Untreated Multiple Myeloma Patients Research Articles

Monoclonal whole IgG impairs both fibrin and thrombin formation: hemostasis and surface plasmon resonance studies.

Monoclonal gammopathies frequently associate with hemostatic alterations. Thrombotic events occur with high incidence particularly upon treatment, while in rarer cases hemorrhagic diathesis can be observed. Thepathology of these tendencies could be caused by thrombocytopenia or hyperviscosity burden of circulating monoclonal antibodies. Studies also suggest interference ofmonoclonal antibodies with primary hemostasis. We isolated monoclonal whole IgG paraproteins from two myeloma patients to observe their effect on thrombin formation and fibrin polymerization. Monoclonal whole IgG was prepared from sera of two newly diagnosed untreated multiple myeloma patients and control normal plasma samples. Fibrin formation was measured using thrombin time and dilute prothrombin time tests and thrombin formation was detected with a fluorimetric thrombin generation assay. In addition, molecular interactions were investigated by surface plasmon resonance (SPR). Thrombin time was prolonged upon addition of monoclonal IgG even at 30 g/L by 12 %, increasing up to 36 % at 60 g/L concentration. Dilute prothrombin time was prolonged by 20 % even at 30 g/L. Thrombin generation assay indicated an impairment in thrombin formation at the presence of monoclonal IgG compared to polyclonal at equivalent concentration. By an SPR assay we determined that both clonality IgG preparations interacted with fibrinogen, however interaction with human thrombin was only detected with monoclonal immunoglobulins (KD=1.03×10-7 M). Here we provide evidence that isolated monoclonal whole IgG from myeloma patients can impair both fibrin and thrombin formation and we demonstrate by SPR assay that it interacts with components of the final phase of the coagulation system.

Low factor XIII levels and altered fibrinolysis in patients with multiple myeloma

BackgroundAcquired factor FXIII (FXIII) deficiency can be immune- or non-immune mediated and may cause severe bleeding symptoms. The incidence of acquired FXIII deficiency and its etiology in patients with multiple myeloma (MM) are poorly understood. ObjectivesTo assess FXIII levels and the balance of fibrinolysis in newly diagnosed, untreated MM and monoclonal gammopathy of undetermined significance (MGUS) patients. MethodsFXIII activity, mixing studies, FXIII-A2B2 antigen, total FXIII-B antigen were measured in platelet-poor plasma from 17 untreated MM patients, 33 untreated MGUS patients, and 30 age and sex-matched healthy controls. Besides routine laboratory measurements, the balance of coagulation and fibrinolysis was evaluated using quantitative fibrin monomer (FM) test, thrombin-antithrombin assay, α2-antiplasmin activity, plasmin-α2-antiplasmin (PAP) complex, D-dimer, plasmin generation assay, clot lysis assay, and ClotPro-TPA test. ResultsFXIII-A2B2 levels were significantly lower in MM patients compared to controls [median (IQR):14.6 (11.2–19.4) vs. 21.8 (17.1–26.4) mg/L, p = 0.0015], whereas total FXIII-B did not differ between groups. Decrease in FXIII activity was parallel to the decrease in FXIII-A2B2. An immune-mediated inhibitory mechanism was ruled out. Free/total FXIII-B was significantly higher in MM patients compared to MGUS and healthy controls, suggesting an etiology of FXIII-A consumption. In MM and MGUS patients, FM, D-dimer, and PAP complex were significantly elevated compared to controls, indicating hypercoagulability and ongoing fibrinolysis. ConclusionsLow FXIII levels due to consumption were observed in MM patients at diagnosis. Hypercoagulability and ongoing fibrinolysis were detected in MM and MGUS, indicating that a disturbed hemostasis balance is already present in the latter benign condition.

Circulating serum microRNAs as biomarkers of drug resistance in multiple myeloma patients treated with bortezomib-based regimens – pilot study

Despite advances in multiple myeloma (MM) treatment, drug resistance remains a clinical challenge. We aimed to develop a prognostic model for bortezomib resistance based on miRNA expression profiling. The study included 40 previously untreated MM patients receiving bortezomib-based regimens (20 treatment-sensitive, 20 resistant). Pretreatment venous blood samples were analyzed for miRNA expression. Differential expression analysis revealed upregulated miR-27b-3p (FC 1.45, p = 0.017) and let-7b-5p (FC 1.44, p = 0.025) in the resistant group. Univariate analysis identified let-7b-5p (OR 3.17, 95%CI: 1.19–11.4, p = 0.04) and miR-27b-3p (OR 4.73, 95%CI: 1.4–26.6, p = 0.036) as risk factors for resistance. The final multivariate model included miR-27b-3p (OR 23.1, 95% CI: 2.8–452, p = 0.015), let-7b-5p (OR 4.38, 95% CI: 1.28–22.2, p = 0.038), and miR-103a-3p (OR 15.3, 95% CI: 1.33–351, p = 0.049). These miRNAs may serve as biomarkers of treatment response in MM. However, external validation is necessary to confirm the clinical utility of our model.

