Monoclonal Gammopathy Of Undetermined Significance Patients Research Articles

IS IT POSSIBLE TO PREDICT TUMOR PROGRESSION THROUGH GENOMIC CHARACTERIZATION OF MONOCLONAL GAMMOPATHY AND SMOLDERING MULTIPLE MYELOMA?

The study of monoclonal serum proteins has led to the generation of two major theories: one proposing that individuals who had monoclonal proteins without any symptoms or evidence of end-organ damage have a benign condition; the other one suggesting that some individuals with asymptomatic monoclonal proteins may progress to multiple myeloma and thus are affected by a monoclonal gammopathy of undetermined significance (MGUS). Longitudinal studies of subjects with MGUS have supported the second theory. Subsequent studies have characterized and defined the existence of another precursor of multiple myeloma, smoldering multiple myeloma (SMM), intermediate between MGUS and multiple myeloma. Primary molecular events, chromosome translocations, and chromosome number alterations resulting in hyperploidy, required for multiple myeloma development, are already observed in myeloma precursors. MGUS and SMM are heterogeneous conditions with the presence of tumors with distinct pathogenic phenotypes and clinical outcomes. The identification of MGUS and SMM patients with a molecularly defined high risk of progression to MM offers the unique opportunity of early intervention with a therapeutic approach on a low tumor burden.

Decreased neutrophil function in newly diagnosed multiple myeloma patients is restored with lenalidomide therapy.

Bacterial infections are common and a major cause of morbidity and mortality in multiple myeloma (MM). We have investigated the function of polymorphonuclear leukocyte (PMN), the immune system's first line of defense against bacteria, in peripheral blood (PB) and bone marrow (BM) samples from patients with newly diagnosed MM (NDMM), smoldering MM (SMM), monoclonal gammopathy of undetermined significance (MGUS) and healthy controls. Phagocytosis and oxidative burst in PMN cells from patients and healthy donors were investigated using PhagoTest and PhagoBurst assay. PMN from NDMM, SMM, and MGUS patients had reduced phagocytosis and oxidative burst ability compared with healthy controls. The dysfunction was most prominent in BM samples from MM, SMM, and MGUS patients. Importantly the reduced phagocytosis in MM patients was restored in patients on lenalidomide therapy. Consistently the ability of Escherichia coli stimulated oxidative burst in BM was reduced for the MM, SMM, and MGUS cohort in contrast to the healthy controls and the patients on lenalidomide treatment. Our results show that MM patients have neutrophil dysfunction that could contribute to susceptibility for bacterial infections and that lenalidomide therapy was associated with restored PMN function.

Abstract 6848: Analysis of the bone microenvironment cells by single cell transcriptomics reveals altered features in multiple myeloma patients compared to pre-malignant monoclonal gammopathies

Abstract Multiple myeloma (MM) is a malignant plasma cell dyscrasia that can be preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). How the bone microenvironment (BME) changes and contributes to tumoral progression in MM remains unresolved. Published data on BME are mainly produced in vitro and few data are on MGUS/SMM patients. Up to date, no single-cell RNA sequencing database of the BME cells has been described in MM. The aim of this project was to characterize for the first time at single cell level, the BME in patients with newly diagnosed MM (MMD) and MGUS/SMM to identify alterations involved in tumoral progression. From 15 bone biopsies of MMD, MGUS and SMM patients, we depleted CD235a+, CD45+, CD31+, and CD138+ cells to enrich the rare BME non-hemopoietic cells. The CD45-CD31-CD235a-CD138- cells were analyzed by scRNAseq. Data were generated on Chromium 10X Genomics. Cellranger and Seurat pipeline in R software has been used. Cell identities were assigned by manual curation. The trajectory analysis has been made by Monocle3. Decouple R and FindMarkers were used to compare pathways, and differential gene expression (DGE) of the samples throughout the different conditions. A total of 35723 cells were profiled. We identified 13 BME cell type clusters: 7 mesenchymal stromal cells (MSCs) clusters (proliferating MSC, MSC DDK-OPG-, MSC DKK+, MSC RUNX2-, MSC OPG+, MSC Osteogenic, Perivascular MSC) and 6 osteoblasts (OBs) clusters (OB precursor, OB precursor LEPR+, OB BGLAP+, OB SPP1+, OB NFkB+, OB late). Psdeudotime analyses highlighted a complex trajectory of differentiation from more immature cell clusters (proliferating MSC) to late OB cluster, confirming the ability of the dataset to snapshot the in vivo complexity of BME and we highlighted an alteration of the MSCs clusters trajectories starting in SMM samples till a disruption in MM. Pathway analyses, in MMD compared to MGUS/SMM, revealed an upregulation of VEGF and PI3K activity in proliferating MSC, perivascular MSC and OB clusters. TRAIL pathway activity, known to suppress MM cell proliferation, was upregulated in MSC osteogenic and OB BGLAP+ in MGUS. Finally, DGE analyses revealed the modulation of different genes, many not yet described in MM. MGUS MSCs clusters showed an upregulation of FBLN1, SERPNF1 and MMD MSCs clusters dramatically downregulated WISP2 and upregulated SRGN, involved in MM cell proliferation. Interestingly, in SMM samples, we reported an upregulation of genes involved both in the promotion of osteoblastogenesis (SCARA3, SPON2) and inhibition (GREM1), likely underling the attempt to contrast the establishment of altered BME, characteristic of MM. Our approach is able to dissect the complex organization of the BME and to highlight at single cells level the alterations of the BME in patients with MM compared to MGUS and SMM. Citation Format: Paola Storti, Tommaso Torelli, Cristina Manferdini, Denise Toscani, Alessandro Guidi, Rosanna Vescovini, Nicolas Thomas Iannozzi, Benedetta Dalla Palma, Matteo Scita, Federica Librale, Stefania Ricci, Vincenzo Raimondi, Oxana Lungu, Giannalisa Todaro, Gabriella Sammarelli, Emanuela Aleo, Gina Lisignoli, Luca Agnelli, Nicola Giuliani. Analysis of the bone microenvironment cells by single cell transcriptomics reveals altered features in multiple myeloma patients compared to pre-malignant monoclonal gammopathies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6848.

The effects of short-term, progressive exercise training on disease activity in smouldering multiple myeloma and monoclonal gammopathy of undetermined significance: a single-arm pilot study.

