Progression Of Monoclonal Gammopathy Of Undetermined Significance Research Articles

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 <0.05) and allowed perfect distinction of MGUS from HCs after PSO (Fig. 1; P <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.

Immune Profiling of Plasma Cell Dyscrasias Reveals a Therapy Related T-Cell Modulation in Multiple Myeloma Patients

Multiple Myeloma (MM) is a hematological malignancy always preceded by a not malignant precursor state defined monoclonal gammopathy of undetermined significance (MGUS) and by a asymptomatic MM (smouldering MM, sMM). Immune dysfunction plays a key role in the pathogenesis of the disease, as demonstrated by the prognostic role of immunoparesis in the progression of MGUS and sMM into active MM (aMM). The aim of this study is to analyze the immune subsets distribution into plasma cells dyscrasias. A total amount of 895 bone marrow samples (170 MGUS, 188 sMM, 586 aMM) of 714 patients affected by plasma cells dyscrasias were studies at the different time point by flow cytometry for CD3, CD4, CD8, CD16, CD19, CD56, CD57, HLA-DR and Tgd antigens.

Distinct Nuclear Organization of Telomeresand Centromeres in Monoclonal Gammopathyof Undetermined Significance and Multiple Myeloma.

Both multiple myeloma (MM) and its precursor state of monoclonal gammopathy of undetermined significance (MGUS) are characterized by an infiltration of plasma cells into the bone marrow, but the mechanisms underlying the disease progression remain poorly understood. Previous research has indicated that 3D nuclear telomeric and centromeric organization may represent important structural indicators for numerous malignancies. Here we corroborate with previously noted differences in the 3D telomeric architecture and report that modifications in the nuclear distribution of centromeres may serve as a novel structural marker with potential to distinguish MM from MGUS. Our findings improve the current characterization of the two disease stages, providing two structural indicators that may become altered in the progression of MGUS to MM.

Od monoklonalnej gammapatii o nieokreślonym znaczeniu (MGUS) do szpiczaka plazmocytowego i amyloidozy — opis przypadku

We present a 69-year-old patient, in whom in March 2016 a monoclonal gammopathy of undetermined significance (MGUS) was diagnosed. The disease was found during the diagnostics of thrombocytopenia (from 2009 the number of platelets varied between 90–140 G/l). In serum immunofixation assay the presence of monoclonal IgG kappa protein was confirmed. In July 2017, when the patient presented with nephrotic syndrome and acute renal failure, 14.5% of plasma cells were found in the bone marrow. Moreover, a biopsy of abdominal fat revealed the presence of amyloid composed of kappa light chains. The diagnosis of IgG kappa plasma cell myeloma (PCM) complicated with amyloidosis of the kidneys, heart, liver and spleen was established. The PCM progression complicated with amyloidosis was very aggressive. Despite the treatment with bortezomib and dexamethasone the patient died early. Sudden deterioration of health (e.g. in the form of acute renal failure) in a patient with MGUS may indicate not only the progression of MGUS to PCM, but also the development of amyloidosis, which is associated with turbulent disease and poor prognosis.

Evaluating the role of Tregs in the progression of multiple myeloma

The role of regulatory T-cells (Treg) and Th17 cells in the progression of multiple myeloma has been unclear. There are conflicting reports of the Treg and Th17 frequency being increased, decreased, and unchanged as compared with controls. In this study, we sought to characterize the T-cell subsets including Treg function in both blood and marrow compartments of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). The Treg/Th17 ratio is skewed toward the suppressive phenotype in MGUS and MM. There are more activated and memory Tregs in the myeloma marrow. Although the myeloma Tregs are functional, they are less suppressive than Tregs in chronic lymphocytic leukemia where they drive disease progression. None of the T-cell subsets were found to have a clinical correlation with time to progression in MGUS or progression-free survival in myeloma. Tregs are important but unlikely major players in the progression of MGUS to MM.

Metabolomic Profiling of Serum from Myeloma and MGUS Patients - a Novel Strategy to Identify Potential Biomarkers of Myeloma Development and Progression

