signals sustains MM cell growth and contributes to bone homeostasis alterations. Protein kinase CK1α (CSNK1A1, casein kinase 1α) belongs to a family of monomeric Ser/Thr kinases that regulate cellular processes important in cancer biology such as membrane biogenesis, transport of molecules, signal transduction, transcription, translation and the DNA damage response. CK1α is also an essential regulator of the function of the Wnt, Hh and NF-κB signalling pathways. It is known that osteogenic differentiation is dependent on the activation of Wnt/β-catenin and that CK1α exerts a negative regulative role on this pathway. Thus, CK1α could play a role in MMABD representing a rational therapeutic target. We recently demonstrated that CK1α supports MM plasma cells growth, in part through the regulation of AKT and β-catenin. CK1α is overexpressed in MM patients compared to healthy controls, and its inhibition/silencing in plasma cells causes apoptosis and cell cycle arrest (Manni et al ., 2017, Oncotarget 8(9):14604). To investigate if this kinase could regulate osteogenesis, we analyzed the generation of osteocytes from stromal cells in conditions of CK1α inactivation in BM stromal cells (BMSC). We found that at variance from malignant plasma cells, CK1α silencing in bone marrow stromal cells did not impact on cell survival but it enhanced the differentiation potential towards bone formation, inducing an osteoblastogenic transcriptional program. CK1α silencing in BMSC, led to the stabilization of β-catenin, increased mRNA expression of the osteoblast markers RUNX2, SPP1 and BGLAP, and it increased calcium deposits of stromal cells pushed to differentiate towards osteoblasts/osteocytes. Moreover, we observed that the immunomodulatory drug lenalidomide, by inducing CK1α degradation in both MM and stromal cells, potentiated the effects of CK1α silencing in BMSC, further supporting osteoblast formation. Previous papers reported that MM cells overexpress RUNX2, leading to a higher expression of RANKL, which in turn could be responsible of the inhibition of osteoblast activity in the bone marrow microenvironment (Giuliani et al., 2005 ; Blood 106, 2472). Moreover, elevated expression of RUNX2 in MM cells enhanced tumour growth and bone disease (Trotter et al., 2015, Blood 125, 3598). To investigate the effects of CK1α on RUNX2 levels in MM cells, we created a model whereby the BM microenvironment dependent INA-6 cell lines were stably transduced with lentiviral shRNA vectors to obtain CK1α silencing by addition of IPTG and then were cultured in the presence of BM stromal cells. At variance with BMSC, CK1α silencing in MM cells determined a reduction of RUNX2 in MM cells with a consequent RANKL-induced increase of RUNX2 in stromal cells. Therefore, CK1α could have a positive regulative role on RUNX2 expression in MM cells participating in the impairment of osteoblastogenesis and yet a negative regulative role still on RUNX2 levels in BMSC. Altogether, our data provide a mechanistic framework for the function of CK1α in BMSC biology and in the MM-BMSC crosstalk and support a dual beneficial therapeutic role of CK1α inhibition in MM, with the potential of targeting not only the malignant plasma cells but also the MMABD. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Evaluation of MEDI2228, a BCMA-Targeting Pyrrolobenzodiazepine-Linked Antibody Drug Conjugate for the Treatment of Multiple Myeloma

Despite remarkable progress in treatment over the past two decades, multiple myeloma (MM) remains an incurable disease. B-cell maturation antigen (BCMA, TNFRSF17) is a suitable therapeutic target for the treatment of MM due to its restricted expression on normal plasma cells and universal expression in myeloma cells. Targeting BCMA with an antibody-drug conjugate (ADC) is an attractive therapeutic option, combining the specificity of a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer.Here we describe MEDI2228, a novel ADC that targets BCMA. MEDI2228 is composed of a fully human antibody site-specifically conjugated to