High levels of physical activity are associated with reduced risk of the blood cancer multiple myeloma (MM). MM is preceded by the asymptomatic stages of monoclonal gammopathy of undetermined significance (MGUS) and smouldering multiple myeloma (SMM) which are clinically managed by watchful waiting. A case study (N = 1) of a former elite athlete aged 44 years previously indicated that a multi-modal exercise programme reversed SMM disease activity. To build from this prior case study, the present pilot study firstly examined if short-term exercise training was feasible and safe for a group of MGUS and SMM patients, and secondly investigated the effects on MGUS/SMM disease activity. In this single-arm pilot study, N = 20 participants diagnosed with MGUS or SMM were allocated to receive a 16-week progressive exercise programme. Primary outcome measures were feasibility and safety. Secondary outcomes were pre- to post-exercise training changes to blood biomarkers of MGUS and SMM disease activity- monoclonal (M)-protein and free light chains (FLC)- plus cardiorespiratory and functional fitness, body composition, quality of life, blood immunophenotype, and blood biomarkers of inflammation. Fifteen (3 MGUS and 12 SMM) participants completed the exercise programme. Adherence was 91 ± 11%. Compliance was 75 ± 25% overall, with a notable decline in compliance at intensities > 70% V̇O2PEAK. There were no serious adverse events. There were no changes to M-protein (0.0 ± 1.0g/L, P =.903), involved FLC (+ 1.8 ± 16.8mg/L, P =.839), or FLC difference (+ 0.2 ± 15.6mg/L, P =.946) from pre- to post-exercise training. There were pre- to post-exercise training improvements to diastolic blood pressure (- 3 ± 5 mmHg, P =.033), sit-to-stand test performance (+ 5 ± 5 repetitions, P =.002), and energy/fatigue scores (+ 10 ± 15%, P =.026). Other secondary outcomes were unchanged. A 16-week progressive exercise programme was feasible and safe, but did not reverse MGUS/SMM disease activity, contrasting a prior case study showing that five years of exercise training reversed SMM in a 44-year-old former athlete. Longer exercise interventions should be explored in a group of MGUS/SMM patients, with measurements of disease biomarkers, along with rates of disease progression (i.e., MGUS/SMM to MM). https://www.isrctn.com/ISRCTN65527208 (14/05/2018).

Clinical Significance of Serum Free Light Chain in Monoclonal Gammopathy

To investigate the expression and clinical significance of serum free light chain (sFLC) in patients with monoclonal gammopathy (MG). The peripheral blood of 98 patients with MG and 30 healthy volunteers were collected. The level of sFLC was detected by immunoturbidimetry, and the value of sFLC in diagnosis, disease severity, and efficacy evaluation was analyzed. Among 98 MG patients, there were 58 males and 40 females, 45 cases of monoclonal gammopathy of renal significance (MGRS), 33 cases of monoclonal gammopathy of undetermined significance (MGUS), 20 cases of hematological malignancy (HM), 58 cases of IgG type, 26 cases of IgA type, 7 cases of IgM type, 5 cases of light chain type, 2 cases of non-secreting type, 35 cases of κ type, 63 cases of λ type, 53 cases of renal insufficiency, 45 cases of normal renal function. The expression levels of sFLC-κ and sFLC-λ in MG patients were significantly higher than those of the control group (P <0.01). The expression levels of sFLC-κ and sFLC-λ in HM patients were significantly higher than MGRS and MGUS patients, and in MGRS patients were also significantly higher than MGUS patients (P <0.05). Patients with abnormal renal function had higher expression levels of sFLC-κ and sFLC-λ than patients with normal renal function (P <0.01). sFLC-κ and sFLC-λ were positively correlated with the expression level of globulin (r =0.392, r =0.435) and β2-MG (r =0.403, r =0.468) in MG patients, as well as serum creatinine in patients with abnormal renal function (r =0.586, r =0.631), while no significant correlation was found with age, sex, albumin, lactate dehydrogenase, and serum calcium. After treatment, the levels of sFLC-κ and sFLC-λ were significantly decreased (P <0.01). sFLC is significantly elevated in MG patients and can be quickly detected with high sensitivity, which is helpful for the diagnosis of disease type, judgment of disease severity, and evaluation of therapy.

Establishing Monoclonal Gammopathy of Undetermined Significance as an Independent Pre-Disease State of Multiple Myeloma Using Raman Spectroscopy, Dynamical Network Biomarker Theory, and Energy Landscape Analysis.

Multiple myeloma (MM) is a cancer of plasma cells. Normal (NL) cells are considered to pass through a precancerous state, such as monoclonal gammopathy of undetermined significance (MGUS), before transitioning to MM. In the present study, we acquired Raman spectra at three stages-834 NL, 711 MGUS, and 970 MM spectra-and applied the dynamical network biomarker (DNB) theory to these spectra. The DNB analysis identified MGUS as the unstable pre-disease state of MM and extracted Raman shifts at 1149 and 1527-1530 cm-1 as DNB variables. The distribution of DNB scores for each patient showed a significant difference between the mean values for MGUS and MM patients. Furthermore, an energy landscape (EL) analysis showed that the NL and MM stages were likely to become stable states. Raman spectroscopy, the DNB theory, and, complementarily, the EL analysis will be applicable to the identification of the pre-disease state in clinical samples.

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.

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.

Outcomes of Septuagenarians and Octogenarians with Monoclonal Gammopathy of Undetermined Significance Admitted for Acute Pulmonary Embolism

Introduction: Monoclonal gammopathy of undetermined significance (MGUS) is clinically defined with a serum monoclonal immunoglobulin of less than 3 g/dL and the presence of less than 10% of plasma cells in the bone marrow, in the absence of any end-organ damage that is typically seen in patients with multiple myeloma. The incidence of MGUS mostly involves the elderly, with a median age estimated at 70 years. While some studies have linked MGUS with a hypercoagulable state, there are no large studies investigating the impact and outcomes of MGUS in the elderly with pulmonary embolism(PE). We thus propose a national analysis to investigate. Methods: Our retrospective study via the National Inpatient Sample (NIS) focused on adults admitted with a primary diagnosis of Acute Pulmonary Embolism. We excluded all cases of ages &amp;lt;70 years and &amp;gt;90 years. Patient characteristics in acute PE cases with and without MGUS such as age, sex, race, and presence of various comorbidities such as diabetes, hypertension, obesity, among many, were explored via Chi-Square tests, t-tests, and Mann-Whitney U tests as appropriate. Differences in outcomes for PE in MGUS and non-MGUS patients were estimated via logistic regression models on SPSS 29.0. A p-value of &amp;lt;0.05 was considered as statistically significant. To comply with the database provider's (HCUP) guidelines, we did not report values &amp;lt;11. Results: Among the 327090 septuagenarians and octogenarians admitted primarily for acute PE in our study, there were 1095 (0.3%) patients with a diagnosis for MGUS that also reported a higher mean Charlson Comorbidity Index (CCI) score (4.26 vs. 2.90, p&amp;lt;0.01) (Table 1). While 51.1% of MGUS cases with acute PE were males, the non-MGUS group mainly consisted of females (56.1%, p&amp;lt;0.01). Medicare covered 91.3% and 90.8% of all MGUS and non-MGUS cases in our study, and 68.9% and 66.3% of all admissions involved Urban-teaching centers, respectively. Around 11.0% of MGUS cases also had a history of prior pulmonary embolism, while it was 8.0% in the non-MGUS cohort (p&amp;lt;0.01). The MGUS cohort also showed a lower prevalence of hypertension (41.1% vs.46.7%, p&amp;lt;0.01) while having a higher prevalence of peripheral vascular disease (11.4% vs. 5.1%, p&amp;lt;0.01), smoking status (40.6% vs. 37.8%, p=0.049), lipid disorders (56.6% vs. 50.6%, p&amp;lt;0.01), and chronic kidney disease (35.6% vs. 20.0%, p&amp;lt;0.01) than the non-MGUS group. MGUS patients showed lower odds of experiencing in-patient events such as myocardial infarction (aOR 0.345, 95% CI 0.221-0.538, p&amp;lt;0.01), acute kidney injury (aOR 0.843, 95% CI 0.719-0.990, p=0.037), and respiratory compromise (aOR 0.749, 95% CI 0.652-0.860, p&amp;lt;0.01). While a higher odds of cardiogenic shock (aOR 1.903, 95% CI 1.316-2.753, p&amp;lt;0.01) was also noted, patients with and without MGUS had no differences related to in-hospital mortality (aOR0.721, 95% CI 0.513-1.013, p=0.060) or the need for IVC filters (aOR 1.156, 95% CI 0.924-1.446, p=0.206). MGUS patients also experienced a longer stay (mean length of stay 4.91 vs. 4.62 days, p&amp;lt;0.01). Conclusion: This nationwide data analysis suggests a higher prevalence of a history of previous PE in MGUS cases admitted for PE than those without MGUS. Furthermore, while odds of events involving in-patient myocardial infarction, acute kidney injury, or respiratory compromise in MGUS patients were lower, they had a higher risk of cardiogenic shock. It is thus vital for physicians to be on the watch-out of such complications and provide adequate testing and care in a timely manner to improve their prognosis and outcomes.