Prothrombotic Profile in Patients with Active Multiple Myeloma and the Impact of Immunomodulators: The Roadmap-MM Study

Backround: Thromboembolism occurs in about 10% of patients with multiple myeloma (MM). The incidence of VTE is higher in newly diagnosed MM patients as compared to those with relapsed or refractory disease and it is higher during the first 3 to 6 months after the initial diagnosis and treatment initiation. Some of the treatments administered to patients with MM are independent risk factors for VTE. Immunomodulatory agents (IMiDs) among anti-myeloma treatments stand out as having a considerable prothrombotic effect. Recognizing the significant risk associated with the use of immunomodulatory agents (the International Myeloma Working Group (IMWG) 2014 statement, and the European Myeloma Network Guidelines in 2015 both included guidance on the prevention of VTE in MM patients who receive IMiDs. The prospective, longitudinal observational study ROADMAP-MM CAT was designed to explore alternative strategies for the development of risk stratification tools in patients with multiple myeloma. To this target we evaluated the baseline profile of hypercoagulability in multiple myeloma patients with various disease status. Blood borne hypercoagulabity partly consisted of enhanced thrombin generation, a common phenomenon in patients with malignancies . It remains to be seen whether this phenomenon appears for multiple myeloma patients during the physical course of the disease. Aim: We conducted a study to explore the relationship between stages of MM and alterations of various thrombosis-related biomarkers in patients with MM and their relationship with MM therapy. Materials and Methods Patients with MM (n=162) were recruited and stratified to the following groups: 59 newly diagnosed treatment-naïve patients (ND), 49 patients receiving IMiDs (IM), 52 in complete remission (CR) and 12 patients in partial remission on IMiDs (PR/IM). Patients on anticoagulant treatment were excluded from the study. The control group (CG) consisted of 30 healthy age and sex-matched individuals. Samples of platelet-poor plasma (PPP) were assessed for thrombin generation (TG) with the TF 5pM PPP-Reagent® on Calibrated Automated Thrombogram (Diagnostica Stago, France). Plasminogen activator inhibitor-1 (PAI-1), soluble endothelial protein C receptor (sEPCR), and soluble vascular cell adhesion molecule-1 (sVCAM-1), Procoag-PPL were measured using antibody-based ELISA kits (Invitrogen International Inc., CA, USA, and Diagnostica Stago, France ). TF expression MPC-derived microparticles (MPC-dMPs) were detected using a Zymuphen MP-TF activity kit (Hyphen BioMed, Neuville sur Oise, France). The upper and lower normal limits (LNL and UNL) were calculated by the mean±2 SD. Results A total of 162 patients were enrolled (age 66.0±12.0 yrs; 53% male). Distribution of disease stage was as follows: 32% ISS I, 23% ISS II, 45% ISS III. Bone disease was present in 71% of patients and 19% of patients had high risk cytogenetic lesions. Patients with ongoing MM (ND, IM, PR/IM) had significantly lower Peak, ETP and MRI as compared to the CG. In contrast, patients in CR had Peak, ETP, MRI values similar to the CG. Patients with PR had lower ETP and MRI values as compared to the CR group. In ND 9% had TG >UNL and 22% had TG<LNL. In IM 10% had TG>UNL and 67% had TG<LNL. None in PR/IM had TG>UNL and 35% had TG<LNL. In CR, 23% had TG>UNL and 12% had TG<LNL. MP-TF levels and PPL were higher in untreated MM (CR) patients compared to control. MP-TF and PPL levels decreased significantly after chemotherapy in PR/IM but MP-TF remained high at CR. The plasma concentrations, PAI-1, sEPCR, and sVCAM-1 were higher in MM patients than controls. , Treatment with IMDs reduced all biomarker levels gradually from PR/IM to CR (Table 1). Conclusion Patients with active MM showed attenuated TG which was enhanced in the presence of IMiDs treatment. Complete remission was associated with normalization of TG. The plasma concentrations, TF-MP , PAI-1, sEPCR, and sVCAM-1 were increased in MM patients suggesting vascular aggression which is followed by release of these markers that could contribute to the procoagulant environment. Attention should be paid to therapy-related thrombosis indeed the effect the suggested role of IMiDs in hypercoagulability does not seem to be confirmed and requires further studies .

Single-cell analysis of the CD8+ T-cell compartment in multiple myeloma reveals disease specific changes are chiefly restricted to a CD69- subset suggesting potent cytotoxic effectors exist within the tumor bed.

Multiple myeloma (MM) is an incurable disease of the bone marrow (BM) characterized by the uncontrolled proliferation of neoplastic plasma cells. While CD8+ T cells have an established role in disease control, few studies have focused on these cells within the MM tumor microenvironment (TME). We analyzed CD8+ T cells in the BM and peripheral blood (PB) of untreated patients with MM and non-myeloma controls using flow cytometry, mass cytometry and single-cell RNA sequencing, using several novel bioinformatics workflows. Inter-tissue differences were most evident in the differential expression of Granzymes B and K, which were strongly associated with two distinct subsets of CD8+ T cells delineated by the expression of CD69, accounting for roughly 50% of BM-CD8+ T cells of all assessed cohorts. While few differences were observable between health and disease in the BM-restricted CD8CD69+ T-cell subset, the CD8+CD69- T-cell subset in the BM of untreated MM patients demonstrated increased representation of highly differentiated effector cells and evident compositional parallels between the PB, absent in age-matched controls, where a marked reduction of effector cells was observed. We demonstrate the transcriptional signature of BM-CD8+ T cells from patients with MM more closely resembles TCR-activated CD8+ T cells from age-matched controls than their resting counterparts.

Application of an artificial intelligence-based tool in [18F]FDG PET/CT for the assessment of bone marrow involvement in multiple myeloma

