Monoclonal Gammopathy Of Undetermined Significance Subjects Research Articles

Bone marrow adipocytes provide early sign for progression from MGUS to multiple myeloma.

Multiple Myeloma (MM) is the second most common hematological malignancy and is characterized by clonal expansion of malignant plasma cells in the bone marrow. In spite of recent advances in the field of MM, the disease has remained incurable. MM is preceded by a premalignant state known as monoclonal gammopathy of undetermined significance (MGUS), with a risk of progression to MM of 1% per year. Establishing a scalable approach that refines the identification of MGUS patients at high risk of progression to MM can transform the clinical management of the disease, improve the patient's quality of life, and will have significant socioeconomic implications. Here, we provide evidence that changes in the bone marrow adipose tissue (BMAT) provide an early sign for progression from MGUS to MM. We employed AI-assisted histological analysis of unstained bone marrow biopsies from MGUS subjects with or without progression to MM within 10 years (n = 24, n = 17 respectively). Although the BMAT fraction was not different between the two groups, bone marrow adipocyte (BMAd) density was decreased in MGUS patients who developed MM, compared to non-progressing MGUS patients. Importantly, the distribution profile for BMAd size and roundness was significantly different between the two groups, indicating a shift toward increased BMAd size and roundness in MGUS patients who developed MM. These early changes in the BMAT could serve as valuable early indicators for the transition from MGUS to MM, potentially enabling timely interventions and personalized treatment strategies. Finally, the AI-based approach for histological characterization of unstained bone marrow biopsies is cost-effective and fast, rendering its clinical implementation feasible.

Peripheral Cellular Immunome Reveals Heterogeneity Spanning Myeloma Spectrum Diseases

INTRODUCTION: Progression from precursor states, termed monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), to multiple myeloma (MM) is facilitated by cross-talk between malignant plasma cells and cellular/soluble components of the immunosuppressive bone marrow milieu, which promotes angiogenesis, bone destruction and immune-impairment. In support of this view, the frequencies of several peripheral immune cell subsets have been shown to be affected by the disease state. We hypothesized that beyond numerical alterations, activation and differentiation state of circulating innate and adaptive immune cell subsets determines progression from pre-malignant to malignant stage.METHODS: We compared multiple compartments of immune cell subsets in the peripheral blood samples of patients with MGUS (n=7), SMM (n=4) and MM (n=7) enrolled on an IRB-approved prospective biospecimen collection protocol of plasma cell disorders. Peripheral blood mononuclear cells obtained from each subject were immunophenotyped using two 14-color flow cytometry panels. Broadly, immune subsets surveyed included NK cell (mature and immature), NK-T cell, γδ T cell (δ9+γ2- and δ9-γ2- subsets), conventional CD4 and CD8 T cell (effector, effector memory and central memory) and regulatory T cell. Expression of activation, inhibition and maturation markers including NKG2A, NKG2D, KIR3D4, KIR2DS4, CD25, CD38, PD1, and Tim3 was analyzed in all relevant subsets. In total 62 immune variables (cell lineage and functional markers) were generated. For comparisons between MGUS, SMM and MM, one-way ANOVA was used. Unsupervised hierarchical cluster analysis was then applied to all samples with selected variables that were differentially expressed among three groups (p<0.1 by ANOVA) using WPGMA and Euclidean distances. Pair-wise comparisons between MGUS+SMM versus MM or MGUS versus SMM+MM were performed using Welch's t-test.RESULTS: Overall, distribution and functional state of three main immune cell lineages - NK cell, CD4+ T and CD8+ T cell - were altered across MGUS, SMM and MM. Compared to subjects with MGUS and SMM, MM patients had fewer circulating NK cells (p=0.007) with a noticeable shift in NK maturation (Immature/mature NK ratio approximately 3 to 1 for MGUS and SMM vs. 1 to 1 for MM, p=0.015). Mature NK phenotype was confirmed by higher expression of NKG2D on NK cell in MM patients (p=0.041). MGUS and SMM subjects shared an increased expression of immune checkpoints Tim3 (p=0.005) on peripheral CD4+ T effector cells and PD1 (p= 0.012) on functional memory cells, compared with MM patients. Levels of circulating activated CD8 T effector cells were increased in SMM and MM patents compared with MGUS subjects (p<0.001). Unsupervised hierarchical clustering with ten immune variables that were differentially expressed among three groups separated 7/7 MM patients and 9/11 MGUS and SMM subjects (Figure).CONCLUSION: Transition from MGUS and SMM to MM is associated with systemic immune alterations in the functional state of innate (NK) and adaptive (CD4 and CD8 effectors) immune cell subsets. This proof of concept study suggests a role of the peripheral cellular immunome as a possible biomarker that might allow for identification of a subset of MGUS and SMM subjects with a high risk of progression to active MM. We are evaluating this approach prospectively in a larger cohort of patients, including the clonality of the T cell repertoire. [Display omitted] DisclosuresBhutani:Prothena Therapeutics: Research Funding; Amgen: Speakers Bureau; BMS: Speakers Bureau; Takeda Oncology: Speakers Bureau. Voorhees:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Oncopeptides: Consultancy; Janssen: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Usmani:Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Array BioPharma: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Honoraria, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Quantitative polymerase Chain reaction profiling of microRNAs in peripheral lymph-monocytes from MGUS subjects