Immune Cells, Cytokines, and Gut Microbiota Landscape Along Monoclonal Gammopathy of Undetermined Significance (MGUS) to Multiple Myeloma (MM) Evolution

Introduction: Multiple myeloma (MM) is an incurable hematologic cancer characterized by abnormal plasma cell growth in the bone marrow (BM). It is preceded by two premalignant conditions: monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Understanding the transition from MGUS to MM is crucial for better risk stratification, early interventions, and targeted treatments. This study aims to gain insights into immunological changes and fecal microbiota variations along the MGUS to MM transition. Methods: To comprehensively evaluate the immune composition (myeloid/lymphoid T/B/NK subpopulations and immune checkpoint distribution) in both bone marrow (BM) and peripheral blood (PB), we designed six 10-color custom and two 8-color flow cytometry panels. We analyzed samples from 13 MGUS, 12 SMM, and 63 newly diagnosed MM patients. Additionally, we evaluated cytokine/chemokine abundance in BM and PB using a 48-plex Luminex plate on 72 samples from the same MGUS, SMM, and MM patients, along with 4 healthy donors (HD). Furthermore, we performed fecal microbiome profiling on samples from 3 MGUS, 6 SMM, and 9 MM patients to investigate potential relationships between bacterial composition and MM evolution. Statistical analysis was performed in R by using specific packages (such as FlowCT). Results: Through unsupervised analysis of T cells by flow cytometry, we identified a significant increase in circulating TEMRA CD8 T cells, mostly for CD57+ cells (mean, 7.5% for MGUS vs 20.1% for SMM vs 21.6% for MM; p&amp;lt;0.05) and a significant reduction in naïve CD8 T TIGIT+ cells (mean, 12.4% for MGUS vs 5.98% for SMM vs 4.16% for MM; p&amp;lt;0.001) and naïve CD8 T TIGIT+ TIM3+ (mean, 2.18% for MGUS vs 1.24% for SMM vs 1.17% for MM; p&amp;lt;0.001). Regarding CD4 T subsets, we observed a reduction in BM effector memory phenotype along disease evolution (mean, 7.07% for MGUS vs 4.54% for SMM vs 3.72% for MM; p&amp;lt;0.05). Interestingly, we confirmed the increase of BM CD4 T cells production of IL17 (mean: 6.7% for MGUS vs 22% for MM; p&amp;lt;0.05) in MM patients compared to those with MGUS. In the context of myeloid cell subsets, circulating non-classical monocytes expressing HLA-DR and CD11c (mean, 1.45% for MGUS vs 0.95% for SMM vs 0.69% for MM; p&amp;lt;0.05) as well as granulocytes (mean, 74.18% MGUS vs 73.86% SMM vs 65.59% MM; p&amp;lt;0.05) were found to be reduced along MM progression. No major differences were observed for other evaluated lineages. Regarding cytokines and chemokines, a concurrent decrease in GM-CSF, IFN-a2, IFN-g, IL-1b, IL-2, IL-2Ra, IL-3, IL-10, IL-13, and MCP-1/CCL2 levels within the BM plasma throughout MM progression was observed, thus supporting a global reduction in myeloid function as well as in T cell effector activity. Moreover, PDGF-BB, IFN-a2, IL-16, and IL-8 decrease was observed in PB plasma along MM progression. Lastly, while no differences were observed in term of alpha and beta diversity, we observed an overall increase in total abundance of fecal bacteria along MM evolution (maybe related to loss of myeloid/lymphoid control according to what previously described), with several bacterial genus significantly over-represented in MM patients, including Bariatricus, CAG-83, Alistipes, Bifidobacterium, Streptococcus, Ruminococcus D, Slackia A, and Coprococcus A, while others (such as Parabacteroides B) was found to be more represented in MGUS and SMM patients. The analysis of phyla abundance indicated Firmicutes D and Lactobacillales as more abundant in MM patients, while Actinobacteriota dominated in SMM patients, and Proteobacteria and Bacteroidota were more prominent in MGUS patients. Conclusions: Overall, this study demonstrated a progressive impairment of both myeloid and lymphoid compartment in term of cellular composition, cytokine production and release and control over gut microbiome. Recovering from T cell exhaustion and IL17 production remains a major challenge for these patients.

Stable MGUS and SMM Are Characterized By Distinct Senescent Features in the Pre-Malignant Plasma Cells and the Proximate Bone Microenvironment