Purpose[18F]FDG PET/CT is an imaging modality of high performance in multiple myeloma (MM). Nevertheless, the inter-observer reproducibility in PET/CT scan interpretation may be hampered by the different patterns of bone marrow (BM) infiltration in the disease. Although many approaches have been recently developed to address the issue of standardization, none can yet be considered a standard method in the interpretation of PET/CT. We herein aim to validate a novel three-dimensional deep learning-based tool on PET/CT images for automated assessment of the intensity of BM metabolism in MM patients.Materials and methodsWhole-body [18F]FDG PET/CT scans of 35 consecutive, previously untreated MM patients were studied. All patients were investigated in the context of an open-label, multicenter, randomized, active-controlled, phase 3 trial (GMMG-HD7). Qualitative (visual) analysis classified the PET/CT scans into three groups based on the presence and number of focal [18F]FDG-avid lesions as well as the degree of diffuse [18F]FDG uptake in the BM. The proposed automated method for BM metabolism assessment is based on an initial CT-based segmentation of the skeleton, its transfer to the SUV PET images, the subsequent application of different SUV thresholds, and refinement of the resulting regions using postprocessing. In the present analysis, six different SUV thresholds (Approaches 1–6) were applied for the definition of pathological tracer uptake in the skeleton [Approach 1: liver SUVmedian × 1.1 (axial skeleton), gluteal muscles SUVmedian × 4 (extremities). Approach 2: liver SUVmedian × 1.5 (axial skeleton), gluteal muscles SUVmedian × 4 (extremities). Approach 3: liver SUVmedian × 2 (axial skeleton), gluteal muscles SUVmedian × 4 (extremities). Approach 4: ≥ 2.5. Approach 5: ≥ 2.5 (axial skeleton), ≥ 2.0 (extremities). Approach 6: SUVmax liver]. Using the resulting masks, subsequent calculations of the whole-body metabolic tumor volume (MTV) and total lesion glycolysis (TLG) in each patient were performed. A correlation analysis was performed between the automated PET values and the results of the visual PET/CT analysis as well as the histopathological, cytogenetical, and clinical data of the patients.ResultsBM segmentation and calculation of MTV and TLG after the application of the deep learning tool were feasible in all patients. A significant positive correlation (p < 0.05) was observed between the results of the visual analysis of the PET/CT scans for the three patient groups and the MTV and TLG values after the employment of all six [18F]FDG uptake thresholds. In addition, there were significant differences between the three patient groups with regard to their MTV and TLG values for all applied thresholds of pathological tracer uptake. Furthermore, we could demonstrate a significant, moderate, positive correlation of BM plasma cell infiltration and plasma levels of β2-microglobulin with the automated quantitative PET/CT parameters MTV and TLG after utilization of Approaches 1, 2, 4, and 5.ConclusionsThe automated, volumetric, whole-body PET/CT assessment of the BM metabolic activity in MM is feasible with the herein applied method and correlates with clinically relevant parameters in the disease. This methodology offers a potentially reliable tool in the direction of optimization and standardization of PET/CT interpretation in MM. Based on the present promising findings, the deep learning-based approach will be further evaluated in future prospective studies with larger patient cohorts.

FAM46C-mediated tumor heterogeneity predicts extramedullary metastasis and poorer survival in multiple myeloma.

Cancers originate from a single cell according to Nowell's theory of clonal evolution. The enrichment of the most aggressive clones has been developed and the heterogeneity arises for genomic instability and environmental selection. Multiple myeloma (MM) is a multiple relapse plasma cell cancer generated from bone marrow. Although there were accumulating researches in multiple myeloma pathogenesis, the heterogeneity remains poorly understood. The participants enrolled in this study were 4 EMP+ (EMP, Extramedullary plasmacytoma) and 2 EMP- primarily untreated MM patients. Single cell RNA sequencing and analysis were conducted for the single cell suspension, which was sorted by flow cytometry from peripheral blood mononuclear cells or bone marrow cells. In our research, the results of single cell RNA sequencing show that FAM46C determines MM tumor heterogeneity predicting extramedullary metastasis by influencing RNA stability. Further, we integrated and analyzed 2280 multiple myeloma samples from 7 independent datasets, which uncover that FAM46C mediated tumor heterogeneity predicts poorer survival in multiple myeloma.

Patients with multiple myeloma and monoclonal gammopathy of undetermined significance have variably increased thrombin generation and different sensitivity to the anticoagulant effect of activated protein C

BackgroundPatients with multiple myeloma (MM) are at high risk of thrombosis especially when receiving immunomodulatory therapy. Thrombotic risk in patients with monoclonal gammopathy of undetermined significance (MGUS) may also be increased. Although activated protein C (APC) resistance has been linked to an increased risk of thrombosis in MM, little is known about how APC influences thrombotic risk in MGUS. We compared thrombin generation (TG) in MM and MGUS patients to that of healthy controls (HCs) and investigated the exogenous effect of APC on TG in these groups. MethodsHemostasis tests including factor VIII (FVIII) and von Willebrand factor (vWF) levels were measured in platelet-poor plasma in 14 untreated MM patients, 34 MGUS patients, and 30 age and sex-matched HCs. TG assay was performed with or without the addition of APC. ResultsPeak thrombin and velocity index were significantly higher in MM and MGUS patients compared to HCs, while MM patients also had elevated endogenous thrombin potential (ETP). In MGUS cases, ETP and peak thrombin values significantly correlated with FVIII and vWF levels. In the presence of APC, peak thrombin and ETP were reduced in MGUS and control plasmas whereas lagtime and time to peak were significantly prolonged. In contrast, adding APC to MM plasma had no effect on any TG parameters. ConclusionsHypercoagulability was observed in MM and even in MGUS cases with very low monoclonal protein concentration. In MM patients, APC had no effect on TG, but it attenuated TG in MGUS patients.

Myocardial Injury in Multiple Myeloma Patients With Preserved Left Ventricular Ejection Fraction: Noninvasive Left Ventricular Pressure-Strain Myocardial Work.