Monoclonal gammopathy of undetermined significance (MGUS) is a pre-malignant abnormality of plasma cells, with increased serum levels of immunoglobulins. Patients with MGUS may evolve to multiple myeloma through a multistep process including deregulated gene expression. microRNAs are small non-coding RNA molecules involved in post-transcriptional regulation of crucial biological processes, such as morphogenesis, cell differentiation, apoptosis, and cancer.This study aimed to evaluate microRNA expression on peripheral lymph-monocytes from MGUS subjects compared with healthy controls using qPCR arrays.Blood samples were collected by venipuncture from fifteen, newly diagnosed MGUS patients and fifteen healthy subjects. A further group (validation group) of six newly diagnosed MGUS patients and five healthy control were enrolled for the validation of miRNAs and their mRNAs target. The study was conducted performing miProfile miRNA qPCR arrays, followed by validation of miRNAs and related mRNA targets through RT-qPCR. The functional interaction between microRNAs and target gene were obtained by Ingenuity Pathways Analysis (IPA). IPA network analysis identified only molecules and relationships experimentally observed in peripheral lymphomonocytes.The following miRNAs :133a-3p, 16−5p, 291−3p, 23a-3p, 205−5p, 17−5p, 7a-5p, 221−3p, 30c-5p, 126a-3p,155−5p, let-7a-5p and 26a-5p, involved in the regulation of genes with a role in lymphocyte homeostasis, cell proliferation, apoptosis, and multiple myeloma (MM) progression, were differently expressed in MGUS with respect to healthy subjects.This miRNA signature and its relative targets could be considered for the formulation of new therapeutic strategies in the prophylaxis or treatment of monoclonal gammopathies.

Platelet RNA Splicing Profiles Can Distinguish IgM MGUS Patients from Healthy Individuals

