Monoclonal Gammopathy Patients Research Articles

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.

Multi-Center Clinical Validation of a Mass Spectrometry Immunoassay for the Diagnosis and Monitoring of Multiple Myeloma and Associated Disorders

Introduction: Mass spectrometry (MS) technology holds great promise for the investigation of monoclonal proteins (M proteins) in peripheral blood. We present results from a multi-center clinical validation study using quantitative immunoprecipitation MS (QIP-MS). QIP-MS combines isotype-specific immunopurification with matrix-assisted laser-desorption ionization MS, and offers automated, sensitive detection, isotyping and quantification of M proteins. It reports the mass/charge ratio (m/z) of the involved light chain, which serves as molecular fingerprint for monitoring the M protein. Methods: The study included 460 diagnosed monoclonal gammopathy (MG) patients (160 multiple myeloma (MM), 112 smoldering MM (SMM), 120 monoclonal gammopathy of undetermined significance (MGUS), 47 Waldenström's Macroglobulinemia (WM), and 21 AL amyloidosis), and 170 disease controls for assessing diagnostic sensitivity and specificity. Sixty-four MM patients with 439 follow-up samples and 10 WM patients with 91 follow-up samples were included to evaluate the ability of QIP-MS to detect M protein changes related to treatment. Median follow up was 19 and 15 months, respectively. Serum samples were retrospectively analyzed at three sites. QIP-MS was carried out using the automated EXENT® solution (in development, The Binding Site, part of Thermo Fisher Scientific). The assay's diagnostic sensitivity and specificity were calculated based on categorizing results as positive or negative. A positive result was defined in baseline samples as the presence of an M protein which was either an intact immunoglobulin ≥0.200 g/L or a light chain only. Results by QIP-MS were also compared to serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). QIP-MS response categories were defined based on M protein changes per international guidelines criteria, and compared to response categories assigned by the treating physician. Complete response (CR) was defined as absence of the M-peak that was observed at baseline, using isotype and m/z value of ±4 as criterion for identity. Results: The overall diagnostic sensitivity of QIP-MS in this study was 95.0%: 93.3% for MGUS; 100.0% for SMM; 94.4% for MM; 100.0% for WM; and 71.4% for AL amyloidosis. The diagnostic specificity of the assay was 68.2%. QIP-MS identified an M protein in more MG patients compared to SPE: 437 (95.0%) vs 398 (86.5%). The positivity rate by QIP-MS vs SPE was 93.3% vs 84.2% in MGUS; 100% vs 92.9% in SMM; 94.3% vs 85.0% in MM; 100% vs 100% in WM and 71.4% vs 47.6% in AL amyloidosis. Method comparison demonstrated a Passing-Bablok slope of 0.8 to 1.2 between QIP-MS and SPE for the quantification of M proteins for each disease group and for each isotype, except for monoclonal IgM and in WM (slope of 1.58). In SPE-positive MG patients, the overall concordance between QIP-MS and IFE for M protein isotype was 97%. The overall concordance rate between QIP-MS response categories and standard response assignment was 55% for MM and 56% for WM: 48% for progressive disease (PD); 63% for stable disease (SD); 46% for minimal response (MR); 71% for partial response (PR); 66% for very good partial response (VGPR); and 25% for CR in MM patients. Among 73 responses categorized as CR, QIP-MS produced a positive result for the original clone in 55 (75.3%) cases. Concordance rates in WM patients were 71% for PD%; 30% for SD; 38% for MR; and 74% for PR; no VGPR or CR were reported by either method. Conclusions: In this study, QIP-MS demonstrated the potential for same or superior diagnostic sensitivity compared to SPE, high concordance with IFE for the M protein isotype and good quantitative agreement with SPE measurements of the M proteins. It reported higher IgM values compared to SPE, likely due to reliance on turbidimetric immunoglobulin measurement for quantitations. Diagnostic specificity was impacted by the identification of minor M proteins, not detectable by SPE, and whose clinical significance requires further investigation. QIP-MS demonstrated “moderate” to “fair” agreement for response assignment in MM and WM, respectively, mostly due to the detection of residual M proteins in a significant proportion of patients in CR, in line with its enhanced analytical sensitivity. These data support the use of QIP-MS as an aid in the diagnosis and monitoring of MGs.

Improved Screening of Monoclonal Gammopathy Patients by MALDI-TOF Mass Spectrometry.

Monoclonal gammopathies are a group of blood diseases characterized by presence of abnormal immunoglobulins in peripheral blood and/or urine of patients. Multiple myeloma and plasma cell leukemia are monoclonal gammopathies with unclear etiology, caused by malignant transformation of bone marrow plasma cells. Mass spectrometry with matrix-assisted laser desorption/ionization and time-of-flight detection is commonly used for investigation of the peptidome and small proteome of blood plasma with high accuracy, robustness, and cost-effectivity. In addition, mass spectrometry coupled with advanced statistics can be used for molecular profiling, classification, and diagnosis of liquid biopsies and tissue specimens in various malignancies. Despite the fact there have been fully optimized protocols for mass spectrometry of normal blood plasma available for decades, in monoclonal gammopathy patients, the massive alterations of biophysical and biochemical parameters of peripheral blood plasma often limit the mass spectrometry measurements. In this paper, we present a new two-step extraction protocol and demonstrated the enhanced resolution and intensity (>50×) of mass spectra obtained from extracts of peripheral blood plasma from monoclonal gammopathy patients. When coupled with advanced statistics and machine learning, the mass spectra profiles enabled the direct identification, classification, and discrimination of multiple myeloma and plasma cell leukemia patients with high accuracy and precision. A model based on PLS-DA achieved the best performance with 71.5% accuracy (95% confidence interval, CI = 57.1-83.3%) when the 10× repeated 5-fold CV was performed. In summary, the two-step extraction protocol improved the analysis of monoclonal gammopathy peripheral blood plasma samples by mass spectrometry and provided a tool for addressing the complex molecular etiology of monoclonal gammopathies.