IntroductionOver one-half of patients with multiple myeloma (MM) die of heart failure or arrhythmia. Left ventricular ejection fraction (LVEF) is used to describe left ventricular systolic function. However, depressed LVEF means advanced stage of left ventricular dysfunction in patients with MM. Left ventricular pressure-strain-derived myocardial work (LVMW) is a novel and noninvasive method for evaluating LV function related to LV dynamic pressure load. MW is assessed by LV MW index (LVMWI), constructive work, wasted work, and LV MW efficiency (LVMWE). In this study, we aimed to investigate the value of LVMW in cardiac function assessment and clinical prognosis of MM patients with preserved LVEF.MethodsA total of 72 subjects, including 40 untreated MM patients with preserved EF (including the thick wall and normal wall groups) and 32 non-MM patients, were enrolled in this study. Laboratory data and clinical history of all the patients were collected. All the patients underwent comprehensive echocardiographic examinations and then LVMWI and LVMWE were calculated. Moreover, cardiac adverse events (CAEs) were observed in MM patients treated with bortezomib-based therapy after 6 months and the prognostic value of MW was assessed.Results(1) LV myocardial global work index (GWI), myocardial global work efficiency (GWE), and global longitudinal strain (GLS) were lower in the thick wall group of patients with MM compared with the normal wall group and controls. Cardiac segmental analysis of LVMWI in patients with MM showed an apical sparing pattern; (2) The area under the curve (AUC) of GWE for judging the disease severity based on the Revised International Staging System (R-ISS) was 0.835 (95% CI: 0.684–0.933, p < 0.05); (3) GWE, LgdFLC, and arrhythmia were independent risk factors of CAEs. The AUC of GWE for predicting CAEs in MM patients treated with bortezomib-based therapy for 6 months follow-up was 0.896 (95% CI: 0.758–0.970, p < 0.05).ConclusionMM Patients with preserved EF had subclinical LV systolic dysfunction, which was worse in the thick wall group. LVMWI was presented as “apical sparing” in patients with MM. A lower LVGWE may have a predictive value for CAEs in patients with MM after 6 months of follow-up.

Prognostic value of 18F-fluorodeoxyglucose positron emission tomography/computed tomography at diagnosis in untreated multiple myeloma patients: a systematic review and meta-analysis.

Multiple myeloma is a clonal B-lymphocyte tumor of terminally differentiated plasma cells. 18F-FDG PET/CT can provide valuable data for the diagnosis, restaging, and evaluate prognosis of multiple myeloma (MM). This meta-analysis aimed to evaluate the prognostic value of pre-treatment 18F-FDG PET/CT at diagnosis in MM patients. Related researches came from Embase, PubMed, and Cochrane Library databases through a systematic search, and the last one was updated on April 26, 2021. Cochran Q test and I-squared statistics were used to test for heterogeneity among the studies analyzed. The fixed model and random model were used to combine results when appropriate. Stata 12.0 was used to perform statistical analysis, and p < 0.05 was considered statistically significant. A total of 16 articles with 2589 patients were included in this study. Our results indicated PET/CT has an excellent prognostic role in MM, that higher SUVmax, more FL and EMD were associated with poor OS and PFS. SUVmax: OS (HR 1.89, 95% CI 1.47-2.44), PFS (HR 1.34, 95% CI 1.18-1.51); Fl: OS (HR 2.65, 95% CI 1.83-3.79), PFS (HR 1.61, 95% CI 1.40-1.86); EMD: OS (HR 2.11, 95% CI 1.41-3.16), PFS (HR 2.18, 95% CI 1.69-2.81). Furthermore, similar results were observed in most subgroup analyzes. Conclusion Pre-treatment 18F-FDG PET/CT examination has prognostic value for myeloma patients and has guiding significance for clinical treatment.

Targeting the Mitotic Checkpoint in Myeloma with OSU-13, a Novel Mps1/Ttk Inhibitor

Despite recent advances, there is still a lack of treatment options for patients with high-risk multiple myeloma (MM), warranting the need for novel therapeutic targets. Monopolar spindle 1 (Mps1), also known as TTK protein kinase, is localized at the kinetochores and centromeres essential for the mitotic spindle checkpoint and centrosome duplication. In cancer cells, high levels of Mps1 help to support aneuploidy, a common malignant trait. In fact, high MPS1 expression correlates to unfavorable prognosis in colon and breast cancers. Here, we report the importance of Mps1 in MM and perform functional characterization of a novel Mps1 inhibitor, OSU-13.To assess the relevance of Mps1 in MM prognosis, we analyzed the CoMMpass database and examined the association of MPS1 expression with clinical outcome and genetic alterations in MM patients (n=769). Elevated MPS1 expression correlates with decreased overall survival (p-value = 0.0001) and decreased event-free survival (p-value < 0.0001). In addition, MPS1 expression is higher in high-risk MM with specific genetic alterations, such as deletion of 17p (p-value = 0.0003), Myc translocation t(8;14) (p-value = 0.02), and gain of 1q21 (p-value = 0.0001).We therefore compared MPS1 expression in eight different MM cell lines and primary CD138 - and CD138 + cells isolated from bone marrow (BM) of four recently diagnosed, untreated MM patients. Gene expression levels assessed by quantitative qPCR were normalized to 18S mRNA internal control, and relative quantification was performed using the ΔΔCt method. MPS1 expression was higher in all MM cell lines compared to the average expression level in primary CD138 + BM plasma cells, with a minimum of 3.2-fold increase in L363 and a maximum of 16.2-fold increase in MM1S. In addition, MPS1 expression was 2-fold higher in CD138 + MM cells compared to CD138 - counterparts from the BM of the same patient, suggesting its selectivity as a target.Next, we characterized the effects of OSU-13, a novel Mps1 inhibitor. In an Mps1 target engagement assay (NanoBRET™) in HEK293 cells, we measured the relative levels of OSU-13-mediated inhibition of Mps1-NanoLuc binding to a fluorescent tracer in comparison to MM clinical candidates. OSU-13 showed EC 50=10 nM, almost 10-fold lower than other agents tested.Endpoint measurement by MTT (3-[4,5- dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate cytotoxicity of OSU-13. MM cell lines were treated with increasing doses of OSU-13 for 72h and cell viability was measured. OPM-2 was the most sensitive cell line (IC 50 = 610 nM), followed by H929 (IC 50 = 1440 nM), RPMI-8226 (IC 50 = 4196 nM), and U266 (IC 50 = 5071 nM).To investigate the kinetics and the mechanisms used by OSU-13 to cause cell death, we treated H929 and OPM-2 cell lines with 500 nM of OSU-13 for 24h, 48h, 72h, 96h, and 120h and assessed both Annexin V-FITC and Zombie-Aqua staining by flow cytometry. Treatment with OSU-13 caused an increase in Annexin V staining as early as 24h in H929 (2.1-fold) and OPM-2 (1.6-fold). Maximum increase of apoptotic cells (Annexin V single positive) was observed after 72h, with a 5-fold increase in OPM-2 and 8.6-fold increase in H929 cell line. Cell death peaked at 120h, with Zombie staining 74% of OPM-2 and 60% of H929 cells. The activity of caspases 3/7 was further assessed in H929 myeloma cells at five time points after treatment with different concentrations of OSU-13. Caspases 3/7 activation started after 48h with 500nM OSU-13, and peaked after 72h post-treatment with the same dose, in agreement with the Annexin staining kinetics results.Finally, we evaluated the in vivo effect of OSU-13 in tumor growth in a H929 MM subcutaneous xenograft model in CB.17 SCID mice. Mice were treated with 5 mg/kg or 10 mg/kg daily dose of OSU-13 per oral gavage for 21 days. Treatment with 10 mg/kg daily dose of OSU-13 produced a 22% tumor growth delay (p<0.05).Collectively, we show here that elevated MPS1/TTK expression correlates with poorer prognosis in MM, and OSU-13, a novel Mps1/TTK inhibitor, induces apoptosis and cell death in MM cell lines, and decreases tumor growth in mice containing H929 cell xenografts. Together, our findings reveal that targeting the mitotic checkpoint using OSU-13 is a potential novel strategy, particularly for high risk MM. DisclosuresNo relevant conflicts of interest to declare.