Multiple Myeloma (MM) progresses from monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM). Despite similar primary genetic events as MM, MGUS and SMM PCs are non- or low-proliferative. Since oncogene activation and DNA damage drive senescence growth arrest to promote immune clearance of potentially malignant cells, we hypothesized that MGUS and SMM pre-malignant PCs exhibit senescence features. To test this, we performed gene set enrichment analysis (GSEA) of a published human PC gene array dataset (GSE5900) comparing normal, MGUS, and SMM PCs for custom senescence phenotyping gene sets (Senescence Up, SenUp; Senescent Cell Anti-Apoptosis Pathways, SCAPs; Senescence Growth Arrest, SenGA; Inflammatory Senescence Associated Secretory Phenotype, iSASP; Interferon SASP, IFN-SASP) versus biological aging gene sets (SenMayo; CellAge). MGUS and SMM PCs exhibited significant enrichment (NES&amp;gt;1.5, q&amp;lt;0.05) for senescence gene sets SenUp, SenGA, and SCAPs compared to normal PCs, with no enrichment for aging gene sets. MGUS PCs were also enriched for iSASP (NES=1.4, q=0.11), whereas SMM PCs were enriched for IFN-SASP (NES=2.3, q&amp;lt;0.05), characteristic of late-senescence. Thus, both MGUS and SMM PCs show enrichment of senescence gene sets compared to normal PCs with differential SASP profiles. Next, we evaluated MGUS, SMM, and MM patient PCs isolated by magnetic sorting for histological senescence features. PCs were immunostained for LMNB1 and HMGB1, and fluorescent in situ hybridized for α-satellite. Cellular senescence was defined as loss of ≥50% nuclear membrane LMNB1, loss of HMGB1, and ≥2 senescence-associated distensions of satellite (SADS) per nuclei. PCs from MGUS patients (N=12) exhibited increased percentage of senescent PCs (26.9±4.1%, Mean±SEM, p&amp;lt;0.0001) compared to SMM (11.0±1.9%, N=19, p&amp;lt;0.0001) and MM (11.6±1.5%, N=11, p&amp;lt;0.001). A subset analysis of SMM patients for which disease stability was known (≥2 years, N=6-8) showed no differences. These results demonstrate that the percentage of senescent PCs is increased in MGUS but not SMM PCs compared to proliferative disease. To assess PC senescence in the native bone marrow microenvironment (BMME), we evaluated trephine bone biopsies from MGUS and SMM patients that progressed or not to MM ≤10 years, as well as newly diagnosed MM. Biopsies were immunostained for CD138, LMNB1, and HMGB1. Whole biopsy images were analyzed using the artificial intelligence-based software HALO. Cells were classified as senescent if they were double negative and non-senescent if they were double positive for LMNB1 and HMGB1. The percentage of non-senescent PCs increased with progression to MM and positively correlated with total PC burden (R=0.52, p&amp;lt;0.0001), reflective of proliferative disease. Senescent PCs correlated with total senescent BMME (R=0.87, p&amp;lt;0.0001), consistent with a role for senescent cells to drive paracrine senescence. We performed proximity analysis to evaluate the cellular composition within 25um of senescent and non-senescent PCs. As expected, non-senescent PCs exhibited a significantly greater percentage of proximate non-senescent BMME in all groups (p&amp;lt;0.0001). In contrast, senescent PCs in stable MGUS (N=16) and SMM (N=14) were equally surrounded by senescent and non-senescent BMME. In progressing MGUS (N=21) and SMM (N=20), as well as MM (N=18), senescent PCs exhibited significantly reduced senescent BMME in their proximate environment (p&amp;lt;0.05). This suggests that senescent PCs in progressive disease, in contrast to stable disease, fail to drive local paracrine senescence responses. These data demonstrate that PCs from stable MGUS and SMM patients exhibit senescence features, with isolated PCs from stable MGUS showing increased senescence gene expression and histologic features. While stable SMM also exhibits senescence gene expression, they lose morphological features of senescence and show a preferential IFN SASP. Within the bone compartment, senescent PCs in stable MGUS and SMM exhibit the expected proximity to senescent BMME. In contrast, progressing MGUS and SMM, as well as NDMM, show a loss of proximate senescent BMME. Given the role of senescence to drive inflammation to clear potentially tumorigenic cells, these data support that failure to drive paracrine senescence may be key to the immune escape of pre-malignant cells in MGUS and SMM.

Monoclonal Gammopathy of Undetermined Significance and Associated Cardiovascular Outcomes in a Hospital Setting - a Fresh Perspective

Introduction: A growing body of evidence has confirmed the association between monoclonal gammopathy of undetermined significance (MGUS) and a variety of diseases including bone disease, renal impairment, autoimmune disorders, neuropathies, secondary immunodeficiencies, and infections. Recently, there has been an emerging focus on cardiovascular (CV) outcomes in patients with MGUS. However, they have not been investigated well, especially among hospitalized patients. In our study, we investigated the association between MGUS and cardiovascular outcomes in a hospital setting using the National Inpatient Sample database. Methods: MGUS patients were sampled using ICD-10 codes. Patients less than the age of 18 years, those with missing data, and those with concomitant multiple myeloma, smoldering multiple myeloma, B-cell lymphomas, non-Hodgkin lymphomas, amyloidosis, and Waldenstrom's macroglobulinemia were excluded . Patients were stratified into two cohorts based on the presence or absence of MGUS. Comorbidities and CV outcomes were collected using ICD 10 DM codes. CV outcomes were evaluated before and after 1:1 matching for age, gender, and race. Furthermore, a sensitivity analysis was performed where all individuals with a history of diabetes mellitus (DM), prior myocardial infarction, hypertension, chronic kidney disease (CKD) stage 3-5, dialysis-dependence, obesity, metabolic syndrome, cancer (all-cause), antiplatelet or oral anticoagulant use were excluded. CV outcomes were then reevaluated using multivariate logistic regression to adjust for smoking, dyslipidemia, and aspirin use. P-values &amp;lt; 0.05 was considered statistically significant. Results: Out of a total of 17,357,556 patients included in the study, 23,435 patients had MGUS (0.1%). MGUS patients tended to be older, male, smokers, and Caucasian. Additionally, MGUS patients tended to have more DM (39.2% vs 27.9%), CKD (33.6% vs 8.6%), dialysis-dependence (13.3% vs 4%), obesity (18% vs 16.5%), dyslipidemia (39.1% vs 23.3%), metabolic syndrome (18.9% vs 10.4%), and cancer (12.9% vs 8.1%) when compared to non-MGUS patients. Aspirin, antiplatelets, and anticoagulant use were more prevalent in the MGUS patients when compared to non-MGUS patients. MGUS patients had more heart failure (36.8% vs 17.2%;p&amp;lt;0.001), atrial fibrillation (10% vs 5.9%;p&amp;lt;0.001), venous thromboembolism (VTE) (2.8% vs 1.6%;p&amp;lt;0.001), aortic aneurysm (2.2% vs 1%;p=0.005), aortic stenosis (4.0% vs 1.6%;p&amp;lt;0.001), aortic regurgitation (1.2% vs 0.5%;p&amp;lt;0.001), mitral stenosis (0.3% vs 0.1%;p=0.009), mitral regurgitation (3.5% vs 1.5%;p&amp;lt;0.001), conduction disorder (6.6% vs 3.3%;p=0.002), cor-pulmonale (0.4% vs 0.2%;p&amp;lt;0.001), peripheral vascular disease (PVD) (14.4% vs 5.6%;p&amp;lt;0.001), and acute myocardial infarction (MI) (4.5% vs 3.6%;p&amp;lt;0.001). There were no significant differences in ischemic stroke/transient ischemic attack (TIA) and aortic dissection rates among both groups. The matched model corroborated several of our unmatched findings that MGUS was associated with higher heart failure, atrial fibrillation, VTE, aortic stenosis, mitral regurgitation, conduction disorder, cor-pulmonale, and PVD. Acute MI and ischemic stroke/TIA rates were negatively associated with MGUS in our matched model. Finally, sensitivity analysis showed that MGUS was still associated with higher rates of heart failure, atrial fibrillation, VTE, aortic regurgitation, and PVD, but decreased ischemic stroke/TIA and acute MI rates. Conclusion: In conclusion, our study found that patients with MGUS are at an increased risk of a wide spectrum of cardiovascular diseases in a hospital setting. MGUS patients may benefit from cardiovascular screening tools like electrocardiograms and echocardiograms at the hospital as this disease is independently associated with cardiovascular outcomes. Additional studies are needed to corroborate these findings as this will have implications on monitoring and screening of cardiovascular complications in MGUS patients.