The Clusterin/Von Willebrand Factor Ratio Is Significantly Lower in Marrow Plasma from AL λ-Type Than from λ-Isotype Monoclonal Gammopathy Patients

Background: Our primary objective is to develop methods that enable early diagnosis of AL or risk of AL during the prodromal decade prior to symptomatic presentation (Weiss et al. J Clin Oncol 2014;32:2699). There are parameters that increase risk of AL such as the presence of a monoclonal gammopathy of undetermined significance (MGUS) or smoldering multiple myeloma (SMM) or having AL-related IGVL genes encoding light-chain production in clonal plasma cells (Zhou et al. J Clin Oncol 2019;37:8010; Kourelis et al. Blood 2017;129:299; Kyle et al. N Engl J Med 2007;356:2582). Previous studies have shown that clusterin (apolipoprotein J) may play a variety of roles in protein folding disorders, possibly binding and clearing misfolded proteins. Like apolipoprotein E (apoE), clusterin is found in amyloid deposits of all types including the arterial deposits of cerebral amyloid angiopathy; moreover, it interacts cooperatively with apoE in Alzheimer's (Endo et al. Acta Neuropathol Commun 2019;7:12; DeMattos et al. Neuron 2004;41:193). Furthermore, serum levels of clusterin are reduced in patients with AL cardiac involvement (Filippini et al. Glia2021;69:681; Greene et al. Biochemistry 2015;54:268; Greene et al. Am J Pathol2011;178:61; Lee et al. Biochem Biophys Res Commun2009;388:256). A previous study has also shown that von Willebrand factor (vWF) levels are significantly elevated in AL patients, possibly due to endothelial cell dysfunction (Kastritis et al. Blood 2016;128:405); AL amyloidosis commonly involves the vasculature. Seeking additional parameters for likelihood of AL we asked whether the ratio of clusterin to vWF (C/v) in marrow plasma differed among AL, MGUS and SMM patients. Materials & Methods: λ-isotype MGUS or SMM patients from multiple sites in the USA with a difference between involved and uninvolved FLC > 23mg/L and a κ-to-λ ratio below normal and no evidence of amyloid on prior studies consented to participate in a clinical study screening for AL (NCT04615572) and had bone marrow aspirates sent overnite to Tufts. In addition, newly diagnosed AL patients consented to have blood and marrow aspirates obtained for research on an IRB-approved study. All samples were heparinized and had plasma separated initially and aliquoted for storage at -20o C. We also obtained peripheral blood plasma from normal individuals, ages unknown (Precision for Medicine; Norton, MA). Clusterin and vWF ELISA were performed on thawed plasma following manufacturers’ instructions (R&D Systems; Minneapolis, MN). GraphPad PRISM V5 was used for statistical analyses. Results: Twelve MGUS and 16 SMM patients, all λ-isotype, enrolled on NCT04615572, met FLC criteria and had marrow shipped to Tufts; 2 of the SMM patients were found to have AL and are included with 24 AL λ-type patients whose marrow aspirates were obtained in the same time period. There were no differences in age or gender, but the percentage of marrow plasma cells in SMM and AL patients were significantly greater than in MGUS patients. Comparisons of AL, SMM and MGUS iFLC λ levels did not achieve statistical significance. Medians were 338, 182 and 346 mg/L respectively. Marrow plasma from AL patients had significantly lower C/v ratios than those in either MGUS or SMM specimens (Figure 1). There was no correlation between λ FLC and the C/v ratio (r = -0.20, P > 0.05). Compared to normal individuals (n=20) with a median C/v ratio of 16.9x104 (range, 2.7-56.5), the C/v ratio in peripheral blood in newly diagnosed AL patients (n=15) was significantly lower at a median of 1.5x104 (0.8-2.0) (P<0.01, Mann-Whitney two-tailed). Conclusion: These preliminary data suggest that the C/v ratio is a potential parameter of interest in the construction of a likelihood algorithm for AL in patients with monoclonal gammopathies. The rationale for the ratio is that with progression during the prodromal period prior to significant organ damage clusterin may decrease while vWF may increase. Whether peripheral blood may provide a useful source of plasma is unclear and whether ratios below a threshold or changes in the ratio over time may signal risk of AL or be significant remain questions for further exploration. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Reduced Immune Response and Neutralizing Antibody Activity to the SARS-CoV-2 Vaccination in Patients with a History of Solid Organ Transplant.

ObjectiveThree SARS-CoV-2 vaccinations and boosters are available. We determined whether solid organ transplant patients mounted an immune response to the vaccinations and whether the antibodies had neutralizing activity compared to healthcare worker controls and monoclonal gammopathy patients.MethodsRemnant plasma was obtained from vaccinated solid organ transplant, allogeneic stem cell transplant, monoclonal gammopathy patients, and healthcare worker controls. Samples positive on a SARS-CoV-2 IgG assay (detects spike protein and nucleocapsid) were run on a SARS-CoV-2 in vitro neutralizing antibody assay and a nucleocapsid-specific SARS-CoV-2 IgG assay.ResultsOnly 25% of solid organ transplant patients produced antibodies to SARS-CoV-2 vaccination. Of these, 90% had neutralizing activity against wild type virus, but reduced activity to the variants compared to monoclonal gammopathy patients and healthcare worker controls, particularly the delta variant, for which only 50% had neutralizing antibody activity. ConclusionSolid organ transplant patients should consider protecting themselves against future SARS-CoV-2 infection.