The Potential Role of Proinflammatory Cytokines and Complement Components in the Development of Drug-Induced Neuropathy in Patients with Multiple Myeloma.

The launch of novel chemotherapeutic agents—in particular, proteasome inhibitors and immunomodulatory drugs—dramatically changed multiple myeloma (MM) therapy, improving the response rate and prolonging progression-free survival. However, none of the anti-MM drugs are deprived of side effects. Peripheral neuropathy (PN) seems to be one of the most pressing problems. Despite extensive research in this area, the pathogenesis of drug-induced peripheral neuropathy (DiPN) has not yet been fully elucidated. In the present study, we aimed to assess the potential relationship between proinflammatory factors and the development of PN in MM patients with particular emphasis on the application of VTD (bortezomib, thalidomide, dexamethasone) regimen. Our analysis identified increased concentrations of CCL2, IL-1β, and IFN-γ in plasma of MM patients during treatment, both with and without symptoms of PN, compared with untreated neuropathy-free MM patients. At the same time, the plasma concentration of IL-1β in patients with neuropathy was significantly increased compared with patients without PN before and during treatment. Moreover, the results were enhanced at the transcript level by performing global mRNA expression analysis using microarray technology. The most significant changes were observed in the expression of genes responsible for regulating immunological and apoptotic processes. An in-depth understanding of the mechanisms responsible for the development of DiPN might in the future reduce the incidence of PN and accelerate diagnosis, allowing the choice of neuropathy-free treatment strategies for MM.

CD200 Is a Potent New Growth Factor in Multiple Myeloma through IGF-1R Interaction

Multiple myeloma (MM) is the second most common hematologic malignancy characterized by the accumulation of clonal plasma cells within the bone marrow.We previously identified that CD200 is expressed in Multiple Myeloma Cells (MMCs) of 78% of newly MMdiagnosed patients and this expression is associated with a bad prognosis. CD200 is a type I immunoglobulin superfamily transmembrane glycoprotein that is expressed by various cell types including B cells and T cells. In tumors cells, interaction of CD200 with its known receptor CD200R, inhibits T-cell mediated immune response and induces immunosuppressive responses. However, the role of CD200 in tumorigenesis is poorly understood. Recently, a study shown that CD200 can induce neuritogenesis and promote neuronal survival of primary neurons through interaction and activation of the fibroblast growth factor receptor.Here, we investigated for the first time the role of CD200 in MM cell biology. First, we identified that CD200 protein expression is present only in 3 out of 9 Human Myeloma Cell lines (HMCLs). Then, we confirmed that CD200 protein is strongly expressed in MMCs of 73% of the 35 previously untreated MM patients and that CD200 is never expressed in the normal plasma cell counterparts.CD200 overexpression using lentiviral vector significantly increased HMCLs cell growth in a serum free culture medium (SYN-H) compared to GFP overexpression (fold increasing: 1.6 and 1.4 at day 4; 2.5 and 2.1 at day 7 for LP1 and XG1 respectively). Furthermore, CD200 overexpression results in significant increasing of the number of colonies for HMCLs CD200positive compared to HMCLs CD200negative in clonogenic assays (4.3 fold and 13.1 fold for LP1 and XG1 respectively, p<0.001). We confirmed also the growth advantage linked to CD200 overexpression in coculture model. These effects are significantly inhibited by a monoclonal antibody (MoAb) against CD200 compared to treatment with a no-relevant anti-IgG1. We also confirmed the significant inhibition of proliferation with the anti-CD200 MoAb using clonogenic assays. The anti-CD200 MoAb has no effect on the GFP transduced HMCLs.To investigate the implication of CD200R in CD200-induced MM cell growth, inducible CD200R depletion was analyzed in CD200positive and CD200negative, using lentiviral vectors. After doxycycline addition, the shCD200R reduced CD200R expression by 95-100% but had no significant effects on the proliferation advantage mediated by CD200 demonstrating that the growth advantage supported by CD200 overexpression is independent of CD200R.Thus, to investigate the possibility of another receptor activated by CD200 interaction, we added different essential MM growth factors to the serum free medium. Addition of fetal calf serum, IGF1 or insulin in SYN-H medium abrogated the growth advantage provided by CD200 overexpression in HMCLs. These data suggest that CD200 could act as a growth factor through interaction and activation of IGF1R. In addition, overexpression of CD200 in a IGF-1R negative XG12 HMCL did not stimulate its cell growth or clonogenic activity. Furthermore, anti-IGF-1R monoclonal antibody completely abrogated the CD200-dependent growth activity at day 4 and at day 7 in LP1CD200positive cell line compared to non-relevant antibody. The anti-IGF-1R MoAb has no effect on control LP1GFPpositive cell line cultured in serum free culture medium.In order to identify a direct interaction between CD200 and IGF-1R in serum free culture conditions, IGF-1R immunoprecipitation in denaturing conditions was performed at day 4 using LP1GFP and LP1CD200 cell lines. CD200 and IGF-1R interaction was identified by mass spectrometry and western-blotting.According to these data, MM patients with CD200high and IGF1Rhigh gene expression have a poor prognosis (OS) compared to patient with only CD200high or IGF1Rhigh expression (P=0.03, 52.7 vs 61.9 months in HM series; and P=0.02, 58.9 vs 68.1 months in TT2 series) and compared to patients with CD200low and IGF1Rlow gene expression (P=0.002, 52.7 vs 71.6 months in HM series; and P=0.01, 58.9 vs 70.2 months in TT2 series). Same results are observed for EFS.Altogether, these data demonstrated a new role of CD200 as a MM growth factor through interaction with IGF1R. Our results suggest that CD200 and IGF1R inhibition by monoclonal antibodies could represent a promising strategy in MM. DisclosuresCartron:Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Celgene: Consultancy, Employment; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding. Hose:Sanofi: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; EngMab: Research Funding.