Introduction: Drivers that underlie the progression of MGUS (Monoclonal gammopathy of undetermined significance) to multiple myeloma are yet largely unknown. Because of the vast number of potential players, these drivers may not necessarily aimed to transform plasma cell or the bone marrow niche, but rather remodel a supporting microenvironment. Metabolic alterations have been linked to cancer development and resistance to chemotherapy. It has been suggested that high rates of glycolysis and glutaminolysis are necessary in malignant cells to allow sensitive and precise control of the pathways that generate metabolic intermediates for macromolecular biosynthesis, hence cancer progression and metastasis. Experimental evidence indicates that glutamine, the most abundant amino acid in the blood and tissues, is the major respiratory fuel for cancer cells, and its conversion to glutamate is the first step in a series of reactions that generate essential metabolic intermediates. Thus, physiologic concentrations of circulating glutamine are required for optimal growth of malignant cells. This study attempts to identify circulating metabolites in myeloma patients which could potentially relate to disease progression.Methods: We employed hydrophilic interaction liquid chromatography (ZIC-pHILIC)-high resolution mass spectrometry (Q-Exactive) to profile metabolites in cell-free serum samples from 10 MGUS and 10 myeloma patients with written consent. The data were analysed using IDEOM software and its quality was assessed by calculating % RSD (relative standard deviation) of peak median and internal standards within groups, repeated analysis of pooled quality control samples, and inspecting the heatmap for outlier samples. The fold changes of metabolites were measured in the MGUS versus myeloma cohorts using the ratio of the mean peak intensities. Significant features generated after statistical analyses were used for metabolic pathway analysis.Results: In excess of 600 metabolite features were reproducibly detected, with 76 metabolites confidently identified based on authentic standards and the remainder putatively identified by accurate mass. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis determined that amino acids, carbohydrate and glycerolipid metabolism were affected by myeloma. L-glutamate, L-alanine and L-phenylalanine amino acids showed 0.63-, 0.71- and 0.70-fold decreases, respectively, in myeloma patients compared to MGUS individuals (p<0.05). Consistent with the literature, the circulating level of glutamine was maintained, confirming that glutamine's uptake and release by various organs and skeletal muscle is proportional to its usage by cancer cells for glutaminolysis that supplies them with L-glutamate. With regard to carbohydrate metabolism, D-gluconic acid, pentitol, glycerol and citrate with 0.57-, 0.60-, 0.68- and 0.80-fold decreases, respectively, (p <0.05) were among the compounds with the highest confidence that contributed to the discrimination between MGUS and myeloma. The only markedly decreased lipid-associated metabolite was sn-Glycerol 3-phosphate (p value: 0.043). Exploring the metabolic pathways employed by myeloma cells for energy and biomass generation, Pentose Phosphate Pathway (PPP) appeared to be highly perturbed as D-gluconic acid, D-ribose, deoxyribose and D-glucono-1,5-lactone all showed significantly decreased levels ranging from 0.38 to 0.70 fold. The involvement of the tricarboxylic acid (TCA) cycle, to which both L-glutamate and glucose can contribute, was also evident as suggested by decreases in citrate, malate (0.48-fold), cis-aconitate (0.68-fold) and succinate (0.76-fold).Conclusions: As proof of concept, our results showed significant differences in a number of circulating metabolites between myeloma and MGUS, which requires further validation through a quantitative metabolomics study. Here, the analysis of non-invasive sampling and easily traceable circulating metabolites exemplifies how detailed metabolic profiling could be potentially utilized to predict progression of MGUS to myeloma and to identify potential targets for treatment interventions. DisclosuresSpencer:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; BMS: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria.

Update on Severe Isotype-Matched Immunosuppression By Hevylite® Confirms Its Predictive Value in MGUS Patients: A Real-Life Study

Several studies have now shown that monoclonal gammopathy of undetermined significance (MGUS) constantly precedes all multiple myelomas (MM). Risk adapted follow-up may increase chances of early diagnosis of progression and, therefore, increase the chances of a better outcome for the patient, as also shown by two long-term follow-up studies. However, in real-life patients it is still difficult to identify MGUS cases at very high risk of progression. On a previous study we showed that Severe Isotype-Matched Immunosuppression (IMI) by Hevylite® was a potential risk factor of progression for MGUS patients. We have now expanded the median follow-up time by an additional year.Methods:The clinical records of 154 MGUS patients for which a Hevylite determination was available together with monoclonal protein quantification by gel electrophoresis (EP) and free light chains by Freelite were reviewed. Samples were analyzed with Hevylite® (The Binding Site, UK) in a BNII analyzer (Siemens) according to the monoclonal protein isotype. Normal ranges were those provided by the manufacturer. Values 50% below the lower normal ranges of the immunoglobulins determined by Hevylite were defined as severe IMI. Statistical analysis done using Graph pad V5. Progression free survival (PFS) curves calculated by Kaplan-Meyer method and curve differences by Log-rank (Mantel Cox) test.Results:The median follow-up of the 154 MGUS patients (150 IgG, 20 IgA, and 27 IgM) was 944 days (range:27-3471), during which 16 progressions to MM, 3 progressions to smoldering (by plasma cell infiltration >10%) and 1 progression to Non-Hodgkin lymphoma were registered. These numbers represent an increase in 366 days of follow-up and 3 extra progressions, with respect to the previous study. The median time to progression of the cohort is now 9,5 years.Severe IMI determined by Hevylite was observed in 14% of the patients while severe immunoparesis (i.e. more than 50% suppression of the immunoglobulins not associated to the isotype of the tumor cells; e.g. suppression of IgA or IgM in an IgG MGUS) was observed in 7% of the patients.The frequency of IMI was very similar among patients of different isotypes: IgG=60%, IgA=69%, IgM=57%, (p=0.37).When patients were analyzed according to the presence/absence of severe IMI or severe immunoparesis (IP), only the presence of severe IMI was observed to be predictive of shorter time to progression (p=0.009; HR=6.3, 95% IC=1.6-24.9) as shown in Fig.1A. Patients with severe IP were as likely to progress as patients without (p=0.604; Fig.1B). Additionally, patients showing simultaneously severe IMI and involved HLC >1.5g/dL showed a median time to progression 4 times shorter than the remaining group (Fig.2).ConclusionsThe results obtained strengthen the conclusions obtained previously showing that severe IMI is a risk factor for MGUS progression, mainly if combined with an involved HLC greater than 1.5g/dL. This means that, potentially, with one assay, two risk factors could be easily and quickly assessed for, virtually, every MGUS patient.The limitations of this study are its retrospective nature and a moderate over-representation of higher risk MGUS groups, which is expected since, being a real-life study, these are the patients that are most likely to maintain the follow-up with the specialist. DisclosuresBarbosa:The Binding Site: Employment. Pais:The Binding Site: Employment. Campos:The Binding Site: Employment.