Multicenter Descriptive Study of Monoclonal Gammopathy of Undetermined Significance in Patients with Heart Failure

Introduction: Monoclonal gammopathy of undetermined significance (MGUS) is the presence of a monoclonal immunoglobulin without concurrent lymphoproliferative disease. It is found in about 5% of people aged 50 and older in the general population. Prevalence rates of MGUS increase with age, especially in men and individuals of Black ethnicity compared to women and White individuals, respectively. The diagnostic criteria for MGUS includes a serum M-protein level &amp;lt;3 g/dL, &amp;lt;10% clonal plasma cells in the bone marrow, and the absence of a myeloma-defining event. Though historically considered benign, emerging evidence suggests that MGUS patients, even without lymphoproliferative disorders, face increased risks of various diseases like bone diseases, recurrent infections, autoimmune disorders, peripheral neuropathy, renal complications, and cardiovascular disease (CVD). This study aims to investigate the prevalence and characteristics of individuals with both MGUS and heart failure (HF) and examine potential racial disparities between Black patients and non-Black patients with MGUS and HF. Methods: This is a retrospective multicenter study that includes patients diagnosed with both MGUS and New York Heart Association grade II or higher HF from January 2010 to January 2023. Patients with potential confounding conditions of MGUS were excluded, such as non-MGUS plasma cell dyscrasias, hematologic malignancies, autoimmune/inflammatory disorders, and viral illnesses. Laboratory data was recorded at the time of MGUS diagnosis, and HF data was derived from imaging studies performed closest to the time of MGUS diagnosis or at the initial diagnosis of HF if it was diagnosed after MGUS. Findings were summarized using descriptive statistics. Results: We analyzed a total of 128 patients with MGUS and HF, excluding 24 patients with potential MGUS confounders. Among the remaining 104 patients, 41 (39.4%) were female, and 63 (60.6%) were male. The cohort consisted of 80 (76.9%) Black patients and 24 patients of non-Black ethnicity. For the overall cohort, the mean age at MGUS diagnosis was 72, and the mean time from HF diagnosis to MGUS diagnosis was 22.5 months with a median of 9.1 months. In 24% of patients, HF and MGUS were diagnosed within less than 1 month of each other. The predominant monoclonal proteins observed were IgG Kappa, IgG Lambda, and IgA Kappa, found in 54 (51.9%), 30 (28.8%), and 13 (12.5%) patients, respectively. The average M spike was 0.7 +/- 0.6 (SD, standard deviation), and the average free light chain ratio was 2.3 +/- 2.3 (SD). The most common type of HF diagnosed was heart failure with reduced ejection fraction (HFrEF, EF &amp;lt;40%), occurring in 63.5% of the cohort. The etiology of HF in the cohort was attributed to ischemic cardiomyopathy in 43.3%, non-ischemic cardiomyopathy in 51%, and a combination of both in 5.7%. Table 1 presents the MGUS variables for the overall cohort and stratification by race. Table 2 displays the characteristics of study subjects based on the type of HF, including HFrEF, mid-range ejection fraction (HFmrEF, EF 40-49%), and preserved ejection fraction (HFpEF, EF &amp;gt;50%). Conclusion: This study did not find a significant association between MGUS variables and HF, likely due to the limited sample size. However, a larger study by Schwartz et al. using a Danish nationwide database with over 8,000 participants suggested that MGUS patients had a higher risk of incident CVD, even after adjusting for other comorbidities. They also noted a higher prevalence of CVD risk factors in patients with MGUS. These findings indicate a possible link between MGUS and CVD, though the true nature of this association remains uncertain - whether it's causal or coincidental. An interesting observation from our study is that MGUS is often incidentally detected in patients already diagnosed with HF. This suggests that physicians may come across MGUS while investigating the underlying causes of HF, raising the possibility of shared underlying factors or pathological processes between both conditions. Considering our findings, it's essential to recognize that MGUS patients may have a higher mortality risk independent of developing plasma cell disorders. Large-scale studies are needed to better understand the association between MGUS and CVD, potentially influencing monitoring and follow-up strategies for MGUS patients, especially concerning potential cardiovascular implications.

Monoclonal Gammopathy of Undetermined Significance and Risk of Cardiovascular Disease in US Veterans

Background: Monoclonal gammopathy of undetermined significance (MGUS) has been associated with worse overall survival. The cause of this association is unclear but may result from increased risk of cardiovascular disease (CVD) events. Population-based studies have demonstrated associations between MGUS and CVD, but these were in largely Non-Hispanic White (NHW) populations. As such, we aim to examine the association between MGUS and CVD risk in the US Veterans Health Administration (VHA) which has the largest population of non-Hispanic Black patients (NHB). Additionally, this database can capture patient outcomes with an extensive electronic medical record system. The identification of a novel risk factor for CVD will improve our ability to prevent CVD events further. Methods: This was a population-based retrospective cohort study of US Veterans using linked data from the national Veterans Health Administration and Medicare. Veterans free from CVD events were included in the analytic cohort and were followed from age 50 until they either develop CVD, they expire, or the date 12/31/2022 is reached. Veterans who died or were lost to follow up within 3 years after age 50 were excluded. The exposure is presumed MGUS diagnosis before CVD event or ever. The MGUS diagnosis was captured by a natural language processing (NLP) algorithm (previously developed by our group) based on the presence of a monoclonal protein less than 3 gm/dL and bone marrow plasma cells less than 10%. The outcome of interest was CVD event after age 50 which was identified with International Classification of Diseases (ICD)-9 and -10 codes atherosclerotic cardiovascular diseases and congestive heart failure. A multivariable logistic regression adjusting for sex, race/ethnicity, region of residence, income level, marital status, type 2 diabetes before CVD diagnosis or ever, and BMI at age 50 was performed to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Results: The analytic cohort contained 523,604 Veterans: 503,098 without MGUS, among whom 25.8% developed CVD and 20,506 with MGUS, among whom, 40% developed CVD (p&amp;lt;0.0001). The CVD events included heart failure with 18.6% (n=3,804) in the MGUS group and 8.7% (n=43,955) in the group without MGUS (p&amp;lt;0.0001). The mean age of MGUS (n=20,506) was 69.2 years and 66.8 in patients who had a CVD event (n=137,918). Compared to individuals without MGUS, individuals with MGUS had a higher proportion of being male (95.5 versus 86.5%, p&amp;lt;0.0001), non-Hispanic Black (29.7 versus 22.0%, p&amp;lt;0.0001), non-Hispanic White (55.2 versus 50.0%, p&amp;lt;0.0001), divorced/separated/widowed (36.2 versus 19.6%, p&amp;lt;0.0001), single/never married (10.6 versus 6.0%, p&amp;lt;0.0001), diabetes mellitus (38.4 versus 22.0%, p&amp;lt;0.001). The odds ratio of having a CVD in the setting of MGUS was 1.23 (95% CI: 1.19-1.26). Conclusions: MGUS was associated with an increased risk of diagnosis of cardiovascular disease for individuals aged 50 years or older. Clinicians should consider evaluation of patients with MGUS for CVD risk factors. Further studies are required to evaluate whether addressing modifiable CVD risk factors is associated with decreased risk of developing CVD in MGUS patients.