Introduction Within the general population over 50 years, 3% is affected with a monoclonal gammopathy of undetermined significance (MGUS). Although MGUS generally demands no treatment, interest in treating premalignant stages to prevent progression to overt malignancies has grown. Better insight in processes underlying progression of MGUS is therefore warranted. Platelet RNA profiles have been shown to reflect information on the topical tumor environment in various cancer types, including multiple myeloma (MM). Our aim was to investigate whether IgM MGUS patients can be distinguished from healthy controls (HCs) using platelet RNA splicing profiles, and to relate IgM MGUS splicing profiles to genes associated with Waldenström's Macroglobulinemia (WM). Methods Twenty-four IgM MGUS patients with presence of a paraprotein confirmed through immunofixation consented to participation. The thromboSeq protocol for platelet RNA sequencing (RNA-Seq) based classification (Best et al. Nat Protoc 2019) was used to acquire platelet RNA profiles from all MGUS subjects. RNA was extracted from platelets isolated from whole blood. After reverse transcription and SMARTer amplification, 100 bp single-read sequencing was performed on the Illumina Hiseq 2500 platform. Raw sequencing data were processed with Trimmomatic, aligned to a reference genome with STAR and mapped with HTSeq. Mapping data of 29 age and blood storage time matched HCs were also included in the analysis. Differential expression of spliced RNAs expressed as log2 fold change (logFC) was assessed with a likelihood-ratio ANOVA and visualized in a heatmap constructed with differentially spliced RNAs that passed the particle swarm optimized (PSO) false discovery rate (FDR) threshold. RNA profiles were further explored and related to WM and MM by DAVID gene ontology enrichment analyses. Results Mean age in the MGUS and HC groups was 67.2 (SD 7.7) and 66.9 (SD 6.7) years. Thirteen of 24 MGUS patients suffered from IgM mediated anti-myelin associated glycoprotein (MAG) neuropathy; the other 11 were asymptomatic. Of 3,426 high-abundant spliced RNAs detected, 1,371 demonstrated significant differential expression (FDR &lt;0.05) and allowed perfect distinction of MGUS from HCs after PSO (Fig. 1; P &lt;0.0001). The two distinct MGUS phenotypes (neuropathy vs. asymptomatic) could not be distinguished. Top 50 overexpressed RNA isoforms were mainly involved in apoptotic processes, transcription regulation, cellular defense response and immune response regulation. WM associated genes (logFC in parentheses) BCL7C (1.35), CASP8 (1.75), CD79A (2.65), CD81 (1.58), DUSP1 (2.32), HLA-DRB1 (1.83), JAK3 (0.65), NFKBIA (3.59), NFKB2 (1.63), REL (1.35) and TNFRSF14 (0.75), as well as MM associated JUND (4.61) and CCNL1 (3.11), were overexpressed in MGUS. TNFAIP3, negatively regulating the NF-κB pathway, was highly overexpressed (logFC 4.07). JAK1 and JAK2 mRNAs were mildly though significantly depleted in the MGUS group. We also found overexpression of CCNL2 (1.81), MS4A1 (2.17) and WNK1 (0.79), which have been shown to be enriched in WM relative to IgM MGUS. Top 50 depleted RNAs were mainly involved in protein phosphorylation, intracellular signal transduction and response to tumor necrosis factor. CASP3 (-1.43), CASP4 (-0.62) and TNFSF4 (-0.99) mRNAs, of which overexpression is associated with WM, were depleted in MGUS. Conclusions For the first time, we have demonstrated widespread differential expression of spliced RNAs in platelets of MGUS patients as compared to healthy controls. We found evidence of overexpression of various genes that have been associated with WM. The differences were mainly in pathways related to apoptosis, signaling and immune response regulation. This is in line with findings that balanced elevation of free light chains, possibly due to a chronic inflammatory state, increases risk of developing a monoclonal gammopathy (Kumar et al. Blood Cancer J 2019). Overexpression of NFKBIA, NFKB2 andTNFAIP3 in MGUS possiblysuggests activation of the NF-κB pathway, a key step in MYD88 mediated progression to WM. JAK subtype expression levels may reflect that the JAK/STAT pathway, also important for progression to WM, has not (yet) been aberrantly activated in our MGUS subjects. Future research could be focused on studying the development of MGUS and MGUS progression to WM and MM using differentially expressed splice junctions in platelets. Disclosures Minnema: Amgen: Honoraria; Celgene Corporation: Honoraria, Research Funding; Gilead: Honoraria; Jansen Cilag: Honoraria; Servier: Honoraria.

Hemorheological parameters in Monoclonal Gammopathy of Undetermined Significance (MGUS).

There is scarcity of information about the hemorheological pattern in subjects with Monoclonal Gammopathy of Undetermined Significance (MGUS). This preliminary research is focused on the behaviour of whole-blood and plasma viscosity, haematocrit and erythrocyte deformability in the above clinical condition. We enrolled 21 MGUS subjects (10 women and 11 men; mean age 66.4 ± 11.6 years). In fasting venous blood we examined whole-blood and plasma viscosity at high and low shear rates, haematocrit, the ratios between whole-blood viscosity (at high and low shear rate) and haematocrit × 100, the ratio between plasma viscosity at low and high shear rate, and the erythrocyte deformability expressed as elongation index. By comparing normal controls to MGUS subjects a significant increase in whole-blood viscosity at high shear rate and in plasma viscosity at low shear rate were observed. In MGUS subjects the ratios between the high and low shear rate blood viscosity and haematocrit × 100, as well as the ratio between the low and high shear rate plasma viscosity were significantly higher. In MGUS subjects a marked decrease in erythrocyte deformability was also observed. The alteration of the hemorheological profile found in these subjects might be involved in the pathogenesis of thromboembolic events, which occur with a high frequency in MGUS.