Immunoglobulin Pattern in Monoclonal Gammopathy Disorders

Introduction: The presence of abnormal monoclonal proteins, the M band, is a frequent characteristic feature of plasma cell dyscrasias and is usually detected as a discrete band in the γ or β region in serum or urine protein electrophoresis. It is characterized and confirmed by immunofixation electrophoresis (IFE). Accurate detection and quantification of monoclonal immunoglobulins are important for the diagnosis and management of monoclonal gammopathies.&#x0D; Objectives: To find out the pattern of immunoglobulin in monoclonal gammopathy cases and evaluate the role of IFE in the detection of them.&#x0D; Materials and Methods: This cross-sectional descriptive study was conducted in the Department of Haematology, Armed Forces Institute of Pathology (AFIP), Dhaka from July 2015 to December 2015. Thirty diagnosed cases of monoclonal gammopathies of both sexes were selected. Bone marrow examination, serum protein electrophoresis, skeletal survey, relevant biochemical test and IFE were performed for all the cases.&#x0D; Results: Out of 30 monoclonal gammopathy cases, M band was identified in 24(80%) cases by serum protein electrophoresis but by the IFE M band was found in all 30(100%) cases. Among the M band pattern of immunoglobulin was characterized by IFE and the result was; 15(50%) cases IgG Kappa, 09(30%) cases IgG Lambda, 02(6.7%) cases IgA Kappa, 02(6.7%) cases IgM Kappa and 02(6.7%) cases light chain kappa monoclonal protein.&#x0D; Conclusion: Though the number of the patient was limited, it is evident that in 20% gammopathy cases M band was missing by conventional serum protein electrophoresis but IFE could identify M band in all the cases. It is recommended that IFE should be carried out in all monoclonal gammopathy patients.&#x0D; Journal of Armed Forces Medical College Bangladesh Vol.14 (2) 2018: 160-163

Noninvasive assessment of corneal alterations associated with monoclonal gammopathy.

Aggregation of monoclonal immunoglobulin can lead to organ damage. However, the necessity of invasive examination such as biopsy has hampered better understanding of the pathophysiology. Corneal crystalline deposition is a rarely reported but known ocular manifestation of multiple myeloma. It is unclear whether the cornea is a common target of monoclonal immunoglobulin deposition. We conducted a prospective clinical case-control study to objectively quantify monoclonal gammopathy-associated corneal changes as well as any therapeutic response. Using an ophthalmic Scheimpflug camera imaging for noninvasive corneal assessments, we quantified densitometry values in 30 patients. Although none had crystalline keratopathy, corneal transparency in monoclonal gammopathy patients was significantly impaired compared to that in age-matched controls, based on noninvasive Scheimpflug camera imaging. Furthermore, treatment for multiple myeloma seemed to eradicate the diffuse aggregation of monoclonal proteins. Our results indicate that exposure to monoclonal immunoglobulin may induce the accumulation of monoclonal immunoglobulin in the cornea, and ophthalmic examinations such as corneal densitometry measurements with a Scheimpflug camera may be useful for noninvasive evaluation of monoclonal immunoglobulin deposition diseases.

Systematic overestimation of human serum albumin by capillary zone electrophoresis method due to monoclonal immunoglobulin interferences

BackgroundThe capillary zone electrophoresis method of albumin measurement is frequently used in monoclonal gammopathy patients but some studies suggest poor performances of the method in this population. The aim of this study was to analyse the impact of serum monoclonal immunoglobulins on human serum albumin determination by capillary zone electrophoresis method compared to other available methods. MethodWe prospectively measured albumin in 100 freshly collected non-frozen serum samples in a monoclonal gammopathy patients population, by using four different methods: the capillary zone electrophoresis method, the bromocresol purple dye method, the nephelometric method and the turbidimetric method. Differences in albumin values between the different methods were analysed with respect to serum monoclonal immunoglobulin concentration. These differences were further investigated by measuring albumin levels in human serum samples spiked with exogenous monoclonal immunoglobulins. ResultsHuman serum albumin difference values between capillary zone electrophoresis compared to immunonephelometry method are significantly correlated with increasing monoclonal immunoglobulins concentrations: regression analyses revealed a correlation coefficient r2 = 0.60 and a slope of 0.14 (0.12–0.17, 95% confidence interval). The capillary zone electrophoresis method overestimated serum albumin levels by up to 67% (12 g/L) when monoclonal immunoglobulin level was 63 g/L. The determination of albumin levels in human serum samples spiked with exogenous monoclonal immunoglobulins showed an overestimation of human serum albumin measurement by the capillary zone electrophoresis method proportional to the amount of monoclonal immunoglobulin added in the serum with a slope of 0.19 (0.18–0.20, 95% confidence interval). ConclusionMonoclonal immunoglobulins directly interfere with serum albumin measurement by the capillary zone electrophoresis method leading to a systematic overestimation of serum albumin concentrations proportional to the serum monoclonal immunoglobulin level.

Monoclonal gammopathy in rheumatic diseases.