Psychological Impact in Individuals with Monoclonal Gammopathy of Undetermined Significance and Smoldering Multiple Myeloma

BACKGROUND: Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) are medical conditions characterized by asymptomatic clonal plasma cell disorders that have the potential to develop into multiple myeloma (MM). The diagnosis of MGUS and/or SMM may be linked to psychological effects that can have a detrimental impact on patients' quality of life. The objective of our study was to evaluate the prevalence of adverse psychosocial effects among individuals with MGUS and/or SMM. METHODS: From August 2015 to February 2023, we conducted prospective interviews with all patients referred to the University of Arkansas for Medical Sciences who had newly diagnosed plasma cell dyscrasias, such as MGUS and SMM, before their consultation with the oncologist. Trained social workers performed an initial psychosocial evaluation and prepared a comprehensive narrative report for each patient referred during this period. Results: Our study consisted of 246 patients, with 115 having MGUS and 131 having SMM. The average age was 68 years, and 51.2% were male, with 80.5% identifying as white. Most patients (91%) were diagnosed incidentally through blood and/or urine tests, and 61.8% had a family history of cancer. Anxiety was found in 19.1% of patients, while 33.7% had a previous psychiatric disorder. Among those with a history of psychiatric disorders, 78.3% were taking medication, and 21.7% were receiving treatment from a psychiatrist/therapist. Anxiety or nervousness was reported by 22% of MGUS patients and 17% of SMM patients. About one third of patients had a history of psychiatric disorders, with 79% on medication and 26% seeing a psychiatrist/therapist. The presence of anxiety or a history of psychiatric disorders did not significantly differ between MGUS and SMM patients (p &amp;gt; 0.05). In our analysis, age, race, working status, and history of psychiatric disorders were significant risk factors for anxiety (p = 0.001, 0.033, 0.045, and &amp;lt;0.001, respectively). However, in the multivariable model, only race and history of psychiatric disorders remained significant (p = 0.037 and p = 0.006, respectively). White patients had 5 times higher odds of experiencing anxiety, while patients with a history of psychiatric disorders had 2.6 times higher odds (odds ratio: 5.1 and 2.7, respectively). The most common themes expressed by patients during interviews were 'fear, stress, and/or anxiety,' followed by 'emotional initial reaction' and ‘feeling upset.‘ Conclusion: In our study, about 20% of patients felt anxious about their diagnosis of MGUS or SMM, and around 33% reported previous psychiatric disorder. African American patients were less likely to feel anxious or nervous, while patients with a history of psychiatric disorders were more likely to feel anxious or nervous. Given that anxiety can negatively affect patients' well-being and quality of life, healthcare providers should prioritize comprehensive care that includes addressing and managing anxiety alongside the medical aspects of the disease.

Single Cell Multi-Omic Analysis of Neoplastic Plasma Cells across the Disease Spectrum Identifies Novel Pathobiologic Mediators and Potential Therapeutic Targets in Multiple Myeloma (MM)

Background: A challenge to developing a full understanding of MM pathobiology using bulk profiling techniques is the large degree of intra-tumoral heterogeneity from minor subclones that, despite their low representation, nonetheless contribute to therapy resistance and disease progression. Moreover, our current therapies tend to be targeted against pathways common to both neoplastic and normal plasma cells (PCs), indicating a need to define novel tumor biology modifiers that could potentially serve to improve risk stratification and as therapeutic targets. Methods: To overcome these limitations, we simultaneously evaluated individual PC single-cell transcriptome and B-cell receptor variable (VDJ) sequences from freshly enriched bone marrow biopsies. These included 33 patients with precursor conditions, 17 each with newly diagnosed symptomatic or relapsed/refractory disease (RRMM), and 3 controls using the 10x Genomics platform. A total of ~320,000 PCs were analyzed, yielding median per sample cell counts of 4,626 and ~300 million total reads. Identical immunoglobulin VDJ sequences defined monoclonal PCs, while diverse VDJ sequences at low frequencies defined polyclonal cells, allowing each patient to serve as their own control. Results: The percentage of monoclonal PCs in active MM was &amp;gt;84%, while precursor stages were more variable (40-100% in smoldering MM; 0.3-82% in monoclonal gammopathy of undetermined significance (MGUS)). Though we could not identify monoclonal PCs in two MGUS cases, we identified them by scRNA-seq in four MGUS patients whose bone marrow showed no clonal cells by standard clinical multiparametric flow cytometry. In differential gene expression analysis where samples were grouped based on the diagnosis, 501 dysregulated genes were identified in monoclonal PCs from symptomatic MM compared to polyclonal PCs. These included genes previously identified as disease modifiers, including Hepatocyte growth factor, Lactate dehydrogenase A, Dickkopf WNT signaling pathway inhibitor 1, CD27, and Lysosomal associated membrane protein family member 5, providing validation for our approach. Next, we identified 279 genes that were commonly dysregulated in monoclonal PCs from symptomatic vs. asymptomatic MM. We also performed unsupervised hierarchical clustering of the top 1000 most variable genes and defined three diagnostic categories. Significant ontology terms characterizing these categories broadly involved antigen processing and recognition in Group 1 (enriched in precursor stages); transcription and translation control in Group 2 (enriched in active MM); and cell division and DNA metabolism in Group 3 (enriched with RRMM) ( A). We focused on dysregulated targets that: showed differential expression in monoclonal vs. polyclonal PCs; expression in most patients within a diagnostic category; a correlation with outcomes in the CoMMpassstudy; and novelty in MM. Among these were Mitotic arrest deficient 2 like 1 (MAD2L1), a component of the mitotic spindle assembly checkpoint, and Methionine adenosyltransferase 2A (MAT2A), which catalyzes the production of S-adenosylmethionine, a key methyl donor in multiple cellular processes, from methionine and ATP. Notably, MAD2L1 inhibition with M2I1, and MAT2A targeting with the allosteric inhibitor AG-270, both produced a dose- and time-dependent inhibition in cell proliferation and reduction in myeloma cell line viability. Importantly, the effects of AG-270 ( B) were associated with induction of markers of apoptosis, including Caspase cleavage, and a reduction of Histone H3 dimethylation. Moreover, they occurred at clinically relevant concentrations based on the known pharmacokinetics of this drug in Phase I testing. Conclusions: Our combined single-cell transcriptomics and VDJ sequencing allowed a greater understanding of heterogeneity among neoplastic PCs compared to each patient's own polyclonal PCs. These analyses can identify putative novel mediators of disease pathobiology that impact prognosis, and that can serve as potential new therapeutic targets using either novel agents, or drug repurposed from other therapeutic areas, setting the stage for their translation to the clinic.