a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker. Flow cytometry studies show that MEDI2228 is rapidly internalized and trafficked to lysosomes. Upon release, the warhead binds to the minor-groove and cross-links DNA, leading to DNA damage and apoptotic cell death.The anti-tumor activity of MEDI2228 was assessed in vitro in 10 MM and plasma cell leukemia (PCL) cell lines encompassing several cytogenetic subtypes. MEDI2228 was highly active in 8 of 10 MM cell lines tested (IC50 range 6 to 210 ng/mL) including cell lines with high (~19,000 receptors/cell) or low (~930 receptors/cell) expression of BCMA. In addition, MEDI2228 is active in the presence of bone marrow stromal cells, which have been shown to play a role in chemotherapy resistance, and in cell models resistant to Lenalidomide. In vivo antitumor activity was evaluated in 4 separate MM and PCL subcutaneous xenograft models and 2 PCL disseminated xenograft models, where a single injection of MEDI2228 induced tumor regression at doses as low as 0.1 mg/kg.A soluble form of BCMA (sBCMA) has been detected in the serum of MM patients (Sanchez et. al. Br J Haematol 2012) and is comprised of the entire extracellular domain of the molecule (Laurent et. al. Nat Commun 2015). Therefore, sBCMA could diminish the effects of antibody-based therapies. We found that levels of sBCMA are elevated in the sera of MM patients compared with healthy donor controls, with levels ranging from 23 ng/mL to 728 ng/mL. The functional features of sBCMA and recombinant monomeric human BCMA are similar (Laurent et. al. Nat Commun 2015). Therefore, to mitigate the potential effects of sBCMA on the efficacy of our drug, the antibody component of MEDI2228 was selected because it possessed weak binding to recombinant monomeric human BCMA and strong binding to membrane-bound BCMA. The in vitro cytotoxicity of MEDI2228 in the presence of clinically-relevant levels of sBCMA was evaluated using NCI-H929 and MM.1S cells. MEDI2228 was cytotoxic to both MM.1S and NCI-H929 in vitro, killing an average of 95% of tumor cells in the presence of sBCMA at levels up to 720 ng/mL with little impact on IC50. ADCs developed from antibodies that possessed a similar affinity between monomeric BCMA and membrane-bound BCMA exhibited a sBCMA-dose dependent drop in potency, with a 20-fold shift in IC50 in the presence of 720 ng/mL sBCMA.MM is a clonally heterogeneous disease, and previously it has been reported that a small population of CD19+CD138- clonogenic cells can be identified in the bone marrow (BM) of MM patients (Matsui et. al. Blood 2004). We used flow cytometric sorting of CD19 and CD138 populations and outgrowth in methocult to confirm that CD19+CD138- cells are the clonogenic cells in MM BM samples. Furthermore, we found that this population contains the same light chain restriction, genetic translocation, and aberrant phenotype as the MM plasma cells. These cells express BCMA and can be killed by MEDI2228 in methocult outgrowth assays.These data demonstrate that MEDI2228 exhibits potent antitumor activity in preclinical models of MM. Importantly, in vitro experiments suggest that this activity is maintained in the presence of sBCMA. MEDI2228, bearing a potent PBD payload, may effectively target both the bulk myeloma plasma cells as well as the more quiescent, CD19+CD138- clonogenic cells, which may offer an opportunity for more durable clinical response in this genetically heterogeneous disease. DisclosuresKinneer:MedImmune: Employment. Meekin:MedImmune: Employment. Varkey:MedImmune: Employment. Xiao:MedImmune: Employment; Bristol-Myers Squibb: Employment. Zhong:MedImmune: Employment. Breen:MedImmune: Employment. Hurt:MedImmune: Employment. Thomas:MedImmune: Employment. Flynn:MedImmune: Employment. Hynes:MedImmune: Employment. Bezabeh:Salubris Biotherapeutics Inc: Employment; MedImmune: Employment. Chen:MedImmune: Employment. Wetzel:MedImmune: Employment. Chen:MedImmune: Employment. Anderson:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Herbst:MedImmune: Employment. Tice:MedImmune: Employment.