To analyze the clinical spectrum, laboratory characteristics, and outcomes of monoclonal gammopathy (MG) in patients with rheumatic diseases. Screening for the presence of MG was performed in 872 inpatients with rheumatic diseases from January 2010 to July 2017. A total of 41 patients were enrolled. Their clinical and biological features in addition to outcomes were described. For each patient with primary Sjögren syndrome (pSS), 2 age- and sex-matched pSS patients without MG were selected as controls. Risk factors for the presence of MG and malignant hematological neoplasias were assessed. MG was observed in patients with SS, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, primary biliary cirrhosis, polymyositis, hypomyopathic dermatomyositis, psoriatic arthritis, ANCA-associated vasculitis, polyarteritis nodosa, and polymyalgia rheumatic, with SS the most frequent type. Serum M protein was detected in 37 patients. The monoclonal bands identified in serum were 16 IgG (5 κ, 11 λ), 11 IgA (6 κ, 5 λ), 6 IgM (5 κ, 1 λ), and 4 free λ chains. M components were observed in urine in the other 4 patients. High ESR, albumin/globulin inversion, rheumatoid factor positivity, hypergammaglobulinemia, and hypocomplementemia were common features, presented in more than half of the 41 patients. Patients with pSS, when complicated with MG, showed a higher rate of abnormal urine NAG (71.4 vs 15.8%, P = 0.025), higher levels of ESR [55.0 (53.5) mm/h vs 21.0 (31.8) mm/h, P = 0.001], ESSDAI [26.0 (25.0) vs 12.0 (9.0), P = 0.006], and ClinESSDAI scores [24.0 (25.0) vs 10.5 (10.0), P = 0.011]. Multivariate analysis revealed that the disease activity, assessed by either ESSDAI [adjusted OR 1.127 (95%CI 1.015-1.251), P = 0.025] or ClinESSDAI [adjusted OR 1.121 (95%CI 1.011-1.242), P = 0.030], was the only independent risk factor for the presence of MG. During the follow-up, 2 patients had transient serum M protein, 2 had isotype switch, 1 progressed to multiple myeloma (MM), and another 2 experienced renal injuries attributed by monoclonal or polyclonal plasma cell interstitial infiltration. Seven (17.1%) of the 41 MG patients presented hematological neoplasias, 4 with MM, 2 with smoldering multiple myeloma, and 1 with B cell lymphoma of mucosa-associated lymphoid tissue (MALT) type. The presence of light-chain MG was associated with the development of MM [OR 17.5 (95%CI 1.551-197.435), P = 0.041], but not with an increased risk of lymphoma or SMM. MG was observed in patients with various rheumatic disorders, with SS being the most common type. The presence of MG might be associated with higher disease activity. The development of hematological neoplasias including MM and lymphoma was seen in this setting. Therefore, we recommend the screening for MG and close monitoring for potential malignant transformation in patients with rheumatic diseases as needed.

Activity of aldehyde dehydrogenase in B-cell and plasma cell subsets of monoclonal gammopathy patients and healthy donors.

Aldehyde dehydrogenase (ALDH) is highly active in physiological stem cells as well as in tumor-initiating cells of some malignancies including multiple myeloma (MM). Finding higher activity of ALDH in some cell subsets in monoclonal gammopathies (MG) could identify potential source of myeloma-initiating cells (MICs). Bone marrow of 12 MM, 9 monoclonal gammopathy of undetermined significance (MGUS), and 10 healthy donors (HD) were analyzed by flow cytometry. ALDH activity of B-cells and plasma cells (PC) was analyzed using Aldefluor. Similar changes of ALDH activity were found during B-cell development in HD and MG. Decreasing of ALDH activity from immature to naïve B-cells was found. In postgerminal stages, the activity started to increase, and in PCs, the ALDH activity was the same as in immature B-cells. Increased ALDH activity of all PC subsets compared to naïve B-cells was found in MM as well as in HD, while in MGUS, only CD19- PCs have higher ALDH activity. In HD, ALDH activity was higher in CD19+ PCs compared with MG. Our results indicate that changes of ALDH activity are the natural phenomenon in B-cell development; thus, high ALDH activity as a single marker is not appropriate for MICs identification.

Analysis of B-cell subpopulations in monoclonal gammopathies.

Multiple myeloma (MM) is characterized by accumulation of pathological plasma cells (PCs) in bone marrow (BM) as a result of deregulation of B-cell development. To clarify its pathophysiology it is necessary to investigate in detail the developmental stages of B-cells. Enumeration of total CD19-positive (CD19(+)) cells and their subpopulations together with PCs was done in peripheral blood (PB) and BM of newly diagnosed monoclonal gammopathy patients and control subjects. Representation of subsets was compared among groups and relationships between subset percentage and cytogenetic/biochemical findings were analyzed. A lower number of total CD19(+) cells was found in MM, particularly in advanced stages of disease. Reduction of naive (P < .01) and transitional B-cells (P < .05) and increase of switched memory and switched CD27(-) B-cells and germinal center founder cells were detected in PB of MM compared with controls (P < .01). Similar results were found in BM. β2 microglobulin level in MM positively correlated with the number of PCs and negatively with percentage of naive B-cells (P < .05). Our results provided a detailed phenotypic profile and enumeration of B and PC subpopulations in monoclonal gammopathy patients. A reduced number of B-cells and particularly a differentiation shift to more numerous antigen-stimulated forms was observed in MM. This might indicate a potential source of myeloma-initiating cells in one of these subpopulations.