IRF2 Regulates De Novo Active Chromatin Regions and Participates in the Development and Pathogenesis of Multiple Myeloma

Multiple Myeloma (MM) is the second most common hematologic neoplasia, and it remains largely incurable due to the genetic heterogeneity of the patients. We have recently characterized the epigenome of plasma cells from patients with MM, describing the de novo activation of the chromatin as a unifying factor underlying the pathobiology of the MM. Precisely, we identified 1556 de novo active regions, showing the H3K27ac chromatin activation mark, in MM in comparison to different subpopulations of normal B cells. Moreover, those de novo active regions where enriched in Transcription Factor Binding Sites (TFBS). In order to defined the potential role of these regions we have performed a CRISPR-Cas9 screening using guides against 56 TF that presented TFBS in de novo active regions and that are expressed in MM. A CRISPR-Cas9 library containing 10 guides for each gene was transduced in two MM cell lines (MM.1R and KMS-11) and the presences of guides were analyzed at days +1, +14 and +21. Out of the 22 TFs shown to be essential for MM survival, there were 3 members of the IRF TF family (IRF1, IRF2 and IRF5). Single knockdowns identified IRF2 and the previously described IRF4 as the best candidates. To characterize the signalling pathways involved in IRF family regulation of MM, the chromatin regions where IRF1, IRF2 and IRF4 are bound were mapped using CUT&amp;RUN in MM.1R and KMS-11 cell lines. Interestingly, the three TF were located in 573 out of the 1556 de novo active regions. Notably, IRF2 was present in the 86.9% of those regions, and in 40% of the cases, the binding occurred independently of IRF1 or IRF4 ( Fig. 1). Additionally, the genomic distribution demonstrated that the regions where IRF2 binds alone were enriched in promoters compared to those regions where IRF1 and IRF4 also bound, being these enriched mostly in the gene body. Gene Ontology studies showed that IRF2, as well as IRF1/4 were involved in regulation of cell death in response to oxidative stress, while only IRF2 autonomously was involved in the regulation of pathways relevant for MM such as the kinase dysregulation, cell migration and osteoblast regulation. Furthermore, using our previous RNA-seq data, we observed that the majority of the genes regulated uniquely by IRF2 presented an overexpression in MM in comparison to different subpopulations of normal B cells. This overexpression was already identified in plasma cells from MGUS (Monoclonal Gammopathy of Undetermined Significance) patients and SMM (Smouldering MM) stages in contrast to normal bone marrow plasma cells. Finally, we examined the impact of IRF2 and IRF4 expression on the outcome of MM using RNA-seq data from 664 patients from MMRF (MM Research Foundation) CoMMpass (IA18) database. We observed that patients with higher expression of IRF2 presented significantly poorer Progression Free Survival (PFS) and Overall Survival (OS) in univariate analysis. Interestingly, in multivariate analyses taking into account the classic high-risk genetic factors for MM, the expression of IRF2 together with del13q and amp1q resulted in a significantly lower PFS (global p &amp;lt; 0.001) and the expression of IRF2 together with amp1q and del17p resulted in statistically significant worse OS (global p &amp;lt; 0.001). On a multivariate model, MM patients were stratified according to the number of events ( IRF2 expression, del13q and amp1q for PFS and IRF2 expression, amp1q and del17p for OS) into four risk groups both for PFS and OS ( Fig. 2). In conclusion, our study demonstrate that IRF2 plays a significant role in the pathogenesis of MM by participating in the regulation of the de novo active chromatin regions, specifically regions related to kinase pathway. Additionally, IRF2 represents a robust biomarker that in combination with classical genetic markers improves stratification of MM patients. These results establish IRF2 as a promising candidate for new therapeutic strategies in patients with MM.

CRIP1 Upregulated Along with chr1q-Gain Indicates Immune Dysregulation and Portends Poor Outcomes in Multiple Myeloma

Background: The gain of chromosome 1q (chr1q-gain), one of the frequently occurring copy number aberrations in multiple myeloma (MM), is associated with a poor prognosis. However, the specific high-risk genes involved and the underlying molecular mechanisms remain elusive. Method: In this study, we gathered a comprehensive dataset comprising bulk transcriptome data from 1271 multiple myeloma (MM) patients, including 413 patients from the GSE4581 dataset and 858 patients from the MMRF dataset. Additionally, we acquired single-cell RNA sequencing data from various disease stages, which included 12 MM patients, 7 patients with monoclonal gammopathy of undetermined significance (MGUS), 4 patients with amyloidosis (AL), 6 patients with smoldering multiple myeloma (SMM), and 11 healthy control donors from GSE117156. Cox regression and Kaplan-Meier curves were employed to identify and validate significant candidates associated with adverse prognosis among the overexpressed genes in patients with chr1q-gain. Furthermore, the expression profiles of the candidates in malignant plasma cells from MM patients compared to plasma cells in healthy donors were evaluated at single cell resolution. Gene Set Enrichment Analysis (GSEA) was conducted to uncover the pathways enriched in high-CRIP1 groups. To better understand the immune infiltration in MM, we utilized CIBERSORTx to infer the immune cell composition. Results: A total of 541 genes were found to be upregulated in multiple myeloma (MM) patients with chr1q-gain in the GSE4581 dataset, with a false discovery rate (FDR) of less than 0.05 and a log fold change (LogFC) greater than 0.5.We identified five genes (CRIP1, ANP32E, CKS1B, RBFA, TOP2A) associated with poor prognosis through univariate Cox and multivariate Cox regression analyses and successfully validated in MMRF dataset(n=858). Upon characterizing the expression of these candidate genes in malignant plasma cells at a single-cell resolution, we made an interesting observation: CRIP1 was uniquely expressed in multiple myeloma (MM) patients when compared to healthy donors, individuals with MGUS, SMM and AL patients. Moreover, the GSEA results showed significant enrichment of the “Anti-viral Mechanism by IFN-stimulated Genes” (Normalized Enrichment Score, NES=1.98, False Discovery Rate, FDR=0.017) and “Interferon Signaling” (NES=2.11, FDR&amp;lt;0.001) in CRIP1-low MM patients. These findings suggest that CRIP1 may play a role in the development of multiple myeloma by influencing the tumor immune microenvironment through the regulation of these pathways. Analysis of immune cell infiltration in MM showed the expression level of CRIP1 is negatively correlated with CD4 naïve T cells ( R=-0.24, P&amp;lt;0.001) and positively correlated with Monocytes ( R=0.19, P&amp;lt;0.001). Tumors with high CRIP1 expression showed increased infiltration of CD8 T cells, CD4 memory T cells, activated NK cells, M0 macrophages, and Eosinophils. Conclusions: The study showed that CRIP1 upregulated along with chr1q-gain was highly expressed in MM patients compared to healthy individuals and associated with immune dysregulation. The patients with higher CRIP1 expression have poorer survival. CRIP1 could serve as a potential therapeutic target and prognostic biomarker in multiple myeloma.