Diagnostic Relevance of 18F-FDG PET/CT in Newly Diagnosed Patients with Monoclonal Gammopathy of Undetermined Significance (MGUS) : Single- Center Experience

Introduction:The International Myeloma Working Group (IMWG) consensus aimed to provide recommendations for the optimal use of positron emission tomography-computed tomography (PET/CT) in patients with multiple myeloma (MM) and other plasma cell disorders, including smouldering multiple myeloma (SMM) and solitary plasmocytoma (Cavo M et al., Lancet Oncol. 2017). Monoclonal gammopathy of undetermined significance (MGUS) is one of the most common pre-malignant disorders, in which the probability of progression to MM or other lymphoproliferative disorders is 1% per year (Kyle RA. Blood 2003). MGUS patients have an increased risk of developing hematologic and non-hematologic malignancies (Mailankody S, et al. Blood. 2011; Gregersen H et al., Am J Hematol. 2000). Data for the role of ¹⁸F-FDG PET/CT in diagnostic relevance in newly MGUS patients are still limited. We think, that using ¹⁸F-FDG PET/CT imaging in newly MGUS patients may be useful in early detection of other serious pathologies, not only in predicting progression of MGUS to active MM.Patients and methods:We prospectively analyzed the diagnostic relevance of ¹⁸F-FDG PET/CT in 390 newly MGUS patients . All MGUS patients were diagnosed and monitored at the Department of Internal Hematology and Oncology in Brno, Czech Republic from January 2010 to December 2016. The diagnosis of MGUS was made according to updated 2010 International Myeloma Working Group diagnostic criteria (Kyle RA et al., Leukemia 2010). Imaging scans were reviewed by a radiologist and a nuclear medicine physician. The aim of this analysis was to evaluate the benefit of ¹⁸F-FDG PET/CT in early detection of other serious illnesses in newly MGUS patients. This study did not focuse on the MGUS patients re-classified as MM. Patients underwent ¹⁸F-FDG PET/CT within 6 months of MGUS diagnosis. At the time of ¹⁸F-FDG PET/CT imaging the patients showed no specific symptoms indicative of later diagnosed serious illnesses.Results:A total of 390 newly diagnosed MGUS patients were evaluated, and by IMWG definition had no lytic lesions on skeletal survey. On ¹⁸F-FDG PET/CT, presence of focal or diffuse areas of detectable increased tracer uptake on at least two consecutive slices was recorded in 36 (9.2%) of MGUS patients. The most frequent pathology was lymphadenopathy (3.8%), followed by thyreopathy (2.1%), rheumatologic diseases (1.8%) and others solid tumors (1.3%). Primary malignancy was confirmed by biopsy in 17 (4.4%) patients. This included 12 (3.1%) patients with lymphoproliferative diseases and 3 (0.8%) patients with colon cancers. In 1 case prostate and thyroid cancer was diagnosed.On ¹⁸F-FDG PET/CT focal lymph nodes abnormalities were detected in 15 (3.8%) evaluable patients. Twelve (3.1%) patients were reclassified as having lymphoproliferative diseases. Among the remaining 3 patients with lymph nodes abnormalities 1 patient was detected with Sjögren's syndrome, 1 patient with sarcoidosis, and 1 patient with reactive lymphadenopathy ,without detection of malignant cells after biopsy. None of the patients had symptoms typical for lymphoproliferative diseaseas at the time of ¹⁸F-FDG PET/CT imaging. Thyreopathy was the second most common pathology in our study, observed in 8 (2.1%) patients. In 1 patient thyroid carcinoma was diagnosed; among the remaining 7 (1.8%) patients with focal thyreoid abnormalities nontoxic struma was found. All patients' thyroid hormones tested within the normal range at the time of ¹⁸F-FDG PET/CT imaging. Rheumatologic diseases were the third most common pathology, occurring in 7 (1.8%) of all scanned patients. Specifically, there were 4 (1.1%) patients with rheumatoid arthritis, 2 (0.5%) patients with polymyalgia rheumatica and 1 patient with giant-cell temporal arteritis. In 1 patient thymoma and schwannoma of the femoral nerve were identified. For all these patients, all diseases were early-onset without typical symptoms.Summary:In conclusion, using newer sensitive imaging tool we detected 36 (9.2%) patients with serious pathologies before the first symptoms. Therefore we believe, that ¹⁸F-FDG PET/CT imaging can be recommended to be done not only in all MGUS patients with suspected SMM or active MM, but also for early identification of other serious pathologies. DisclosuresNo relevant conflicts of interest to declare.