Whole Exome Sequencing of Plasma Cells Precancerosis MGUS and Malignant AL Amyloidosis Reveals Small Number of Shared Mutated Genes

Background: Immunoglobulin light chain amyloidosis (ALA) is a plasma cell dyscrasia that develops, similarly as multiple myeloma (MM), from clinically indistinguishable precancerous stage, monoclonal gammopathy of undetermined significance (MGUS). The specific mutations causing malignant transformation from MGUS to amyloidogenic immunoglobulin producing ALA are still unknown. The next generation sequencing (NGS) technology is a powerful tool that allows comprehensive description of the whole genome sequence. Previous studies have already shown highly overlapping phenotypic profile between ALA and both MGUS and MM and lack of unifying mutations in ALA patients (Paiva, 2016). Here we present whole exome NGS data of aberrant plasma cells (aPCs) obtained from patients with MGUS (n=10) and ALA (n=9). Further comparison of the exome sequences of the precancerosis and malignant stage will help to identify recurrently occurring mutations, novel oncogenic drivers and tumor suppressor genes. This will offer new prognostic markers to distinguish potential malignant plasma cell clones in MGUS that will ultimately lead to the development of fatal ALA.Aims: Revealing the mutational status of aPCs in MGUS and ALA using the whole exome NGS.Material and methods: aPCs and peripheral blood (to exclude germinal mutations) were collected from 9 ALA and 10 MGUS samples. aPCs were separated using fluorescence activated cell sorting and genomic DNA was isolated using AllPrep DNA/RNA Micro Kit (Qiagen) and amplified with REPLI-g Mini (Qiagen). DNA library was prepared using SureSelect Human All Exon V5 Kit and sequenced on Illumina HiSeq 4000 platform with average coverage 50x. Somatic variants were called against human genome version 38 using VarScan v2.0. Only non-synonymous mutations, indels and splice site alterations were considered for further investigation.Results: Using NGS we describe whole exome profile for 9 ALA and 10 MGUS patients. Total number of mutated genes for ALA was 554 (average 69 SNVs per patient) and for MGUS 558 (average 73 SNVs per patient). The number of shared mutated genes between both groups was only 26. Among these genes, we identified 4 functional groups: A) Killer-Cell Immunoglobulin Like Receptors (KIR2DL1, KIR3DL2), B) Mucins (MUC16, MUC3A, MUC6), C) Poly(A) Binding Cytoplasmic Proteins (PABPC1, PABPC3) and D) Serine Proteases (PRSS1, PRSS2, PRSS3). Genetic profiles in ALA and MGUS did not reveal any already known MM driver mutations (KRAS, NRAS, BRAF, FAM46C, TP53 and DIS3), with the exception of one ALA patient possessing mutated DIS3 gene. Importantly, we have observed a significant heterogeneity between the samples, as the number of mutated genes shared by all MGUS or ALA patients was only 7 and 3 genes, respectively.Conclusions: ALA develops from a precancerous stage MGUS. Whole exome sequencing of the aPCs showed comparable number of mutated genes between both disease stages (554 vs. 558). Nevertheless, the mutated genes in both groups were very distinct. We identified only 26 mutated genes shared in both ALA and MGUS. Interestingly, none of these genes was previously linked to tumorigenesis. Moreover, genetic profiles were very heterogeneous among individual patients. Future analysis of the presented data will include comparison with genomic profiles obtained from MM plasma cells to reveal additional insights into the mechanisms of clonal plasma cells development in monoclonal gammopathies.Acknowledgment: This work was supported by the Ministry of Health (15-29667A; DRO - FNOs/2014, DRO - FNOs/2016) and the Ministry of Education, Youth and Sports (Institutional Development Plan of University of Ostrava-IRP201550). DisclosuresHajek:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Pharma MAR: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Higher Expression of Nuclear Cereblon in Bone Marrow Biopsies of Patients with Multiple Myeloma Included in the HOVON-87/Nmsg-18 Trial May be Associated with Longer PFS and OS