Single-Cell RNA Sequencing of Circulating Tumor Cells in Precursor Myeloma Patients Reveals Mechanisms of Disease Dissemination

Introduction Multiple Myeloma (MM) is preceded by the precursors Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM), where some patients with precursor disease progress to active MM faster than others. BM biopsies are useful to monitor disease progression, however, they cannot be repeated often for continuous monitoring of tumor burden especially in the precursor disease management setting. Numerous studies have identified circulating tumor cell (CTC) levels in peripheral blood (PB) are a powerful biomarker of disease aggressiveness; CTC levels have associated with disease stage, PFS and OS in various trials and even outperformed BM to assess tumor burden in the SMM setting. Genomic profiling also demonstrated CTCs harbor the same copy number abnormalities, translocations and mutations as BM tumor cells, and CTCs can be serially monitored to detect the emergence of high-risk subclones in the blood. Further investigations of the molecular profile of both normal plasma cells (NPCs) and CTCs are needed to improve our understanding of myeloma pathogenesis and mechanisms of CTC dissemination across the MM disease continuum. Methods A cohort of 309 samples including CD138-enriched paired PB and BM aspirates and matched CD138- BM aspirates were collected from 103 individuals, including patients with MGUS (n=11), SMM (n=46), NDMM (n=17) and healthy donors (n=29). Malignant PCs from BM and PB underwent 5' single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics), and CD138- BM immune cells underwent 5' scRNA-seq and single-cell T-cell receptor sequencing (scTCR-seq) to study immune alterations related the tumors' ability to circulate. To differentiate malignant from normal PCs, we used clonal V(D)J rearrangements. Differential expression (DE) and composition analyses were conducted using Wilcoxon's rank-sum tests. Results We successfully captured and profiled 774,647 BM tumor cells and 93,878 CTCs from the PB. The percent malignant plasma cells within the PB increased with disease stage, where NDMM patients had significantly more CTCs compared to MGUS patients (p=0.017) and low-risk SMM patients (p=0.0052). A median of 13.51%, 15.53%, and 18.34% CTCs were present from low (n=20), intermediate (n=10), and high-risk (n=16) SMM patients as defined by the International Myeloma Working Group's 2/20/20 criteria, suggesting sequencing-based CTC enumeration captures prognostically relevant differences in tumor burden. High expression of driver genes upregulated in patients with translocations, including CCND1, NSD2, and MAF, were detected in both BM tumor cells and CTCs in patients with t(11;14), t(4;14), and t(14;16) as identified by fluorescence in situ hybridization (FISH). In 5 patients with normal or inconclusive FISH results, we observed high levels of CCND2 and MAF in both BM and CTCs, indicating scRNA-seq can detect missed prognostically relevant cytogenetic abnormalities. A cytogenetic classifier model was developed to determine the accuracy of translocation calling using minimal numbers of CTCs, where the classifier's performance was able to consistently identify translocations downsampled to 50 CTCs. DE analysis of CTCs vs. BM tumor cells highlighted transcriptional similarity between BM and CTCs, validating their utility as a surrogate for analyzing BM tumor cells. DE analysis also revealed 8 genes significantly upregulated and 3 genes significantly downregulated in CTCs compared to BM tumor cells, providing novel insights into genes involved in PC circulatory potential. Pathway enrichment analysis revealed genes upregulated in CTCs were associated with epithelial mesenchymal transition, interferon response, and inflammation, consistent with CTC studies on MM patients, suggesting these pathways are dysregulated earlier in the disease continuum. Conclusions In the largest scRNA-seq study on CTCs to date, we demonstrate the utility of CTC-based molecular profiling for prognostication of patients with early-stage disease and provide novel insights into PC circulatory potential. Additional analyses are ongoing to gain further insight into intra-patient CTC heterogeneity and define high-risk disease CTC signatures that emerge throughout the MM disease continuum.

Evaluation of Metabolic Profile in Monoclonal Gammopathy of Undetermined Significance and Asymptomatic Multiple Myeloma

Background: Multiple myeloma (MM) is an incurable, heterogeneous neoplasm of clonal plasma cells preceded by the precursor conditions monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic multiple myeloma (AMM). Features of metabolic syndrome including obesity and diabetes mellitus (DM) have been linked to MM development and mortality. A study of serum metabolomics in patients with clinical MM revealed alterations in the citrate cycle, pyrimidine, purine, and amino acid metabolism (Chanukuppa V, et al, 2019). However, the role of dysregulated metabolism in progression of monoclonal gammopathy across the disease spectrum requires further investigation. We performed a pilot study to compare metabolic profiles in patients with MGUS and AMM. Methods: Clinical data and peripheral blood plasma samples were collected on 20 patients with non-immunoglobulin M MGUS and 20 patients with AMM. Global metabolomic studies by liquid chromatography (LC) tandem mass spectrometry (MS) were performed on plasma using a SCIEX TripleTOF 6600+ mass spectrometer. Each sample was run on Hypercarb and Reverse phase LC columns in both positive and negative MS modes. Standard targeted and untargeted metabolomic platforms were used with assessment of changing metabolites between the two groups in a one-to-one comparison. Correlation analysis and differential expression analysis were completed and top changing metabolites determined based on ranking of log fold change and p value cutoff (at least &amp;lt;0.05). Pathway enrichment data was generated with advanced data analysis. Results: Median age was 70 and 67 years in the MGUS and AMM groups, respectively. 65% of patients in the AMM group were male versus (vs) 40% in the MGUS group. Black individuals represented 15% of MGUS and 30% of AMM patients. Mean serum monoclonal (M) protein was higher in AMM patients (0.87 g/dL vs 0.28 g/dL, p=0.009); mean bone marrow clonal plasma cells in AMM was 17% (range 10-40%). Three AMM patients had high risk cytogenetics inclusive of chromosome 1 abnormalities. We observed that more individuals in the AMM group had a diagnosis of DM (35% vs 15%), were on hyperlipidemia (HLD) medications (80% vs 45%) and were on ≥3 hypertension (HTN) medications (35% vs 20%). Increasing weight and hemoglobin a1c values were seen with rise in serum M protein in the combined groups. For global metabolomics, 2,513 metabolites were annotated from targeted and untargeted peak curation and results underwent data normalization and standardization with exclusion of outliers from the data set. Using targeted data, Partial Least Squares-Discriminant Analysis showed separation of the MGUS and AMM groups (Figure 1). There were 8 significantly increased metabolites (dihydrouracil, quinate, melatonin, azelate, phosphonoacetate, malate, L-alanine, and lysine) and 9 significantly decreased metabolites (diethanolamine, xanthosine, nervonate, quinolinate, L-dopa, ethylmalonate, butanoate, carnosine, and thyroxine) in AMM when compared to MGUS. Pathway enrichment analysis revealed several pathways increased in AMM (Figure 2), including D-glutamine and D-glutamate metabolism and the citrate cycle. Pathways decreased in AMM included purine, pyrimidine, and glutathione metabolism. Conclusions: In our study, patients with AMM were more likely to demonstrate features of metabolic syndrome, including higher rate of DM and need for HTN and HLD medications. Additionally, a trend of higher weight and hemoglobin a1c values occurred as serum M protein increased. Global metabolomics identified unique metabolites that differed in AMM relative to MGUS patients. The unsaturated fatty acid nervonate, decreased in AMM in our study, was previously shown to have potential preventive effects on obesity-related metabolic disorders (Oda E, et al, 2005). Our findings in these precursor cohorts are concordant with prior serum metabolomics in MM showing alterations in amino acid metabolism including glutamine (Chanukuppa V, et al, 2019), with glutaminase inhibitors currently under study in relapsed/refractory MM (Bolzoni M, et al, 2016). Additional investigation of metabolic profile in larger groups across the spectrum of monoclonal gammopathy will help further elucidate the role of this dysregulated metabolism in myelomagenesis and could lead to preventative interventions targeting the associated metabolic syndrome earlier in disease course.