Dose intensity and treatment duration of bortezomib in transplant-ineligible newly diagnosed multiple myeloma.

Bortezomib-related peripheral neuropathy (PN) affects a relevant proportion of multiple myeloma (MM) patients treated with melphalan, prednisone, and bortezomib (VMP). Empirical dose modifications have attempted to reduce toxicity without compromising efficacy. We retrospectively evaluated the dose-response and dose-toxicity relationships in 114 unselected untreated MM patients intended for treatment with VMP with subcutaneous bortezomib. Sixty-two patients (54%) completed the 9 scheduled cycles. Median treatment duration was 48weeks (range 1-57), cumulative bortezomib dose was 41.8mg/m2 (2.6-67.6) and median dose intensity was 1.0mg/m2 /wk (0.2-2.6). Median progression-free survival (PFS) and overall survival (OS) for the full cohort were 86weeks (95%CI 77-104) and 209weeks (95% CI 157-259) respectively. Patients who progressed <60days after discontinuing bortezomib had received a significantly inferior mean cumulative dose, 34.6mg/m2 than the remaining individuals, 45.5 (P=.023). PFS was significantly improved for patients achieving a very good partial response (VGPR) or better (P=.00007). Additional variables with a prognostic impact on PFS on univariate analysis included completion of the 9 scheduled cycles (P=.00002), patients with at least 50weeks of treatment (P=.02) and patients receiving a cumulative dose of at least 49mg/m2 (P=.05). Achievement of a VGPR (HR 0.23; 95%CI 0.12-0.46; P=.00002) and a cumulative dose of 49mg/m2 (HR 0.46, 95%CI 0.27-0.78; P=.003) were statistically independent prognostic factors for PFS. Toxicity-related treatment dose reductions occurred in 75 individuals (66%). PN was observed in 50 individuals (44.6%), grade 3 in 9 (8%). The only prognostic factor for emergence of PN in multivariate analysis was the presence of baseline PN. Biweekly full-dose treatment in the first cycles has a major impact in depth of response. Depth of response, cumulative bortezomib dose, and treatment duration had an impact in prolongation of PFS.

Monocyte Subsets and Serum Inflammatory and Bone-Associated Markers in Monoclonal Gammopathy of Undetermined Significance and Multiple Myeloma.

Simple SummaryWe investigated the distribution of different subsets of monocytes (Mo) in blood and bone marrow (BM) of newly-diagnosed untreated monoclonal gammopathy of undetermined significance (MGUS), smoldering (SMM) and active multiple myeloma (MM), and its relationship with immune/bone serum-marker profiles. Our results showed decreased production of BM Mo with decreased counts of classical Mo (cMo) in BM and blood of SMM and MM, but not MGUS. Conversely, intermediate and non-classical Mo were significantly increased in MGUS, SMM and MM BM. In parallel, increased levels of interleukin (IL)1β were observed in a fraction of MGUS and SMM, while increased serum IL8 was characteristic of SMM and MM, and higher serum IL6, RANKL and bone alkaline phosphatase concentrations, together with decreased counts of FcεRI+cMo, were restricted to MM presenting with bone lesions. These results provide new insights in the pathogenesis of plasma cell neoplasms and the potential role of FcεRI+cMo in normal bone homeostasis.Background. Monocyte/macrophages have been shown to be altered in monoclonal gammopathy of undetermined significance (MGUS), smoldering (SMM) and active multiple myeloma (MM), with an impact on the disruption of the homeostasis of the normal bone marrow (BM) microenvironment. Methods: We investigated the distribution of different subsets of monocytes (Mo) in blood and BM of newly-diagnosed untreated MGUS (n = 23), SMM (n = 14) and MM (n = 99) patients vs. healthy donors (HD; n = 107), in parallel to a large panel of cytokines and bone-associated serum biomarkers. Results: Our results showed normal production of monocyte precursors and classical Mo (cMo) in MGUS, while decreased in SMM and MM (p ≤ 0.02), in association with lower blood counts of recently-produced CD62L+ cMo in SMM (p = 0.004) and of all subsets of (CD62L+, CD62L− and FcεRI+) cMo in MM (p ≤ 0.02). In contrast, intermediate and end-stage non-classical Mo were increased in BM of MGUS (p ≤ 0.03), SMM (p ≤ 0.03) and MM (p ≤ 0.002), while normal (MGUS and SMM) or decreased (MM; p = 0.01) in blood. In parallel, increased serum levels of interleukin (IL)1β were observed in MGUS (p = 0.007) and SMM (p = 0.01), higher concentrations of serum IL8 were found in SMM (p = 0.01) and MM (p = 0.002), and higher serum IL6 (p = 0.002), RANKL (p = 0.01) and bone alkaline phosphatase (BALP) levels (p = 0.01) with decreased counts of FcεRI+ cMo, were restricted to MM presenting with osteolytic lesions. This translated into three distinct immune/bone profiles: (1) normal (typical of HD and most MGUS cases); (2) senescent-like (increased IL1β and/or IL8, found in a minority of MGUS, most SMM and few MM cases with no bone lesions); and (3) pro-inflammatory-high serum IL6, RANKL and BALP with significantly (p = 0.01) decreased blood counts of immunomodulatory FcεRI+ cMo-, typical of MM presenting with bone lesions. Conclusions: These results provide new insight into the pathogenesis of plasma cell neoplasms and the potential role of FcεRI+ cMo in normal bone homeostasis.