11C-Pittsburgh B PET Imaging in Cardiac Amyloidosis

ObjectivesThis study sought to investigate the efficacy of 11C-Pittsburgh B (PiB) positron emission tomography (PET)/computed tomography (CT) in the diagnosis of cardiac amyloidosis. BackgroundThe PiB compound has been promising for detection of amyloid deposits in the brain. MethodsA total of 22 consecutive patients were enrolled in this prospective pilot study of monoclonal gammopathy patients with suspected cardiac amyloidosis. The study consisted of a series of 11C-PiB PET/CT, echocardiography, cardiac magnetic resonance, and endomyocardial biopsy within a 1-month period. In addition, 10 normal subjects were recruited to determine the most optimal cut-off value for a positive 11C-PiB PET/CT scan. ResultsAmong the 22 patients, 15 patients were diagnosed as cardiac amyloidosis by endomyocardial biopsy and 5 patients had undergone chemotherapy previously before the 11C-PiB PET/CT. There were no differences in echocardiographic parameters between patients with versus without cardiac amyloidosis, except for a marginal difference in the left ventricular end-diastolic dimension (median 41.0 mm [range 33.0 to 49.0 mm] vs. 50.0 mm [range 38.0 to 55.0 mm], p = 0.066). 11C-PiB PET/CT was positive in 13 of 15 biopsy-proven cardiac amyloidosis patients, whereas none of the patients without cardiac amyloidosis demonstrated positive 11C-PiB PET/CT scan results. The maximal myocardium-to-blood cavity ratio was significantly different between patients with versus without cardiac amyloidosis (median 3.9 [range 1.7 to 19.9] vs. 1.0 [range 0.8 to 1.2], p < 0.001). In association with the significant difference of 11C-PiB uptake in the myocardium between the chemotherapy naïve versus the previous chemotherapy group (median 10.4 [range 1.7 to 19.9] vs. 2.3 [range 1.7 to 3.8], p = 0.014), all except 1 patient among the 5 previously treated patients had responded to chemotherapy by serum free light chain assay results at the time of 11C-PiB PET/CT scan. Conclusions11C-PiB PET/CT may be valuable for the diagnosis of cardiac amyloidosis noninvasively. Whether 11C-PiB PET/CT may be a good surrogate marker of active light chain deposition in the myocardium warrants further investigation in a larger number of patients.

Transcriptome Analysis Reveals Molecular Profiles Associated with Evolving Steps of Monoclonal Gammopathies.

AbstractAbstract 2914 Background:Based on clinical and biological data, a multistep model of disease progression starting in monoclonal gammopathy of undetermined significance (MGUS) continuing through MM, sometimes with an intermediate entity called smoldering MM (SMM), and ending in extramedullary disease, has been proposed. Material and Methods:To gain further insights into the role of the transcriptome deregulation in the transition from normal plasma cells (NPC) to clonal PC and from indolent clonal PC to malignant PC, we performed gene expression profiling (GEP) by Human Gene 1.0 ST Array (Affymetrix) in purified PC from 41 patients with MM, 33 with high-risk SMM and 20 with MGUS. In addition, 5 healthy donors were also included in order to relate the deregulation of GEP of clonal populations to normal condition. Results:Initially, we investigated whether the selected PC population from monoclonal gammopathy patients displayed specific GEP that were clearly distinguishable from NPC. 126 common genes were differentially expressed in MGUS, SMM and MM as compared to NPC (q-value < 10−5). The two most significant molecular and cellular functional categories were cell cycle and cell death (P < 0.01). The top 3 canonical pathways were EIF2 signaling, regulation of EIF4 and p70S6K signaling, and mTOR signaling (P < 0.001). Interestingly, 17 and 9 out of the 126 significant deregulated genes were small nucleolar RNA molecules and zinc finger proteins. GADD45A was the most significant up-regulated gene in clonal PC vs NPC.Subsequently, we looked for genes deregulated in MGUS vs MM, SMM vs MM and MGUS vs SMM, in order to identify gene categories and molecular pathways involved in MM development. A total of 1,184 genes were differentially expressed between MGUS and MM (q-value < 10−5) with an imbalance in favor of antiapoptotic state in MM. Thus, BAG3, GADD45B, AKT1 and AKT2 were overexpressed in MM while APAF1 and BCL2L1 were underexpressed in MM. The molecular and cellular function most significantly affected in this comparison was cell death with 106 genes involved (P < 0.01). One of the top 10 genes upregulated in MM vs MGUS was TERC. In the SMM vs MM comparison, 1,163 genes were deregulated (q-value < 10−5). DNA replication, recombination and repair (ATM, RAD17, RAD50) was the most significant deregulated molecular and cellular function. Telomere extension by telomerase and Myc mediated apoptosis were included within the most significant canonical pathways. The analysis showed that a set of 627 differentially expressed genes was able to differentiate MGUS from SMM (q-value < 10−5). Cell death was identified as the most significant molecular function affected (P < 0.01). The RAC signaling pathway deregulation (P < 0.01) was included within the five most significant canonical pathways. In addition, NFKB signaling was also in the top canonical pathways (P < 0.01).Finally, we also looked for those genes significantly deregulated among the three entities, which in turn were progressively up or downregulated from MGUS to SMM and to MM. We reasoned that these genes could be the most significant for promoting multistep transformation. Surprisingly, only eight out of the 2,974 significant genes exhibited a progressive deregulation (P < 0.0001) in the evolving stages of monoclonal gammopathies (Figure 1). All the genes with a progressive increase from MGUS to SMM and to MM were snoRNA. APAF1, VCAN and MEGF9 showed a progressive downregulation in the transition from MGUS to SMM and to MM. Conclusion:Our data show that although MGUS, SMM and MM are not clearly distinguishable groups according to their GEP, several signaling pathways and genes were significant deregulated in the different steps of transformation process. [Display omitted] Disclosures:Lahuerta:Janssen: Honoraria; Celgene: Honoraria. Oriol:Janssen: Honoraria; Celgene: Honoraria.