IntroductionResponse to treatment in patients with multiple myeloma (MM) is variable. With increasing possibilities of treatment regimens, predictive factors for response are important. Immune modulating agents (IMiDs) require Cereblon (CRBN) for activity. Therefore, the aim of this study is to identify the genes of the CRBN pathway which predict the response to therapy with IMiDs. In this abstract the first results are presented.MethodsParaffin embedded bone marrow biopsies from patients included in HOVON-87/NMSG-18 trial obtained at inclusion were used for this study. In this trial elderly patients with MM were randomized between treatment with Melphalan-Prednisone (MP)-Thalidomide followed by thalidomide maintenance versus MP-Lenalidomide followed by lenalidomide maintenance (Zweegman et al. Blood 2016;127:1109-1116).Bone marrow biopsies were stained with a fully automated dual color, bright-field immunohistochemical (IHC) assay for CRBN, its neosubstrates Ikaros and Aiolos and the downstream targets IRF4 and c-MYC. CD138 was used to identify MM plasma cells in the bone marrow samples. For CRBN, both nuclear and cytoplasmic staining was evaluated. The distribution and intensity of the immunostaining was assessed using the H-score. The H-scores were calculated using the following formula: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)] and range from 0-300 (0-600 for combined cytoplasmic-nuclear CRBN H-score). For the Cox regression analysis H-scores were corrected by dividing these by a factor 100: hazard rates were considered per 100 points increase of the H-score. 113 bone marrow samples were selected so far. Protein levels were compared between patients with complete response (CR) or very good partial response (VGPR) vs partial response (PR) and no change (NC). Statistical analysis was done using univariate Cox regression analysis for progression free survival (PFS) and overall survival (OS), and Mann-Whitney test for comparing response groups. Kaplan-Meier survival curves were generated to illustrate survival.ResultsSo far, bone marrow samples obtained at diagnosis from 34 patients were evaluated. Fourteen patients were treated in the Thalidomide arm vs 20 patients in the Lenalidomide arm. Median age was 72 years [range 60-84]. ISS stages I/II/III were 38%/47%/15% respectively. At the time of analysis, median follow up of the 16 patients still alive was 53 months [range 18-85 months]. Best response on protocol treatment was sCR in 12%, CR in 9%, VGPR in 35%, PR in 29% and NC in 15%.Comparing protein expression across the response groups showed a trend of higher levels of combined cytoplasmic and nuclear CRBN in patients who responded better (sCR/CR/VGPR; median H-score: 360 (90-544)) compared to patients with a worse response (PR/NC; median H-score: 273 (185-471)) Mann-Whitney p-value=0.09). In addition, a trend for increased Aiolos expression to be associated with poorer response was found (Mann-Whitney p=0.08; median H-score sCR/CR/VGPR: 251 (162-260) vs PR/NC: 254 (221-267)).Higher H-score of nuclear staining of CRBN was associated with a longer PFS and OS, with a hazard ratio (HR) of 0.47 for PFS (95% confidence interval (95% CI)=0.24-0.92, p=0.027) and a HR of 0.42 for OS (95% CI=0.19-0.91; p=0.028). Using the median nuclear CRBN expression as an arbitrary cut-off, a significant separation of the lines representing high and low expression could be found for PFS, but not for OS (Figure 1). None of the other markers were associated with survival in the current limited set.Six patients had high-risk FISH (deletion of 17p and/or translocation t(4;14) and/or translocation t(14;16)) out of 24 patients with known FISH status. These patients did not demonstrate a higher or lower expression of any of the markers evaluated, and showed no association with PFS (log rank p=0.2) but a clear association with shorter OS (log rank p=0.008). As expected, no association between arm and survival was seen.ConclusionsIn this study we demonstrate that higher expression of nuclear CRBN in myeloma cells in bone marrow of patients with MM was associated with a superior PFS and OS. These are early data from an ongoing analysis; a total of 113 bone marrow samples were selected up to now. [Display omitted] DisclosuresCouto:Celgene: Employment, Equity Ownership. Ren:Celgene: Employment, Equity Ownership. Wang:Celgene: Employment, Equity Ownership. Qian:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership. Broyl:Amgen: Honoraria; Janssen: Honoraria; Celgene: Honoraria. Zweegman:Amgen: Other: advisory board participation; Takeda: Other: advisory board participation, Research Funding; Janssen: Other: advisory board participation, Research Funding; Celgene: Other: advisory board participation, Research Funding. Sonneveld:Celgene, Amgen, Janssen, Karyopharm, Takeda: Consultancy, Honoraria, Research Funding; Celgene Corporation, Amgen, Janssen, Karyopharm, PharmaMar, SkylineDx: Honoraria; Celgene Corporation, Amgen, Janssen, Karyopharm, SkylineDx, PharmaMar: Consultancy.