Bortezomib use and outcomes for the treatment of multiple myeloma.

The public subsidy in Australia of bortezomib (Velcade) for untreated non-transplant multiple myeloma patients was based on the VISTA trial. To ascertain the health outcomes of bortezomib in 'real world' transplant-ineligible elderly patients, compared to trial data. Patient and treatment data were extracted from an oncology information system, laboratory information system and medical chart audits for three Queensland public hospitals. We identified 74 patients; the median age was 75 years. Our cohort comprised 47% patients who were International Staging System stage III, 45% at stage II and 8% at stage I. Patients who had comorbidities, such as cardiac disease (41%), pulmonary disease (14%), diabetes (22%), peripheral neuropathy (14%) and other comorbidities (41%) at baseline were included. The common regimens prescribed were VMP, CVD and VD, and most patients (n = 73) received bortezomib on a once-weekly or twice-a-week basis. The overall response rate was 81%. Half (53%) of the patients did not complete their planned therapy due to toxicity (30%), suboptimal response or disease progression (15%), or death on treatment (8%). Overall survival was 40.7 months and progression free survival was 17.7 months. Our patients were older, had worse disease characteristics and more comorbidities than patients in the VISTA trial. While response rates were similar, survival outcomes appeared worse. Bortezomib-based treatment in the real world setting still carries a high risk of toxicity in the elderly population.

An open-label, pharmacokinetic study of lenalidomide and dexamethasone therapy in previously untreated multiple myeloma (MM) patients with various degrees of renal impairment – validation of official dosing guidelines

Lenalidomide is a backbone agent in the treatment of multiple myeloma, but dose adjustment is required for those with renal impairment (RI). We evaluated the pharmacokinetics (PK) and safety of lenalidomide and dexamethasone as frontline pre-transplant induction, with doses adjusted at start of each cycle based on creatinine clearance, as per the official dosing guidelines. After 4 cycles, PK studies showed that patients with moderate RI (30 ≤ CrCl < 60 mL/min) receiving 10 mg dosing may be under-dosed and those with severe RI (CrCl <30ml/min) appeared appropriately dosed initially, but sustained significant decreases in maximum serum concentration (Cmax) after repeated dosing, due to rapid clinical improvement and enhanced drug clearance. PK drug monitoring during cycle 1 may facilitate appropriate and timely dose adjustments. Adverse events rates did not vary based on severity of RI. No patient discontinued lenalidomide for toxicity. This supports the feasibility and safety of frontline lenalidomide in transplant-eligible patients with RI.

Efficacy of Bortezomib plus dexamethasone as a first line treatment in newly diagnosed cases of Multiple Myeloma: A Single Centre Study in a Tertiary Care Hospital

Background: Multiple Myeloma (MM) accounts for 1% of malignant tumors and 10%–15% of hematopoietic neoplasms. Bortezomib, a first in class proteasome inhibitor, induces apoptosis and growth arrest and reverse chemoresistence in Myeloma cell and has demonstrated no irreversible adverse effect on haemopoietic stem cell. Dexamethasone increases the response rate. Thus, Bortezomib plus dexamethasone represent highly effective regimen for previously untreated Multiple Myeloma cases and significantly higher response rates approximately 70%– 90% have been observed.This combination thus may serve the basis of future strands of care in Multiple Myeloma patients.&#x0D; Objective: The aim of the study was to assess the efficacy , safety and tolerability of Bortezomib in newly diagnosed cases of Multiple Myeloma patients in Bangladesh.&#x0D; Materials &amp; Methods: This prospective observational study was carried out in the Haematology department of BSMMU from June 2017 to December 2018. Patients received inj. Bortezomib (1.3mg/m2 ) 4 cycles as an intravenous bolus on days 1,4,8,11 in a three week cycle (twice weekly administration) in indoor and same patients as day care basis in outpatients department. Dexamethasone at 40 mg was given intravenously or orally on the day of and day after inj Bortezomib.A self administered questionnaire containing different set of questions regarding Multiple Myeloma were used for data collection.&#x0D; Results: Among the study population, 93% of patients had anaemia followed by bone pain (86%) and renal impairment (39%). Out of 25 patients,complete response achieved in 13 patients (52%), where 4 patients(16%) showed partial response,6 (24%) showed very good partial response and 2 (8%) patients showed no response. The overall response rate was 92% belonged to partial,very goofd partial and no respone respectively. Death occurred in 3 cases (12%). 5 patients (20%) developed Bortezomib induced peripheral neuropathy.Life threatening intracranial haemorrhage occurred in two patients (8%). Death occurred in 3 cases (12%),2 patients due to intracranial haemorrhage and another from cardiac arrest. In this study,S. creatinine, â2 microglobulin and bony lesion variables showed significant association with treatment response.&#x0D; Conclusion: Bortezomib plus dexamethasone is a highly effective and safe regimen for previously untreated multiple myeloma patients. This novel therapy in myeloma represent a new trearment paradigm targeting both tumor and microenvironment which has markedly improve overall response(OR), long progression free survival (PFS) and overall survival (OS)across in all risk groups. Moreover,it can be administered safely in the outpatient setting provided by clinicians.&#x0D; J Dhaka Medical College, Vol. 28, No.1, April, 2019, Page 34-41

MAGE-A inhibit apoptosis and promote proliferation in multiple myeloma through regulation of BIM and p21Cip1.