Neutrophils Of Multiple Myeloma Are Dysfunctional and Immunosuppressive

IntroductionMultiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. However the role and function of neutrophils in MM and monoclonal gammopathy of undetermined significance (MGUS) has been poorly investigated. Methodson neutrophils (N) of 65 MM at diagnosis, 74 MGUS and 30 healthy subjects we evaluated, by flow cytometer, phagocytic activity and surface expression of CD64, CD16, CD62L and CD11b, markers of neutrophil activation. We tested also the immunosuppressive properties of N isolated from MGUS or MM patients, through functional assays, based on in vitro co-culture of N isolated from patients and T-lymphocytes from healthy subjects and we evaluated the expression of the immunosuppressive molecule arginase-1 (Arg-1) by RT-PCR. ResultsDespite no differences in the absolute number of N between MM, MGUS and healthy subjects, we found a functional impairment in MM not evident in MGUS patients.The phagocytic activity of MM-N was significantly reduced compared to healthy subjects-N (p<0.001) and MGUS-N (p<0.0001) and restored after induction chemotherapy (p=0.02).Expression of CD64 was significantly elevated in MM-N compared to MGUS-N or healthy subjects-N (p=0.01 and p= 0.007 respectively) and was inversely correlated with phagocytic activity (p=0,01). No differences were observed among MM, MGUS and healthy subjects for the other surface markers evaluated.MM-N exhibit an increased expression of ARG-1 compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity of ARG-1, positively correlated with advanced disease. After PHA-P stimulation,T-lymphocytes isolated from healthy subjects missed the expression of activation markers such CD71, CD69, CD25, CD3ζ in the presence of MM-N for 72 hours, and in a less extensive way in the presence of MGUS-N. ConclusionCompared to controls, neutrophils obtained from MM patients have a reduced phagocytic activity, a greater expression of Arg-1 and exhibit an immunosuppressive function on T lymphocytes. Taken together, these findings indicated that neutrophils may contribute to impairment of immune function that characterizes MM patients. Disclosures:No relevant conflicts of interest to declare.

Evaluation of serum B-cell maturation antigen in multiple myeloma.

e18549 Background: B cell maturation antigen (BCMA) is a receptor whose expression increases during B-cell development and is found on malignant cells from multiple myeloma (MM) patients; however, it has not been identified in human serum. Methods: Bone marrow (BM) aspirates and peripheral blood were obtained from patients with MM, monoclonal gammopathy of undetermined significance (MGUS) and healthy control subjects following informed consent (Western IRB BIO 001). Serum was isolated and analyzed with a BCMA enzyme‑linked immunosorbent assay. Values represent the mean of triplicate experiments. BM mononuclear cells (MCs) were isolated using density‑gradient centrifugation and cultured for 72 h in RPMI1640 supplemented with 10% fetal bovine serum. Our human MM xenograft (LAGκ-2)was grown in SCID mice and treated with bortezomib (BORT) with cyclophosphamide (CY); tumor volume and BCMA levels were determined. Statistical significance of differences observed was determined using a Student’s t test and analysis was determined using GraphPad prism software. Results: We showed that the supernatants of cultured BMMCs from MM patients had high concentrations of BCMA (median = 2,250 pg/ml) whereas normal subjects showed minimal amounts (56 ng/ml; P &lt; 0.0001). The serum BCMA levels from newly diagnosed MM patients (n = 51) had much higher levels (13.74 ng/ml) than among controls (n = 40; median 2.58 ng/ml; P &lt; 0.0001) and MGUS subjects (n = 26; median 5.40 ng/ml; P = 0.005). Notably, protein levels were much higher among patients with relapsed or refractory (R/R) disease (n = 79; median 20.02 ng/ml) compared to those with responsive (&gt; partial response) disease (n = 80; median 4.14 ng/ml; P = 0.0038). Following treatment, patients with responsive disease showed decreases in BCMA levels whereas those with R/R disease showed increases. Following treatment with BORT and CY, we also showed a marked decrease in tumor volume and serum human BCMA levels in mice bearing the LAGκ-2 xenograft whereas untreated animals showed marked increases in tumor size and serum BCMA. Conclusions: This is the first report identifying serum BCMA in any human disease and suggests that these levels may be a novel biomarker for monitoring disease status and therapeutic response of MM patients.