Monoclonal Gammopathy of Undetermined Significance (MGUS)Monoclonal Gammopathy of Undetermined Significance (MGUS).

Monoclonal gammopathy of undetermined significance (MGUS) is one of the most prevalent premalignant conditions associated with a risk of malignant transformation to multiple myeloma (MM) or other forms of lymphoproliferative disorders with risk of progression of approximately 1% per year. IgG and IgA MGUS are precursor conditions of multiple myeloma (MM), whereas light-chain MGUS is a precursor condition of light chain MM. IgM MGUS is a precursor condition of Waldenström macroglobulinemia (MW) or other lymphoproliferative diseases. Assessment of the risk of progression of patients with asymptomatic monoclonal gammopathies (MG) is based on various factors, including the serum paraprotein (M protein) concentration, isotype of M protein, serum free light chain ratio, infiltration of bone marrow plasmocytes, reduction of one or two noninvolved immunoglobulin subtype levels (immunoparesis), evolving and non-evolving subtype of MGUS, ratio of normal/abnormal plasma cells in bone marrow identified by multiparametric flow cytometry techniques and number of circulating plasma cells in peripheral blood. Three risk stratification models have been constructed that are useful in daily practice for predicting risk of progression of MGUS into malignant forms of monoclonal gammopathy - MAYO, PETHEMA and CMG model. The goal of all three models is to identify correctly prognostic markers that can divide patients into low-risk MGUS and high-risk MGUS groups. This review provides a look at the definition, pathogenesis, diagnostic algorithm, clinical significance and stratification of MGUS patients, followed by recommendations for patient risk dispensarisation intervals.

Computational Model of Progression to Multiple Myeloma Identifies Optimum Screening Strategies.

PurposeRecent advances have uncovered therapeutic interventions that might reduce the risk of progression of premalignant diagnoses, such as monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM). It remains unclear how to best screen populations at risk and how to evaluate the ability of these interventions to reduce disease prevalence and mortality at the population level. To address these questions, we developed a computational modeling framework.Materials and MethodsWe used individual-based computational modeling of MGUS incidence and progression across a population of diverse individuals to determine best screening strategies in terms of screening start, intervals, and risk-group specificity. Inputs were life tables, MGUS incidence, and baseline MM survival. We measured MM-specific mortality and MM prevalence after MGUS detection from simulations and mathematic modeling predictions.ResultsOur framework is applicable to a wide spectrum of screening and intervention scenarios, including variation of the baseline MGUS to MM progression rate and evolving MGUS, in which progression increases over time. Given the currently available point estimate of progression risk reduction to 61% risk, starting screening at age 55 years and performing follow-up screening every 6 years reduced total MM prevalence by 19%. The same reduction could be achieved with starting screening at age 65 years and performing follow-up screening every 2 years. A 40% progression risk reduction per patient with MGUS per year would reduce MM-specific mortality by 40%. Specifically, screening onset age and screening frequency can change disease prevalence, and progression risk reduction changes both prevalence and disease-specific mortality. Screening would generally be favorable in high-risk individuals.ConclusionScreening efforts should focus on specifically identified groups with high lifetime risk of MGUS, for which screening benefits can be significant. Screening low-risk individuals with MGUS would require improved preventions.

Monoclonal IgG in MGUS and multiple myeloma targets infectious pathogens.

Subsets of mature B cell neoplasms are linked to infection with intracellular pathogens such as Epstein-Barr virus (EBV), hepatitis C virus (HCV), or Helicobacter pylori. However, the association between infection and the immunoglobulin-secreting (Ig-secreting) B proliferative disorders remains largely unresolved. We investigated whether the monoclonal IgG (mc IgG) produced by patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM) targets infectious pathogens. Antigen specificity of purified mc IgG from a large patient cohort (n = 244) was determined using a multiplex infectious-antigen array (MIAA), which screens for reactivity to purified antigens or lysates from 9 pathogens. Purified mc IgG from 23.4% of patients (57 of 244) specifically recognized 1 pathogen in the MIAA. EBV was the most frequent target (15.6%), with 36 of 38 mc IgGs recognizing EBV nuclear antigen-1 (EBNA-1). MM patients with EBNA-1-specific mc IgG (14.0%) showed substantially greater bone marrow plasma cell infiltration and higher β2-microglobulin and inflammation/infection-linked cytokine levels compared with other smoldering myeloma/MM patients. Five other pathogens were the targets of mc IgG: herpes virus simplex-1 (2.9%), varicella zoster virus (1.6%), cytomegalovirus (0.8%), hepatitis C virus (1.2%), and H. pylori (1.2%). We conclude that a dysregulated immune response to infection may underlie disease onset and/or progression of MGUS and MM for subsets of patients.

Clinicopathological significance of the p16 hypermethylation in multiple myeloma, a systematic review and meta-analysis.