The type I Melanoma Antigen Gene (MAGE) A3 is a functional target associated with survival and proliferation in multiple myeloma (MM). To investigate the mechanisms of these oncogenic functions, we performed gene expression profiling (GEP) of p53 wild-type human myeloma cell lines (HMCL) after MAGE-A knockdown, which identified a set of 201 differentially expressed genes (DEG) associated with apoptosis, DNA repair, and cell cycle regulation. MAGE knockdown increased protein levels of pro-apoptotic BIM and of the endogenous cyclin-dependent kinase (CDK) inhibitor p21Cip1. Depletion of MAGE-A in HMCL increased sensitivity to the alkylating agent melphalan but not to proteasome inhibition. High MAGEA3 was associated with the MYC and Cell Cycling clusters defined by a network model of GEP data from the CoMMpass database of newly diagnosed, untreated MM patients. Comparative analysis of CoMMpass subjects based on high or low MAGEA3 expression revealed a set of 6748 DEG that also had significant functional associations with cell cycle and DNA replication pathways, similar to that observed in HMCL. High MAGEA3 expression correlated with shorter overall survival after melphalan chemotherapy and autologous stem cell transplantation (ASCT). These results demonstrate that MAGE-A3 regulates Bim and p21Cip1 transcription and protein expression, inhibits apoptosis, and promotes proliferation.

Analysis of Intestinal Microbiome in Multiple Myeloma Reveals Progressive Dysbiosis Compared to MGUS and Healthy Individuals

Background: In addition to acquired genetic changes in neoplastic plasma cells, progression of MGUS to multiple myeloma (MM) is mediated by changes in the proportion of dominant subclones whose expansion is driven by their interaction with the immune microenvironment. Experiments in mouse models of MM have identified a significant contribution to the microenvironment mediated via the gut microbiome and IL-17 responses (Calcinotto et al. Nat Commun 2018;(9)4832:1-7). Despite these preclinical studies, little is known about the composition of the human microbiome in MM at diagnosis, and we hypothesize that it is abnormal and can serve as an axis for immune dysregulation and selection of clonal B cells. In this study we analyzed gut microbial diversity in patients with MGUS and MM and compared these to healthy individuals. Methods: Stool samples were obtained from 10 untreated MGUS and 10 MM patients with ECOG performance status 0-2. Exclusion criteria included those with prior anti-myeloma therapy, primary gastrointestinal disorders, or transient gastrointestinal processes or antibiotic use within the past 3 months. Healthy volunteers from the same geographic region served as controls. 16s rRNA gene sequencing was performed on fecal samples obtained with uBiome Explorer kits processed using Illumina NextSeq500. Qualitative analyses included determination of genera-level operational taxonomic units compared to an established healthy reference range and quantitative assessments of microbial diversity. Cluster analysis was performed using ClustVis (http://biit.cs.ut.ee/clustvis). Results: The mean age at sampling was 74 years for MGUS and 71 for MM. Revised ISS stages I, II, and III for MM patients were 14%, 44%, and 42%, respectively. Median microbial diversity indices showed 84% in MM (range 38-96%), 85% in MGUS (range 42-93%), and 50% in healthy controls (range 23-89%). Supervised clustering of genus-level microbial sequences distinguished specific microbiota separating MM patients from MGUS and healthy controls (Figure). Odoribacter and Lactobacillus were the most enriched genera in MM compared to healthy, conversely Blautia and Faecalibacterium were most reduced. When comparing MM to MGUS, Kluyvera and Bacteroides were found to be most highly represented in MM, whereas Blautia and Parabacteroides showed the greatest decrease compared to MGUS. Data from a MM patient initially enrolled at diagnosis and resampled 4 months after achieving a partial response to induction with VCD showed a decrease in enrichment of Odoribacter from 12.2 to 2.1% as well as Bacteroides from 21.8 to 10.3%. Conclusions: This study identifies distinct changes in the intestinal microbiota that distinguish individuals with MM from its precursor and healthy controls. This is the first time such changes have been identified in untreated MM patients, and some of the genera identified here, such as Faecalibacteria, have been shown to be similarly altered in recent reports of MM patients with persistently active disease despite treatment (Pianko et al. Blood Advances 2019;(3)13:2040-2044). These preliminary studies reveal distinct genus-level differences that exist between MM and MGUS, suggesting increasing dysbiosis with progression to MGUS and MM. We are currently extending these studies to include sequential sampling of additional patients following response to induction chemotherapy. These results provide a rationale for further studying the pathogenic impact of the intestinal microbiome on dysregulation of the MM microenvironment. Figure Disclosures Morgan: Amgen, Roche, Abbvie, Takeda, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: research grant, Research Funding. Braunstein:Celgene: Consultancy, Other: Advisory Boards; Amgen: Consultancy, Other: Advisory Board; AstraZeneca: Consultancy, Other: Advisory Board; Janssen: Consultancy, Other: Advisory Board, Research Funding; Verastem: Consultancy, Other: Advisory Board; Takeda: Consultancy, Other: Advisory Boards.