Association of plasma cell subsets in the bone marrow and free light chain concentrations in the serum of monoclonal gammopathy patients

This study investigated bone marrow plasma cell subsets and monoclonal free light chain concentrations in blood of monoclonal gammopathy patients. 54 bone marrow samples were stained by double immunofluorescence to...

Phenotypic Analysis of CD19+ Subsets In Monoclonal Gammopathy Patients

AbstractAbstract 4998 Background.Monoclonal gammopathy of undetermined significance (MGUS) is the most common plasma cell disorder which can eventually progress into malignant multiple myeloma (MM). Plasma cells (PCs) are the terminal stadium of B cells differentiation, but it is still unclear which population is the source of pathological PCs with malignant transformation and which population is involved in and may give rise to clonogenic myeloma stem cells. Aims.Phenotypic analysis of CD19+ cell subpopulations in monoclonal gammopathy patients and healthy volunteers to asses their frequency and to find differences on cellular level. Methods.Total of 38 samples was analyzed (16 newly diagnosed untreated MM patients, 12 untreated MGUS persons and 10 healthy donors). CD19+ cells were analyzed for surface expression of CD24, CD27, CD38, and IgD by 5-colors immunophenotyping. Subpopulations of pre-plasma cells consist of transitional B cells (CD24+CD38+), naïve B cells (CD38-IgD+), activated B cells (CD38+IgD+), preGC B cells (CD38++IgD+) and memory B cells (CD38-/+IgD-). These were evaluated in whole lysed peripheral blood together with circulating plasmablast/plasma cells (CD38++IgD-). Bone marrow of MGUS and MM patients was analyzed for number of transitional, immature and memory B cells. Results.Flow cytometric analysis shown no statistical difference when compared number of transitional B cells (1.8%; 3.0% and 1.2%) and activated B cells (54.6%; 62.1% and 45.5%) in peripheral blood of healthy volunteers, MGUS and MM patients, respectively. There was found lower number of circulating plasmablast/plasma cells in peripheral blood of healthy volunteers than in MGUS (1.0% vs. 1.7%; p<0.01), but there was no statistically significant difference for MM (1.7%) when compared to others. The highest number of peripheral naive B cells was found in healthy volunteers (21.4%; p<0.001) and the highest number of peripheral memory B cells was found in MM patients (32.9%; p<0.01) when compared to other groups. There was found also higher number of peripheral preGC B cells in MGUS and MM patients (2.7% vs. 1.6% vs. 1.3%; p<0.05) than in healthy volunteers, respectively. Although numbers of transitional and immature B cells in bone marrow were different for MGUS and MM, the only statistically significant difference was found in number of memory B cells (25.4% for MGUS vs. 11.9% for MM; p<0.01). Summary/Conclusions.Our result showed differences in CD19+ subsets when compared peripheral blood of healthy volunteers and monoclonal gammopathy patients as well as in bone marrow of monoclonal gammopathy group. These differences could be a sign of ongoing changes in B cells of monoclonal gammopathy patients. Further analysis will be also focused on changes at DNA level to confirm clonality of selected subpopulations and to find possible myeloma stem cells source.Supported by GACR 301/09/P457, GACR GAP304/10/1395, MSMT LC06027, MSM0021622434, IGA 10408-3, IGA 10406-3. Disclosures:Hajek:Janssen-Cilag: Honoraria; Celgene: Honoraria; Merck, Sharp, and Dohme: Honoraria.

Flow Cytometric Phenotyping and Analysis of CD8 Regulatory and Suppressor Cells In Multiple Myeloma