It is well known that the loss of function of the p16INK4A gene is mainly caused by the hypermethylation of the p16 gene; however, whether or not the inactivation is associated with the clinical significance of multiple myeloma (MM) remains elusive. A meta-analysis was conducted to quantitatively determine the role of the p16 hypermethylation in the clinical significance of MM. We demonstrated that MM patients show much higher hypermethylation rates on the p16 gene in bone marrow compared to normal individuals, as well as monoclonal gammopathy of undetermined significance (MGUS). The difference of aberrant p16 hypermethylation between MM patients in advanced stage and MM patients in early stage is not statistically significant. Interestingly, the survival rate of MM patients with the p16 hypermethylation is much shorter compared to those without the p16 hypermethylation. Our results demonstrate that hypermethylation status of the p16 gene may play a role in the progression of MGUS to MM, as well as worse survival in MM. The p16 hypermethylation, which induces the loss of function of the p16 gene that plays a critical role in the early tumorigenesis of MM.

A first Czech analysis of 1887 cases with monoclonal gammopathy of undetermined significance.

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant condition with a risk of malignant conversion. With the aim to estimate the cumulative risk MGUS progression to hematologic malignancies, we analyzed a nationwide population-basedcohort of 1887 MGUS patients from the Czech Registry of Monoclonal Gammopathies (RMG) between 2007 and 2013. During the follow-up period (median 4years; range 0.6-34.8), progression to hematologic malignancies was observed in 8.6% (162 of 1887) of patients. Factors associated with progression were as follows: M-protein concentration ≥1.5g/dL, pathological sFLC (<0.26 or >1.65) ratio, bone marrow plasma cells (BMPCs) in cytology >5%, immunoparesis, age ≥69years, and the level of serum hemoglobin at baseline <12.0g/dL. Combining these factors, we propose a new risk model (CMG model). The risk of progression at 10years was 1.6%, 16.9%, 22.9%, 39.4%, and 52.3%, respectively, if 0 (reference group), one, two, three, or four to five risk factors are present (P<.001) with HR 63 times higher compared to the reference MGUS group. The new CMG model was established with an advantage for better identification of MGUS patients at low risk.

Obesity and the Transformation of Monoclonal Gammopathy of Undetermined Significance to Multiple Myeloma: A Population-Based Cohort Study.

Multiple myeloma (MM) is one of the most common hematologic malignancies in the United States and is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS). This study investigates the role of obesity in the progression of MGUS to MM. A retrospective identified cohort of patients in the US Veterans Health Administration database diagnosed with MGUS between October 1, 1999, and December 31, 2009, was followed through August 6, 2013. Patient-level clinical data were reviewed to verify MM diagnosis, if any. Survival analyses utilizing interval-censored data were used to investigate the risk of progression of MGUS to MM. Statistical tests were two-sided. The analytic cohort consisted of 7878 MGUS patients with a median follow-up of 68 months. Within the cohort, 39.8% were overweight and 33.8% were obese; 64.1% were of white race. During follow-up, 329 MGUS patients (4.2%) progressed to MM: 72 (3.5%) normal-weight patients (median follow-up = 61.9 months), 144 (4.6%) overweight patients (median follow-up = 69.1 months), and 113 (4.3%) obese patients (median follow-up = 70.6 months). In the multivariable analysis, overweight (hazard ratio [HR] = 1.55, 95% confidence interval [CI] = 1.16 to 2.06) and obesity (HR = 1.98, 95% CI = 1.47 to 2.68) were associated with an increased risk of transformation of MGUS to MM. Moreover, black race was associated with a higher risk of MM (HR = 1.98, 95% CI = 1.55 to 2.54). Obesity and black race are risk factors for transformation of MGUS to MM. Future clinical trials should examine whether weight loss is a way to prevent the progression to MM in MGUS patients.