The Nestin Expression in Monoclonal Gammopathies

Abstract 5111 Background.Monoclonal gammopathies (MG) are the most common plasma cell disorders characteristic by a presence of clonal plasma cells (PCs). The nestin is considered to be a marker of multipotent proliferative precursors, but recently its expression was found in PCs of multiple myeloma (MM) as well. Consequences of the nestin expression still remain unclear. Aim.Phenotypic analysis of the nestin expression in the most frequent MGs and its correlation with other markers. Patients and Methods.Total of 45 samples was analyzed [31 newly diagnosed untreated MM patients, 14 untreated monoclonal gammopathy of undetermined significance (MGUS) subjects and 5 non-MG samples]. Whole bone marrow CD38+CD138+ PCs were studied for intracellular expression of nestin and surface expression of CD19, CD20, CD27, CD28, CD33, CD56, and CD117 by flow cytometry. Results.Flow cytometric analysis shown the nestin expression in 21.4 % (3/14) of MGUS and 45.2 % (14/31) of MM patients, on the other hand no expression of nestin was found in non-MG controls. Expression of the nestin was relatively homogeneous, but not highly intensive. The nestin positive PCs did not express CD19 and/or CD20, but expression of CD27 was found in 29.4 % (5/17) cases, CD28 in 11.8 % (2/17), CD33 in 5.9 % (1/17), CD44 in 35.3 % (6/17), CD56 in 88.2 % (15/17), and CD117 in 29.4 % (5/17) cases. Statistical analysis did not found any significant correlation and/or difference in phenotype when compared the nestin positive and negative samples, although the nestin negative samples expressed CD56 less frequently then the nestin positive samples [46.4 % (13/28) vs. 88.2 % (15/17)], respectively. Summary/Conclusion.Our result showed difference in the nestin expression when compared bone marrow of MGUS subjects with MM patients. Although there was no correlation between the nestin positivity and phenotype of PCs, we suppose that the nestin could associate with unfavorable prognosis but further studies are needed to elucidate its clinical implication in MGs. Disclosures:No relevant conflicts of interest to declare.

Activated Protein C Resistance (APC-R) Analyzed as a Continuous Variable Is Significantly Decreased In Patients with Multiple Myeloma (MM) Compared to Normal Individuals

AbstractAbstract 4988 Background:Increased risk of venous thromboembolism (VTE) has been described in multiple myeloma (MM) patients, particularly when exposed to immunomodulatory drugs. Epidemiological studies have showed that monoclonal gammopathy of undetermined significance (MGUS) patients also have an increased risk of VTE compared with normal subjects. Acquired activated protein C resistance (APC-R) is an independent risk factor for VTE in hematologic malignancies. Methods:We reviewed the records of patients with MM and MGUS for APC resistance by PREFAKIT APC-R test. We excluded from the analysis patients with a documented Factor V Leiden mutation. The PREFAKIT APC-R is a plasma-based functional clotting assay based on the ratio of patient clotting time with and without APC which is standardized and reported as normalized ratio (normalized to results obtained on pooled normal plasma which is performed on each run). Results:APC-R results from 33 MGUS and 75 MM patients were compared with 39 normal subjects. The median APC-R for MM, MGUS and normal subjects were 1, 1.05 and 1.1 respectively. MM patients compared to normal subjects, had significantly lower APC-R (P = 0.0016). No significant difference was observed between MGUS and normal subjects (P= 0.11) (Figure 1). Baseline characteristics from the three groups were similar in terms of age, sex, and performance status. Conclusion:APC-R measured as continuous variable shows a statistically significant decrement in patients with paraproteinemias compared to normal subject and correlates to the underline hypercoagulability observed in patients with MGUS and MM. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis

Progress in understanding the biology of multiple myeloma (MM), a plasma cell malignancy, has been slow. The discovery of microRNAs (miRNAs), a class of small noncoding RNAs targeting multiple mRNAs, has revealed a new level of gene expression regulation. To determine whether miRNAs play a role in the malignant transformation of plasma cells (PCs), we have used both miRNA microarrays and quantitative real time PCR to profile miRNA expression in MM-derived cell lines (n = 49) and CD138+ bone marrow PCs from subjects with MM (n = 16), monoclonal gammopathy of undetermined significance (MGUS) (n = 6), and normal donors (n = 6). We identified overexpression of miR-21, miR-106b approximately 25 cluster, miR-181a and b in MM and MGUS samples with respect to healthy PCs. Selective up-regulation of miR-32 and miR-17 approximately 92 cluster was identified in MM subjects and cell lines but not in MGUS subjects or healthy PCs. Furthermore, two miRNAs, miR-19a and 19b, that are part of the miR-17 approximately 92 cluster, were shown to down regulate expression of SOCS-1, a gene frequently silenced in MM that plays a critical role as inhibitor of IL-6 growth signaling. We also identified p300-CBP-associated factor, a gene involved in p53 regulation, as a bona fide target of the miR106b approximately 25 cluster, miR-181a and b, and miR-32. Xenograft studies using human MM cell lines treated with miR-19a and b, and miR-181a and b antagonists resulted in significant suppression of tumor growth in nude mice. In summary, we have described a MM miRNA signature, which includes miRNAs that modulate the expression of proteins critical to myeloma pathogenesis.