AbstractAbstract 2947In solid tumors and hematological malignancies, elevated proportions of CD4 regulatory (CD4 Tregs) cells were reported and the function was also documented well. In the context of CD8 regulatory cells (CD8 Tregs) existence has been revealed in various solid tumors including colorectal, prostate and ovarian cancers. In hematological malignancies or multiple myeloma (MM) the presence of CD8 Tregs are yet to be revealed. To unveil and understand the existence of CD8 Tregs, we characterized and quantified CD8 Tregs along with CD8 suppressor cells in MM. A total of 91 monoclonal gammopathy patients were screened for CD8 Tregs and suppressor cells in peripheral blood (PB) and bone marrow (BM). For comparison, 21 healthy volunteers (HVs) PB were also analyzed. Clinical diseases of the patients were represented as monoclonal gammopathy of undetermined significance (MGUS)-14% (13/91), untreated MM- 60% (55/91) and relapsed MM (Rel MM)-26% (23/91). A simple three-color flow cytometric analysis was used to phenotype the CD8 Tregs and suppressor cells. CD8 Tregs were determined as CD8+FoxP3+ (Frisullo et al, 2010, Hum Immunol) and CD8 suppressor cells were identified by negative expression of co-stimulatory molecule CD28 (CD8+CD28-). All the results were summarized as percentage median and range. Our results showed strong significant increase in CD8 Tregs and suppressor cells in PB of patient cohort compared to HVs [CD8 Tregs- 0.39% (0.03%–2.41%) vs. 0.18% (0.04%–0.61%); P=0.002 and CD8 suppressor cells- 69.42% (10.99%–95.91%) vs. 27.58% (9.08%–70.52%); P<0.0001]. Additionally, strong significant difference was also noticed between HVs vs. MGUS, MM, and Rel MM for PB CD8 Tregs and suppressor cells; the data with statistical results has been depicted in table 1. More or less similar frequencies of PB CD8 Tregs were found between MGUS vs. MM vs. Rel MM. In term of PB CD8 suppressor cells Rel MM had insignificant increase compared to MGUS and MM. Surprisingly, MM cohort had insignificantly decreased frequency of CD8 suppressor cells compared to MGUS. As similarly to PB, BM CD8 Tregs and suppressor cells did not show any statistical significance in between patient groups. The BM CD8 Tregs frequency was observed respectively as: MGUS-0.32% (0.08%–1.04%), MM-0.44% (0.06%–1.82%) and Rel MM-0.27% (0.11%–1.16%). Respective BM CD8 suppressor cells frequency was noticed as: MGUS-63.32% (24.83%–94.43%), MM-62.15% (6.19%–93.45%) and Rel MM-67.29% (18.55%–87.61%). Subgroup of patients with < 95% of abnormal plasma cells [ANPCs- 59.21% (13.35%–91.83%)] had significantly decreased level of PB CD8 suppressor cells compared to counter group [≥95% of ANPCs- 74.23% (10.99%–95.91%)]; P=0.046. In line with PB, BM CD8 suppressor cells also showed significantly reduced frequency in <95% of ANPCs cohort [56.99% (16.48%–94.43%)] compared to ≥ 95% ANPCs cohort [69.61% (6.19%–93.45%)]; P=0.013. No relevant association was noticed between CD8 Tregs and suppressor cells with other clinical features including β2M, albumin, creatinine, lactate dehydrogenase, hemoglobulin and monoclonal protein level. In conclusion, our results disclose that as similar to CD4 Tregs, CD8 Tregs are also deregulated in MM compared to HVs; this is also in line with CD8 suppressor cells. This data suggest that in MM immune dysfunction is also enhanced by the shift in favor of CD8 immune regulatory and suppressor cells. This study was supported by the respective grants-MSM0021622434, LC06027, IGA NS10406, IGA NS10408, and GACR P304/10/1395. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Timing of acquisition of deletion 13 in plasma cell dyscrasias is dependent on genetic context

Multiple myeloma, monoclonal gammopathy of undetermined significance and smoldering multiple myeloma harbor common chromosomal abnormalities but the prevalence and relative association of aberrations in these diagnostic groups remains controversial. We investigated these aspects in a large series of patients. Chromosome 13 deletion (Delta13), deletion of TP53, ploidy status and immunoglobulin heavy chain (IgH) translocations were evaluated by fluorescence in situ hybridization in patients with monoclonal gammopathy of undetermined significance (n=189), smoldering multiple myeloma (n=127) and multiple myeloma (n=400). Overall, Delta13 (25%, 34% and 47%), 16q23 deletions (6%, 8% and 21%) and 17p13 deletions (3%, 1% and 10%) were less frequent in patients with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma than in those with multiple myeloma. When distinct genetic groups were considered, no differences in the prevalence of Delta13 were found with t(4;14)(p16;q32) and t(14;16)(q32;q23) among the three diagnostic groups; in contrast Delta13 was rarer in t(11;14)(q13;q32) in patients with monoclonal gammopathy (1/28) and smoldering myeloma (2/13) than in those with multiple myeloma (40%). Similar results were seen for the few t(6;14)(p21;q32) cases: 0/3 patients with monoclonal gammopathy or smoldering myeloma had the Delta13, whereas 4/6 (67%) patients with multiple myeloma and this translocation also had the deletion. In multiple myeloma patients with both an IgH translocation and Delta13, the proportions of cells affected by the two abnormalities were similar, as was the case for t(4;14) and t(14;16) monoclonal gammopathy patients positive for Delta13. In contrast, in monoclonal gammopathy patients with t(14;20)(q32;q11), the translocation was present in almost all cells, while the Delta13 was present in only a sub-population. These results indicate that the presence and time of occurrence of Delta13 depends on the presence of specific concurrent abnormalities. The observation that Delta13 was extremely rare in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma with translocations directly involving cyclin D genes (CCND1 and CCND3) suggest a possible role of Delta13 in the progression of the disease specifically in these genetic sub-groups. (clinicaltrials.gov identifier: ISRCTN 68454111; UKCRN ID 1176).

Flow Cytometric Phenotyping and Analysis of T Regulatory Cells in Monoclonal Gammopathy Patients.