Characterization of an In Vitro model of MGUS Progression to Multiple Myeloma

Introduction: Multiple Myeloma (MM) is an incurable cancer characterized by a pre-malignant clonal phase of disease called monoclonal gammopathy of undetermined significance (MGUS). Most patients with MGUS do not develop overt MM and the biology underlying this potential transformation is unclear. Investigations to prevent the development of MM from MGUS are limited by the relative infrequency of MGUS progression. Unfortunately, MGUS cells have historically proven difficult to grow in vitro because of slow rates of proliferation and difficulty in sustaining cell cultures. We present evidence of an in vitro model that generates MM-like plasma cells from patients diagnosed with only MGUS. We further present gene expression patterns of primary patient cells versus the induced MM cells to provide guidance as to important initiating events within our model.Methods: We collected a CD38+ cell fraction and a mononuclear (CD38-) fraction from 4 patients with MGUS using a Miltenyl Biotec column Separator. The CD38- fraction was grown in RPMI with 10% FBS and 1% sodium pyruvate with or without a polyglycolic acid/ poly L-lactic acid 90/10 (PLGA) copolymer scaffold to create 3D culture conditions. The mononuclear layer from healthy donors and the MM cell line, U266, were grown as controls. We then analyzed the initial CD38+ fraction, the initial CD38- fraction, and the CD38- fraction grown in media or 3D co-culture by flow cytometry for expression of kappa, lambda, CD38, CD138, CD45, CD19, and CD56. Gene expression analysis was performed using RNA-sequencing data from the CD38+, CD38-, cultured CD38-, and control cells. Expression of the top 100 ranked differentially expressed genes, which demonstrated the largest variation, were further analyzed using the nCounter® Analysis System (NanoString Technologies).Results: The CD38- fraction from MGUS patients grew into an adherent layer of elongated cells, consistent with bone marrow stromal cells. After several months, the stromal cells were noted to change shape and new, round cells were observed budding off from the stromal layer. Over time, the stromal layer disappeared and the round plasmacytoid cells remained. Characterization of these round cells revealed them to be plasma cells by IHC and flow cytometry. When comparing these in vitro generated cells to the initial CD38+ fraction removed from patients, the new cells showed the re-emergence of CD38 and CD138, increased expression of CD56 and CD19, and decreased expression of CD45. Gene expression analysis revealed 3 distinct populations of cells. The initial CD38- fraction separated with the healthy mononuclear layer. The initial CD38+ fraction clustered independently while the grown plasma cells clustered with the U266 cells. Analysis of the differential gene expression patterns revealed differences in the expression of immunoglobulin genes, as well as alterations in expression of extracellular matrix and cell adhesion markers including PAI-1, MMP2, COL1A2, and GREM1; and alterations in expression of mitochondrial genes.Conclusion: To our knowledge, this is the first in vitro simulation of disease progression from MGUS to MM. Our model induced the growth of plasma cells with an aggressive phenotype as assayed by flow cytometry. The gene expression profile further demonstrates gene expression patterns from our induced plasma cells consistent with MM versus MGUS. The alterations in extracellular matrix proteins as seen in our induced plasma cells are consistent with an epithelial to mesenchymal type transition implicated in disease progression, metastasis, and bone lesions. Additionally, the alterations in mitochondrial gene expression have been implicated in early disease progression in colon cancer and MM. These findings provide further evidence that our model simulates disease transformation and the expression data suggest possible pathways that may be important in myeloma disease progression that can be further evaluated in vivo. DisclosuresNo relevant conflicts of interest to declare.

Severe Isotype-Matched Immunosuppression By Hevylite® Is Predictive of Shorter Time to Progression in MGUS Patients

▪Monoclonal gammopathy of undetermined significance (MGUS) accounts for c.a. 50% of all diagnosed plasma cell dyscrasias and is the precursor state of all multiple myelomas (MM). However, only a small number of cases will progress to a malignant state. The challenge is on how to identify these patients and, specifically, those that are at imminent risk of developing symptomatic disease. Presently, two main bio-marker based models are used in the risk assessment of MGUS patients (Mayo Clinic group and Spanish Myeloma group), however, some patients are very differently assessed by the two models. The recently developed Hevylite assay allowed, for the first time, to easily quantify the uninvolved immunoglobulin associated with the tumor isotype and therefore study the presence of isotype-matched immunosuppression (IMI). In this study we aimed at assessing the utility of this assay for MGUS risk stratification.Methods:309 stored serum samples from 248 MGUS (164 IgG, 44 IgA, and 40 IgM) patients attending different services from our hospital. Patients were previously risk stratified according to the 2010 IMWG guidelines (Kyle et al Leukemia). Samples were analyzed with Hevylite® (The Binding Site, UK) in a BNII analyzer (Siemens) according to the monoclonal protein isotype. Normal ranges were those provided by the manufacturer. Values 50% below the lower normal ranges of the immunoglobulins determined by Hevylite were defined as severe IMI.Results:Ninety percent of the patients showed an abnormal Hevylite Ig’k/Ig’λ ratio (i.e. IgGk/IgGL for an IgG-MGUS) with the greatest percentage observed for IgA and IgM isotypes (95% abnormal ratios). Regarding the isotype-matched immunosuppression, it was observed in 56% of the samples. The frequency of IMI by Hevylite was strongly associated with MGUS risk group (p=0.013) showing a greater prevalence in the highest risk group (Fig.1). Additionally, no significant difference was observed in relation to monoclonal isotype: IgG=60%, IgA=69%, IgM=57%, (p=0.37). Classical immunoparesis (i.e., suppression of IgG and/or IgM in an IgA patient), was less frequent than IMI (43% vs 56%, p<0.0001) but it also increased among higher risk MGUS patients (p=0.0064).Of the 248 MGUS patients, follow-up clinical records were recovered from for 127 (107 IgG, 20 IgA) allowing us to perform a progression free survival (PFS) based on Hevylite first sample results. Severe IMI was able to differentiate two groups of patients with significantly different PFS (p=0.023) (Fig.2). On the contrary, severe suppression of either one of the non-involved immunoglobulins (e.g. classical immunoparesis) was not associated with significant greater risk of progression (p=0.309). Interestingly, there was almost the double of patients with severe IMI in comparison with severe classical immunoparesis.ConclusionsThe results obtained are in line with a previous study from Katzmann et al (Leukemia 2103) where Hevylite pair suppression greater than 50% predicts progression of MGUS, thus reinforcing the potential utility of this parameter as a marker for the risk assessment of monoclonal gammopathies. It also suggests that, in this context, the predictive value of IMI is more useful than that of classical immunoparesis. [Display omitted] [Display omitted] DisclosuresPais:The Binding Site: Employment. Barbosa:The Binding Site: Employment. Campos:The Binding Site: Employment.

Abdominal adipose tissue in MGUS and multiple myeloma.

To determine abdominal adipose tissue parameters on PET/CT in patients with monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) that may serve as predictors of progression of MGUS to MM. We hypothesized that patients with MM had higher abdominal adiposity and higher fat metabolic activity compared to patients with MGUS. Our retrospective study was IRB approved and HIPAA compliant. The study group comprised 40 patients (mean age 64 ± 13years) with MGUS and 32 patients (mean age 62 ± 10years) with recently diagnosed MM (mean time since diagnosis of MM 3.0 ± 3.9months) who had not undergone MM treatment. All patients underwent whole body FDG-PET/CT. Total abdominal adipose tissue (TAT), abdominal subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) cross sectional areas (CSA) (cm(2)) and metabolic activity (SUV) were assessed. Groups were compared using ANOVA. ROC curve analysis was performed to determine cutoff values for abdominal adipose tissue parameters to detect MM. Patients with recently diagnosed MM had higher TAT and SAT CSA (p ≤ 0.03) and higher fat metabolic activity (p < 0.01). VAT metabolic activity showed the highest sensitivity and specificity for identifying patients with MM (area under the curve 0.95 with cutoff value of >0.34, sensitivity 90.6%, specificity 92.5%, p < 0.0001). Patients who were recently diagnosed with MM had higher abdominal fat CSA and higher fat metabolic activity compared to patients with MGUS. These parameters may serve as novel biomarkers of progression of MGUS to MM.

Diagnosis, risk stratification and management of monoclonal gammopathy of undetermined significance and smoldering multiple myeloma.

Monoclonal gammopathy of undetermined significance (MGUS) is one of the most common premalignant disorders. IgG and IgA MGUS are precursor conditions of multiple myeloma (MM), whereas light-chain MGUS is a precursor condition of light-chain MM. Smoldering MM (SMM) is a precursor condition with higher tumor burden and higher risk of progression to symptomatic MM compared to MGUS. Assessment of the risk of progression of patients with asymptomatic monoclonal gammopathies is based on various factors including clonal burden, as well as biological characteristics, such as cytogenetic abnormalities and light-chain production. Several models have been constructed that are useful in daily practice for predicting risk of progression of MGUS or SMM. Importantly, the plasma cell clone may occasionally be responsible for severe organ damage through the production of a M-protein which deposits in tissues or has autoantibody activity. These disorders are rare and often require therapy directed at eradication of the underlying clone. Importantly, recent studies have shown that asymptomatic patients with a bone marrow plasma cell percentage ≥60%, free light-chain ratio ≥100, or >1 focal lesion on MRI (myeloma-defining events) have a 80% risk of developing symptomatic MM within 2years. These patients are now considered to have MM requiring therapy, similar to patients with symptomatic disease. In this review, we provide an overview of the new diagnostic criteria of the monoclonal gammopathies and discuss risk of progression to active MM. We also provide recommendations for the management of patients with MGUS and SMM including risk-adapted follow-up.

Serum Free Light Chain Assays in the Risk Stratification of Monoclonal Gammopathy of Undetermined Significance: Results from a Pharmacoeconomic Evaluation

Monoclonal gammopathy of undetermined significance (MGUS) has a 1% overall risk of progression to symptomatic disease per year. Previously, monoclonal Ig (M-protein) isotype (IgA or IgM) and concentration (≥10g/L) have been used to risk stratify patients. In 2005, Rajkumar et al. reported on three independent risk factors: M-protein concentration ≥15g/L, M-protein isotype IgA or IgM, and an abnormal free light chain ratio (FLCr). An abnormal FLCr, determined by polyclonal antibody-based nephelometric analysis, had a 7.4 fold relative risk for MGUS progression in the absence of the other two factors. The International Myeloma Working Group (IMWG) recommends inclusion of all three factors for MGUS risk stratification, with low risk patients being referred to primary care while, intermediate and higher risk patients have bone marrow biopsies and skeletal surveys at presentation and are then followed annually by a haematologist. Here, we compare costs and resource use associated with the IMWG recommendations with the previous risk stratification approach.The model consists of a decision tree structure from incidental finding of an M-protein level <30g/L to risk stratification, followed by a Markov structure comprising health states for non-stratified, low risk and ≥1 risk factor MGUS, smouldering multiple myeloma, symptomatic disease and death. The distribution of patients in the health states was based on incidence data and the proportion of patients in each risk strata in a US cohort, published by Rajkumar et al. (2005). The proportion of patients stratified as low risk in the previous approach was 33% compared with 39% when following IMWG recommendations.Patients with MGUS experience higher mortality relative to age- and sex-matched healthy individuals; survival was therefore estimated by applying a relative risk of death to general population life tables (Office for National Statistics, 2014). Risk of progression to symptomatic disease was obtained by fitting exponential curves to the published data of Rajkumar et al. (2005) and Kyle et al. (2002) from the Mayo Clinic.Costs of laboratory tests and resources were derived from the 2013-14 NHS and PSSRU 2014 data. Resource use was based on IMWG guidelines and clinician advice. The model used a 5-year time horizon, with outcomes discounted at an annual rate of 3.5%.The model predicts that following IMWG recommendations results in lower costs, fewer bone marrow biopsies, skeletal surveys and haematologist consultations, and more referrals to primary care (Table 1). The pathway following IMWG recommendations increases referrals to primary care; however, the model shows, at Year 5, only 1.5% of those in primary care are predicted to progress to symptomatic disease compared to 3% of those in the previous risk stratification pathway.Cost savings in the diagnostic pathway were driven by fewer skeletal surveys and bone marrow biopsies: -₤13.75 and -₤19.90 per patient, respectively. In the Markov model, savings were driven by the reduction in haematologist consultations, -₤85.91 per patient.One-way sensitivity and scenario analyses indicated that results remained stable unless the proportion of patients referred to primary care in the IMWG pathway was ≤32% or the proportion of patients referred back to primary care in the previous risk stratification approach was ≥39%.Probabilistic sensitivity analysis, based on 10,000 probabilistic simulations, showed that the IMWG recommendations have a 92.5%, 95.4%, 95.4% and 92.1% probability of reducing costs, bone marrow biopsies, skeletal surveys and haematology consultations, respectively, and a 95.4% probability of more patients being referred back to primary care.The UK incidence of newly identified MGUS cases is estimated to be >7000 per year. Applying the IMWG recommendations, including FLCr analysis, therefore leads to substantial cost savings and reduced resource use in both the diagnostic and long-term pathways.Benefits to patients include fewer required bone marrow biopsies and skeletal surveys, while fewer patients identified as low risk are predicted to progress to symptomatic disease.As practice moves towards a more systematic out-of-hospital approach, further benefits are anticipated for the management of patients with MGUS. Haematology clinic resources can then be allocated to other conditions, while maintaining confidence that all MGUS patients are appropriately managed. [Display omitted] DisclosuresPratt:The Binding Site Group Ltd: Other: Member of Medical Advisory Board. Harding:The Binding Site Group Ltd: Employment, Membership on an entity's Board of Directors or advisory committees. Powner:The Binding Site Group Ltd: Employment. Hughes:The Binding Site Group Ltd: Employment. Cook:The Binding Site Group Ltd: Other: Member of Medical Advisory Board.