Abstract 2819Poster Board II-795 Introduction:T regulatory (T reg) cells play an important role in maintenance of self-tolerance, control of immune functions and regulation of homeostasis of T cell populations. T reg cells also influence immune responses against infectious agents and tumor cells. Several recent studies have demonstrated increased counts of T reg cells in patients with solid tumors and hematological malignancies. Monoclonal gammopathies (MGs) are associated with abnormal immune functions, mainly immunoparesis. Abnormal T cell functions have also been reported. We have evaluated the frequency of T reg cells in MG patients in different stages of disease. Patients and Methods:A total of 69 patients (pts) with MGs were enrolled in this pilot study (16% 1169 with MGUS, 7% 569 with asymptomatic myeloma (AMM), 58% 4069 with newly diagnosed symptomatic myeloma (MM), and 19% 1369 with relapsed MM). Twelve healthy individuals were also screened for the frequency of T reg cells in the peripheral blood and used as a control group. Flow cytometric phenotyping of T reg cells was carried by staining for CD4, CD25 and CD127, followed by intracellular staining for Foxp3 was performed. T reg cells were identified by the phenotype of CD4+ CD25hi+ CD127dim Foxp3+. Both peripheral blood (PB) and bone marrow (BM) samples were analyzed for the frequency of T reg cells. Subgroups of pts were compared based on the bone marrow plasma cell (BMPC) infiltration (50 pts ' 10% vs. 19 pts >10%) and serum monoclonal immunoglobulin (M-Ig) level (25 pts ' 30g/L vs. 34 pts >30g/L). Results:Our study showed an overall trend towards the increase of T reg cell frequency in the peripheral blood of MG pts comparing to healthy individuals. The following T reg cell percentages were observed in the PB of different groups: healthy individuals 4.7% (range: 3.4 - 6.7%), MGUS 4.9% (range: 2.1 - 7.3%), AMM 4.5% (range: 3.0 - 7.5%), MM 5.5% (range: 2.7 - 11.7%) and relapsed MM 6.8% (range: 4.8 - 8.7%). Statistically significant differences were observed between healthy individuals vs. relapsed MM (p= 0.008), MGUS vs. relapsed MM (p= 0.043), and AMM vs. relapsed MM (p= 0.035). Similarly, in the bone marrow the frequency of T reg cells was also proportional to the increasing severity of MG. The following percentages of T reg cells were observed in the BM: MGUS 3.5% (range: 2.1 - 6.6%), AMM 3.0% (range: 2.6 - 7.5%), MM 4.7% (range: 2.3 - 7.5%) and relapsed MM 5.6% (range: 3.3 - 7.0%). Differences between the groups did not reach statistical significance. We have found a significant positive correlation between BMPC infiltration and BM T reg cell percentage (r= 0.25, p= 0.030). Increased frequency in the level of T reg cells was observed in the group with >10% of BMPC infiltration comparing to the group with ' 10% of BMPC infiltration (5.2% [3.0 - 7.4%] vs. 4.7% [2.1 - 7.6%] respectively; p= 0.41). Increased number of T reg cells was also observed in both PB and BM of pts with >30g/L of serum M-Ig comparing to pts with ' 30g/L of serum M-Ig (PB: 5.9% [2.7- 11.7%] vs. 5.0% [2.8 - 8.7%]) and (BM: 5.3% [2.3 -7.5%] vs. 4.7% [2.6-7.5%]), although no statistical significant differences have been observed between the two groups. Conclusion:Our results suggest that increases in the frequency of T reg cells in monoclonal gammopathies are associated with advancing disease. The positive correlation between BMPC infiltration and BM T reg cells indicates that T reg cells could play a role in the pathophysiology of monoclonal plasma cell disorders.This work was supported by MSMTLC06027, MSM0021622434 and MSMTNPVII 2B06058. Disclosures:Hajek:Janssen-Cilag: Honoraria.

Monoclonal gammopathy in systemic lupus erythematosus

We studied the prevalence, type and associated features of monoclonal gammopathy in patients with systemic lupus erythematosus (SLE). Patients included in the University of Toronto Lupus Database with an abnormal band on serum electropheresis were identified. Monoclonal gammopathy patients were matched with two controls each from the same database by age at SLE diagnosis, sex and disease duration. Of 1083 patients followed at the Lupus Clinic 59 (5.4%) were identified with monoclonal gammopathy. The gammopathies included 32 with IgG, 14 IgM and 12 IgA, one undefined. Nine (15.3%) malignancies were detected in monoclonal gammopathy and 12 (10.1%) in the controls during the entire course of their disease (P = 0.13). None had multiple myeloma. There was no difference between patients with monoclonal gammopathy and their controls with respect to disease activity, damage, or dose of steroids. The mean ESR and gammaglobulin levels in the monoclonal gammopathy patients were higher than the controls at last visit. We conclude that monoclonal gammopathy is more frequent in SLE patients than in the general population and has a benign course in patients with SLE. There were no differences in disease manifestations, treatment approaches, or malignancies between SLE patients with and those without monoclonal gammopathy.

Characterization of neuropathies associated with elevated IgM serum levels

Background In contrast to the IgM monoclonal gammopathies the neuropathy associated with polyclonal IgM gammopathy has not been well characterized. Objective To characterize the neuropathy in patients with elevated serum IgM. Design Retrospective review. Setting Academically based neuropathy center. Patients 45 patients with elevated quantitative immunoglobulin M were identified. Main outcome measures Patients are described with regard to clinical phenotype, electrodiagnostic features of demyelination or focality, presence of IgM monoclonal gammopathy, and presence of autoantibody activity. Results Elevated IgM levels occurred in 45 (11.5%) of 391 patients. Of these, 24 (53%) had polyclonal gammopathy and 21 (47%) had an IgM monoclonal gammopathy. Anti-nerve antibodies occurred in 14/21 (67%) of patients with monoclonal gammopathy, as compared to 1/24 (4%) with polyclonal gammopathy. Clinically, most patients in all groups had a predominantly large fiber sensory neuropathy. Thirty patients underwent electrodiagnostic testing. Of these, 22/30 (73%) fulfilled at least one published criteria for CIDP, including 92% of the monoclonal gammopathy patients and 59% of the polyclonal gammopathy patients. Fifteen of the 30 patients had evidence of focality or multifocality, with 14 of these 15 showing evidence of demyelination. Conclusions Monoclonal and polyclonal IgM patients have similar distributions of neuropathy phenotypes. Neuropathy in association with elevated serum IgM, with or without monoclonal gammopathy or autoantibody activity, is more likely to be demyelinating or multifocal. Serum quantitative IgM level and immunofixation in neuropathy patients may aid in identification of an immune mediated or a demyelinating component.