Abstract 4026: Growth of monoclonal gammopathy of undetermined significance (MGUS) in a 3-dimensional co-culture in vitro model

Abstract Introduction: Multiple Myeloma (MM) is an incurable cancer with a pre-malignant clonal phase of disease called monoclonal gammopathy of undetermined significance (MGUS). Most patients with MGUS do not develop MM and the biology underlying this potential transformation is unclear. Investigations to prevent the development of MM from MGUS is limited by infrequent MGUS progression. MGUS cells have historically proven difficult to grow in vitro because of slow rates of proliferation and difficulty in sustaining cell cultures. An in vitro model of MGUS that circumvents these challenges by recreating a supportive bone marrow microenvironment will allow further investigation into the pathogenesis of disease progression. Herein, we examine a novel stroma-based in vitro model of MGUS and MM using bone marrow stromal cells to support and maintain a bi-directional relationship within the clonal plasma cell population. Aim: To grow MGUS derived plasma cells by recreating a supportive bone marrow microenvironment using a 3-dimensional in vitro model. Methods: We collected a CD38+ cell fraction and a bone marrow stromal fraction from patients with plasma cell dyscrasias using a Miltenyl Biotec column Separator. We seeded stromal cells from a female, kappa expressing MGUS patient or a healthy donor onto a polyglycolic acid/ poly L-lactic acid 90/10 (PLGA) copolymer scaffold. Then, a CD38+ cell fraction from a male patient with lambda secreting PCL or MM cell line U266 or MM1.s (both male, lambda expressing cell lines) were added to create 3D co-culture conditions. For comparison, a 2 dimensional co-culture condition was created by growing MM patient cells or cell lines over a bone marrow stromal cell layer without scaffold.. After one week, samples were formalin fixed and sectioned for immunohistochemistry (IHC) and stained with CD138, kappa, lambda, and ki-67. Fluorescent in situ hybridization (FISH) was used to label X and Y chromosomes. Results: IHC demonstrated proliferation of CD138+ cells evident by Ki-67 expression in in 3-D co-culture systems. There was no growth of the patient derived CD38+ cells in 2-D co-culture and the proliferation of of cell lines was diminished in 2-D versus 3-D. When seeded on healthy donor stroma in 3-D, the CD38+ cells from all sources were lambda restricted. When seeded on the MGUS stroma, the MM1.s cells were lambda restricted while PCL cells showed both kappa and lambda expression with a dominance of kappa expression, and U266 cells showed kappa restriction. FISH demonstrated a female chromosomal pattern with X,Y in the plasma cells from the 3D co-cultures in the case of U266 or PCL cell 3-D co-culture with MGUS stroma. Conclusion: This is the first demonstration of in vitro growth of plasma cells from a patient with MGUS. We speculate the aggressive plasma cells from the U266 cell line and the PCL sample induced the growth of a CD38- cell fraction and caused differentiation to plasma cells. Citation Format: Brea C. Lipe, Omar Aljitawi, Tara Lin, Joseph Fontes. Growth of monoclonal gammopathy of undetermined significance (MGUS) in a 3-dimensional co-culture in vitro model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4026. doi:10.1158/1538-7445.AM2015-4026